Enhancing Durum Wheat Grain Quality and Safety: The Role of Organic Foliar Fertilization and Selenium Biofortification in Mediterranean Farming Systems

Enhancing Durum Wheat Grain Quality and Safety: The Role of Organic Foliar Fertilization and Selenium Biofortification in Mediterranean Farming Systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing Durum Wheat Grain Quality and Safety: The Role of Organic Foliar Fertilization and Selenium Biofortification in Mediterranean Farming Systems Federica Carucci, Giuseppe Gatta, Massimo Blandino, Valentina Scarpino, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7797922/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives Durum wheat quality and safety are increasingly shaped by agronomic practices that aimed to reduce reliance on synthetic inputs while enhancing nutritional and technological value. Under Mediterranean conditions, where recurrent abiotic stresses linked to climate change pose significant challenges for agricultural production and food security, novel fertilisation strategies are necessary. This study investigates the combined effects of organic foliar fertilization and selenium (Se) biofortification on gluten protein quality, grain nutrient enrichment, and mycotoxin contamination in durum wheat ( Triticum turgidum ssp. durum L.). Methods Field experiments were carried out over two growing seasons (2018 and 2019), using modern (Marco Aurelio, Nadif) and old (Cappelli, old Saragolla) cultivars. Treatments included foliar applications of organic nitrogen (N), sulphur (S), combined N + S, and Se biofortification. Analyses focused on gluten protein polymerization and assembly, grain concentrations of [N], [S], [Se], [Fe], and [Zn], and the profile of major and emerging mycotoxins. Results Organic foliar N and S application enhanced grain [S], [Fe], and [Zn] and improved gluten protein assembly and gluten index. Se biofortification effectively increased grain Se content without compromising other nutrient concentrations and significantly reduced contamination by both deoxynivalenol and emerging mycotoxins (enniatins, moniliformin). The modern cultivar Marco Aurelio combined high concentrations of micronutrients and gluten quality indices, even under water-limited conditions. Conclusion This study provides novel evidence that integrating organic foliar fertilization with Se biofortification can simultaneously improve the nutritional, technological, and safety traits of durum wheat. The findings point to agronomic strategies capable of improving grain quality and reducing mycotoxin risks in Mediterranean environments, thereby supporting more sustainable and resilient wheat production systems with direct relevance for food security. Agronomic Biofortification Nitrogen Sulphur Selenium Gluten Protein Assembly Mycotoxins Micronutrient Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Durum wheat ( Triticum turgidum spp. durum L.) represents about 7% of global wheat production [1]. Still, its economic and nutritional importance is considerable due to its wide use in products such as pasta, couscous, bulgur, and specialty breads [2]. The technological suitability of durum wheat semolina for these products is largely determined by its gluten protein composition [3, 4]. In recent years, however, increasing attention has been paid not only to gluten-related processing traits, but also to broader aspects of grain quality, including nutritional value, food safety, and environmental sustainability. This shift reflects the evolving priorities of both consumers and policymakers, moving beyond yield as the sole breeding and agronomic target. One major challenge in sustainable wheat production is the widespread use of mineral nitrogen (N) fertilizers, which, despite their productivity benefits, are associated with low uptake efficiency (< 40%), high costs, soil degradation, and significant environmental impacts [5, 6, 7]. To mitigate these effects, the European Green Deal is proposing targets to reduce mineral fertilizer use [8]. This goal, however, raises critical issues for high-input crops like wheat, for which fertilization also plays a key role in quality, especially under increasing abiotic stress conditions linked to climate change [9, 10]. Organic fertilizers offer a promising alternative [11, 12], even if their lower nutrient content and slower mineralization can limit durum wheat yield and quality [13, 14]. In organic systems, N is often applied only at sowing, and delayed mineralization during spring can result in insufficient availability during key phenological stages [15, 16]. Foliar application of organic N formulations may represent a viable strategy to improve N use efficiency and guarantee the wheat grain quality target. As well as the need for agronomic sustainability, there is a growing awareness of the nutritional limitations of wheat, which, despite providing about 20% of global dietary protein and calories [17, 18], is inherently low in essential micronutrients such as selenium (Se). In Europe, Se intake is often below recommended levels [19], and this deficiency may worsen due to climate-driven declines in soil Se content [20]. Agronomic biofortification has been shown to improve Se accumulation in cereal grains [21, 22], yet its broader effects on plant physiology, including interactions with N, S, Zn, and Fe uptake, remain underexplored. Durum wheat also faces increasing threats to food safety due to its susceptibility to Fusarium Head Blight (FHB), especially in Mediterranean environments. FHB leads to contamination by deoxynivalenol (DON) and other mycotoxins, posing a major barrier to production and export [23, 24] affecting also the food security. Although DON levels in Southern Italy often remain within regulatory limits [25], recent EU regulations (e.g., EC No. 2024/1022) and the rise in wholegrain consumption [26] have increased the demand for mitigation strategies. Moreover, the presence of emerging mycotoxins such as DON-3-glucoside, enniatins (ENNs), and moniliformin (MON) raises additional concerns [27, 28]. With a planned reduction in pesticide use requested by the legislation and supply chain (Boix-Fayos and de Vente, 2023) [8], agronomic tools with dual physiological and protective effects, such as S and Se, may offer novel solutions. To date, however, to the best of our knowledge, no evidence is available on the combined effects of organic foliar fertilization and selenium biofortification in durum wheat, particularly regarding their impact on gluten protein structure, nutrient enrichment, and mycotoxin contamination. Therefore, this study aims to provide new insights into the role of organic foliar fertilization and Se biofortification in durum wheat. Specifically, we evaluate their effects on gluten protein polymerization and assembly, the accumulation of key nutrients (S, Fe, Zn, Se), and the occurrence of major and emerging mycotoxins under different seasonal conditions and genetic backgrounds. By integrating nutritional, technological, and safety traits, the main goal is to identify sustainable agronomic strategies that simultaneously enhanced durum wheat technological, nutritional and safety quality. 2. Materials and methods 2.1 Field Experiments The field experiments conducted in two consecutive growing seasons, 2017-18 and 2018-19 (namely 2018 and 2019, respectively), have already been described in Carucci et al. [29]. Briefly, two old (Cappelli and old Saragolla) and two moderns (Marco Aurelio and Nadif) Italian durum wheat cultivars were grown in southern Italy (Foggia, 41°46′ N, 16°54′ E) under four different organic foliar fertilization: 1) control (CONT) fertilized with dry blood meal (ORGAZOT®, AGM) in a single application at seeding; 2) CONT plus foliar S (water solution of bio sulfur Fytofert®S, De Sangosse (10% w / v )) application at flag leaf sheath opening stage (BBCH stage 47) [30] (CONT + S); 3) CONT plus N foliar application (water solution of blood meal EUTROFIT®, AGM (5% w / v )) at the beginning of heading (BBCH stage 51) (CONT + N); 4) CONT plus the combination of N and S foliar applications at flag leaf sheath opening stage and at the beginning of the heading, respectively (CONT + NS). Furthermore, two different Se applications were also considered: 1) Se0, control without Se; 2) Se60, with one application of sodium selenate (Na 2 SeO 4 ), at rate of 60 g ha − 1 [31] at the booting stage (BBCH stage 41). A split-split-plot design with three replicates was adopted: the cultivar was the main plot, the organic foliar fertilization was the plot, and the Se application was the sub-plot (10.2 m 2 ) for a total of 96 experimental units. Except for the compared agronomic factor, the standard agronomical management for the growing area and the organic wheat was adopted in all the plots (Table S1 ; additional file). The precipitations and daily temperatures were measured at meteorological stations located near the experimental field. The main meteorological data together with the crop length in the two years are reported in Table 1 . Durum wheat grain was machine-harvested at full maturity using a Wintersteiger Nursery Master Elite plot combine (Wintersteiger Inc., Ried im Innkreis, Austria). The harvested grains were mixed thoroughly, and 1 kg grain samples were taken from each plot to obtain a representative sample for the qualitative analysis. For mycotoxin analysis and determination of the macro and microelement grain concentration, representative sub-samples (300 g) were ground to whole-meal using a laboratory centrifugal mill Cyclotec Sample Mill 1093 (Foss Tecator, Hillerød, Denmark) and then manually sieved through a 1-mm sieve. The semolina flour used for gluten protein polymers and gluten index analysis has been obtained from kernels milled by Bona mill 4 cylinders (sieve 180 µm). Table 1 Meteorological data and crop length related to the two experimental years. Parameters Measurement unit 2018 2019 Crop cycle days 211 207 Grain filling days 66 60 Crop cycle rainfall mm 401 299 Grain filling rainfall mm 203 99 Crop cycle Avg. T °C 13.3 12.6 Avg. T max during grain filling °C 28.8 24.5 30° C < T max 35°C days 3 8 2.2 Analysis of gluten protein parameters and gluten index Size exclusion high performance liquid chromatography (SEC-HPLC) analysis of Sodium Dodecyl Sulfate (SDS) extractable and unextractable proteins was performed according to the method reported in Gagliardi et al. [32]. The SDS-unextractable fraction profile showed one peak containing the largest glutenin polymers, insoluble in SDS solution alone, but rendered soluble by sonication called insoluble large protein (ILP). The SDS soluble fraction profiles were divided into four areas. The first two areas correspond to soluble large (SLP) and medium-sized polymers, respectively, which, together with ILP determine the polymeric protein fraction. The third and the fourth areas represent the amounts of the monomeric protein fraction together. The proportions of each area were calculated as percentages of the total areas of the two chromatograms (SDS-insoluble and SDS-soluble fractions). Finally, the amount of monomeric over polymeric proteins (mon/pol) and the proportion of unextractable polymeric protein (UPP), as the ratio between ILP and the sum of SLP and ILP areas (*100), were calculated. Furthermore, the gluten index (GI), an indicator of gluten strength, was determined on semolina samples using the Glutomatic system according to ICC standard 155 [33]. 2.3 Determination of N, S, Se, Fe and Zn grain concentration N and S grain concentrations (hereinafter called [N] and [S], respectively) of all whole-meal samples were determined in triplicate using a Leco CHNS 628 Analyzer (Leco corporation, St. Joseph, Michigan). The grain [N] was determined on 0.0500 g of whole grain flour, while the grain [S] was determined on 0.1000 g of material. The Se grain concentration (hereinafter called [Se]) was determined using an inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700x, Agilent Technologies Inc., USA) after acid digestion in a microwave oven (MSP 1000, CEM, Matheus, NC), as reported in Carucci et al. [34]. Briefly, 0.5000 g of whole grain flour was digested with nitric acid and hydrogen peroxide. The experimental conditions for ICP-MS analyses were as follows: RF power of 1550 W, sample depth of 8 mm, plasma gas flow rate 15.0 L·min − 1 , auxiliary gas flow rate 0.90 L·min − 1 and conical nebulizer with a flow rate 1.01 L·min − 1 . Se quantification was performed by external calibration with the isotope 78 Se. Regarding the analysis of Fe and Zn grain concentration (hereinafter called [Fe] and [Zn], respectively), around 0.5000 g of wheat grains were accurately weight in an Xpress tube and then digested in a microwave oven (MSP 1000, CEM, Matheus, NC) with 6.0 mL of a mixture of concentrated nitric acid (Scharlab, Spain) and 33% (v/v) hydrogen peroxide (Dismadel, Spain) in a 3:1 ratio. The program of the microwave over for the mineralization of the samples is detailed in previous work (Moreno-Martín et al. 2020). The digested extracts were transferred to 25.0 mL with ultrapure water, filtered through a 0.22 µm syringe Nylon filters (Dismadel, Spain) and analyzed by flame atomic absorption spectrometry (FAAS). The operating conditions of the FAAS were a flame of air-acetylene mixture with a flow rate of 2.5/ 4.0 mL min − 1 , respectively. Fe and Zn were measured in continuous mode with a band pass of 0.2 mm with lamp currents of 30 and 15 mA, and at wavelengths of 248.3 and 213.9 nm, respectively. External calibration using dissolved Fe and Zn ionic standards (Merck, Spain), ranging from 0 to 1 mg L − 1 for Zn and 0 to 5 mg L − 1 for Fe, with an acid content equivalent to that of the samples resulting from the digestion procedure, was utilized to quantify both elements. Analyses were performed in triplicate. 2.4 Determination of Mycotoxin Mycotoxin analysis was performed by a multi-mycotoxin liquid chromatography with tandem mass spectrometry (LC-MS/MS) method as described by Scarpino et al. [35]. The analysis was carried out on a sub-set of the considered treatment for both growing seasons, considering the combination of two cultivars (old Saragolla vs Nadif), two organic foliar fertilizers (CONT + N vs CONT + NS), and two Se application (Se0 vs Se60). Briefly, 5.0000 g of each wholegrain flour was extracted with 20 mL of CH 3 CN/H 2 O/CH 3 COOH (79/20/1, v/v/v), according to the dilute-and-shoot method reported by Scarpino et al. [35], and 20 µL of the diluted filtered extracts was analyzed without any further pretreatment. LC-MS/MS analysis was carried out on a Varian 310 triple quadrupole (TQ) mass spectrometer (Varian, Milan, Italy), equipped with an electrospray ionization (ESI) source, a 212 LC pump, a ProStar 410 AutoSampler and dedicated software. LC separations were performed on a Gemini-NX C18 100 x 2.0 mm i.d., 3 µm particle size, 110 Å, equipped with a C18 4 x 2 mm security guard cartridge column (Phenomenex, Torrance, CA, United States), using water (eluent A) and methanol (eluent B), both acidified with 0.1% v/v CH 3 COOH, as eluents that were delivered at 200 µL min ­1 . The chromatographic and MS conditions and the results pertaining to the linearity range, the limit of detection (LOD), the limit of quantification (LOQ), the apparent recovery RA (%), the matrix effects obtained through the evaluation of the signal suppression/enhancement SSE (%), and the recovery of the extraction RE (%) were described in detail by Scarpino et al. [35]. The results of the mycotoxin concentrations were corrected for the recovery rates and expressed as µg kg ­-1 on dry matter basis. 2.5 Statistical Analysis All statistical analyses were conducted by using the R statistical environment (R Core Team, 2018). The dataset was tested according to the basic assumptions of analysis of variance (ANOVA). The normal distribution of the experimental error and the common variance of the experimental error were verified through Shapiro–Wilk and Levene’s tests, respectively. When required, Box–Cox transformations [36] were applied before analysis. Gluten protein polymers, gluten index, and macro and microelements concentration in grain were analyzed using the split-split-plot linear mixed model ANOVA approach, using the package “nlme” [37]. Cultivar (Cappelli, old Saragolla, Marco Aurelio, and Nadif), organic foliar fertilization (CONT, CONT + S, CONT + N, and CONT + NS), and Se application (Se0 vs Se60) were included in the model as fixed factors, together with their interaction. The growing season was considered as a fixed factor, while the “blocks within growing season”, the “main-plots within blocks within growing season” and the “plots within main-plots within blocks within growing season” were added as random effects to account for blocking units. The statistical significance of the difference among the means was determined using post-hoc Tukey’s test (function “means”) at the 5% probability level. Moreover, to analyze the relationships between gluten protein polymers, GI, and the grain [N], [S], [Se], [Fe] and [Zn], a multivariate analysis was applied to reduce dataset complexity and enhance correlation visualization [38]. Four cultivars, four organic foliar fertilization treatments, and two Se applications were incorporated across two growing seasons as descriptors according to the procedure outlined by Mongiano et al. [39]. Thus, Principal Component Analysis (PCA) was performed after first conducting a correlation analysis. The following experimental traits, proportion of ILP, SLP, monomeric protein, mon/pol ratio, UPP, GI, and grain [N], [S], [Se], [Fe] and [Zn] were used as active quantitative variables, which were centered and scaled prior to PCA, through a procedure which involved diagonalizing the correlation matrix and extracting the associated eigenvectors and eigenvalues. The supplementary qualitative variables, which included factors such as year, cultivar, organic foliar fertilization, and Se application, did not influence the calculation of the principal components. Instead, their coordinates were determined as the barycenter of individuals plotted in the PCA space. Next, we implemented Hierarchical Clustering on Principal Components (HCPC) using Euclidean distance and Ward’s method to identify data clusters with similar behavior. For each experimental factor, the average value within each cluster was tested against the null hypothesis that its distribution remained consistent across all clusters: $$\:u=\frac{{x̄}_{q}-\:x̄}{\sqrt{\frac{{s}^{2}}{{n}_{q}}\:\left(\frac{N-{n}_{q}}{N-1}\right)}}$$ where n q is the number of data points in cluster q, N is the total number of data points, and s is the global standard deviation. We also assessed each cluster's composition, which was characterized considering the representativeness of the qualitative variables used in the PCA, using an alpha level α = 0.05 for all statistical tests. PCA and clustering computations were performed using the FactoMineR package [40]. To assess differences between the resulting clusters, analysis of variance (one-way ANOVA, function “aov”) and the post-hoc Tukey’s test (function “means”) were performed at the 5% probability level. The mycotoxin concentrations in grain were modelled in a linear mixed model using the package “nlme” [37] for data analysis. Cultivar (Nadif, old Saragolla), organic foliar fertilization (CONT + N and CONT + NS), and Se application (Se0 and Se60) were included in the model as fixed factors, together with their interaction. The growing season was considered as a fixed factor, while the “blocks within growing seasons”, the “main-plots within blocks within growing seasons”, and the “plots within main-plots within blocks within growing seasons” were added as random effects to account for blocking units. The ANOVA was performed, and the statistical significance of the difference among the means was determined using post-hoc Tukey’s test (function “emmeans”) at the 5% probability level. Finally, Pearson’s correlations were calculated between the mycotoxin concentration in grain and all the durum wheat traits investigated (gluten protein parameters, gluten index, macro- and micro-elements concentration in grain) on old Saragolla and Nadif cultivars, on CONT + N and CONT + NS organic foliar fertilization, and on the Se application under the two growing seasons, using the “corrplot” package [41]. The package ggplot2 was used [42] for all the visual representation of the data. 3. Results 3.1 Gluten protein parameters and gluten index ANOVA generally showed a significant effect of year, cultivar, foliar fertilization, Se biofortification, and their interactions on gluten protein parameters and gluten index (Table S2, additional file) due to the high number and complexity of the interactions, these will be better explored through multivariate analysis. The two experimental years significantly affected the proportion of gluten polymers and monomers and GI (Table 2 ). In the second year, there was the highest proportion of both SLP and ILP with a consequent significant increase of the polymeric fraction, and the lowest proportion of monomeric proteins, mon/pol ratio, UPP, and GI. The effect of the durum wheat cultivars was also significant (Table 2 ). Among the two old cultivars, Cappelli exhibited the highest proportion of SLP and the lowest GI values, while old Saragolla had the highest proportion of monomeric protein and mon/pol ratio and the lowest proportion of polymeric protein. For the modern cultivars, Marco Aurelio showed the highest proportions of ILP, UPP, and polymeric protein, along with the highest GI value, and the lowest proportions of monomeric protein and mon/pol ratio, while Nadif showed intermediate values for all the parameters considered. Regarding organic foliar fertilization, CONT + S significantly increased the proportion of SLP and GI value. Conversely, CONT + NS significantly decreased the proportion of SLP and significantly increased the proportion of UPP. CONT + N resulted in the lowest GI value. The organic foliar fertilization adopted did not affect the proportion of polymeric and monomeric proteins or their mon/pol ratio (Table 2 ). Finally, Se application did not significantly impact gluten protein parameters or the GI (Table 2 ). Table 2 Effect of year, cultivar, foliar fertilization, and selenium application on protein fraction resulting from analysis of variance (ANOVA). Factor SLP (%) ILP (%) Polymeric protein fraction (%) Monomeric protein fraction (%) mon/pol (-) UPP (%) GI (-) Year 2018 26.7 ± 3.6 b 9.2 ± 3.7 b 46.8 ± 3.5 b 53.2 ± 3.5 a 1.15 ± 0.16 a 25.4 ± 8.8 a 64.2 ± 31.6 a 2019 31.2 ± 2.9 a 10.4 ± 4.6 a 54.6 ± 4.3 a 45.4 ± 4.3 b 0.84 ± 0.14 b 24.3 ± 8.8 b 42.9 ± 31.0 b Cultivar Cappelli 32.9 ± 2.7 a 7.9 ± 3.5 c 52.2 ± 3.0 b 47.8 ± 3.0 c 0.92 ± 0.12 c 19.1 ± 7.7 c 13.9 ± 15.1 d old Saragolla 27.8 ± 2.1 b 7.0 ± 1.4 c 46.9 ± 3.1 d 53.1 ± 3.1 a 1.14 ± 0.15 a 20.1 ± 4.0 c 36.0 ± 12.1 c Marco Aurelio 27.4 ± 4.9 b 14.1 ± 4.2 a 54.5 ± 6.4 a 45.5 ± 6.4 d 0.86 ± 0.22 d 33.8 ± 8.4 a 86.9 ± 12.8 a Nadif 27.8 ± 3.0 b 10.2 ± 2.8 b 49.2 ± 5.5 c 50.8 ± 5.5 b 1.06 ± 0.23 b 26.5 ± 5.3 b 77.5 ± 15.3 b Foliar fertilization CONT 29.0 ± 4.7 b 10.0 ± 4.0 b 50.6 ± 5.5 49.4 ± 5.5 1.00 ± 0.21 25.4 ± 8.8 b 53.6 ± 34.5 b CONT + N 28.8 ± 3.5 b 10.0 ± 5.0 b 50.8 ± 6.0 49.2 ± 6.0 0.99 ± 0.23 24.9 ± 10.0 b 50.7 ± 32.6 c CONT + S 30.1 ± 3.4 a 8.5 ± 4.1 c 50.3 ± 5.6 49.7 ± 5.6 1.01 ± 0.22 21.4 ± 8.1 c 56.5 ± 31.2 a CONT + NS 27.9 ± 4.0 c 10.8 ± 3.3 a 51.1 ± 5.2 48.9 ± 5.2 0.98 ± 0.20 27.7 ± 7.26 a 53.4 ± 34.3 b Selenium application Se0 28.9 ± 4.2 9.8 ± 4.1 50.8 ± 5.6 49.2 ± 5.6 0.99 ± 0.22 25.0 ± 8.8 53.2 ± 34.4 Se60 29.0 ± 3.8 9.7 ± 4.4 50.6 ± 5.5 49.4 ± 5.5 1.00 ± 0.21 24.8 ± 8.9 53.9 ± 31.7 CONT: wheat fertilized with dry blood meal in a single application at seeding; CONT + N: CONT plus N foliar application at the beginning of heading (BBCH stage 51); CONT + S: CONT plus foliar S application at flag leaf sheath opening stage (BBCH stage 47); CONT + NS: CONT plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively; Se0: control without selenium; Se60: application of sodium selenate (Na 2 SeO4), at rate of 60 g ha − 1 at booting stage (BBCH stage 41). SLP: Soluble large polymers; ILP: insoluble large polymers; mon/pol: monomeric/polymeric ratio; UPP: unextractable polymeric protein; GI: gluten index According to Tukey's test, for each factor, values in each column followed by different letters are significantly different ( p -value ≤ 0.05). Data are reported as means ± standard deviation (3 replicates). 3.2 Macro and microelements concentration in the grain The analysis of variance (ANOVA) generally showed a significant effect of year, cultivar, foliar fertilization, Se biofortification, and their interactions on grain macro and microelements concentration (Table S3, additional file). Due to the high number and complexity of the interactions, these will be better explored through multivariate analysis. The two years significantly affected the concentration of macro and microelements in the grain. In the first wetter year, the highest values of grain [N], [Se], and [Fe] were observed. On the contrary, the grain [S] and [Zn] were higher in the second drier year (Table 3 ). Among the cultivars, Cappelli showed a significantly higher grain [N] and [S], the latter together with the cultivar old Saragolla. Old Saragolla differed from the other cultivars for its lowest grain [Se]. As for the two modern cultivars, Marco Aurelio differed for the highest grain [Fe] and Nadif for the highest grain [Se] and the lowest grain [S] and [Zn] (Table 3 ). As for the organic foliar fertilization strategies under study, the highest grain [S] and [Fe] were obtained by CONT + NS fertilization, while grain [Se] significantly decreased using CONT + S or CONT + NS strategies. Finally, the highest grain [Zn] was observed utilizing CONT + NS and the lowest utilizing CONT + N strategies (Table 3 ). The foliar Se application significantly increased the grain [Se] with no effect on the other detected micronutrient grain concentration (Table 3 ). Table 3 Effect of year, cultivar, foliar fertilization, and selenium application on macro and micro nutrient grain concentration resulting from analysis of variance (ANOVA). Grain [N] (%) Grain [S] (%) Grain [Se] (mg kg − 1 ) Grain [Fe] (mg kg − 1 ) Grain [Zn] (mg kg − 1 ) Year 2018 2.1 ± 0.2 a 0.12 ± 0.05 b 1.07 ± 0.61 a 40.4 ± 10.3 a 19.7 ± 2.8 b 2019 1.29 ± 0.15 b 0.20 ± 0.01 a 0.96 ± 0.59 b 31.9 ± 9.2 b 29.2 ± 2.4 a Cultivar Cappelli 1.79 ± 0.36 a 0.18 ± 0.04 a 1.07 ± 0.55 b 33.7 ± 5.9 b 24.8 ± 5.9 a old Saragolla 1.70 ± 0.46 b 0.18 ± 0.03 a 0.77 ± 0.56 c 32.9 ± 10.3 b 24.9 ± 4.8 a Marco Aurelio 1.63 ± 0.52 b 0.15 ± 0.04 b 0.98 ± 0.52 b 46.2 ± 12.8 a 25.3 ± 4.7 a Nadif 1.62 ± 0.39 b 0.13 ± 0.07 c 1.25 ± 0.69 a 31.7 ± 4.0 b 22.9 ± 6.0 b Foliar fertilization CONT 1.65 ± 0.42 0.16 ± 0.05 b 1.12 ± 0.65 a 35.6 ± 10.7 b 24.3 ± 5.7 ab CONT + N 1.68 ± 0.42 0.16 ± 0.06 b 1.12 ± 0.67 a 35.1 ± 9.1 b 23.9 ± 5.7 b CONT + S 1.71 ± 0.47 0.16 ± 0.05 b 0.92 ± 0.53 b 34.5 ± 10.4 b 24.3 ± 5.5 ab CONT + NS 1.70 ± 0.44 0.17 ± 0.05 a 0.90 ± 0.53 b 39.3 ± 11.8 a 25.4 ± 4.7 a Selenium application Se0 1.68 ± 0.43 0.16 ± 0.05 0.50 ± 0.21 b 36.3 ± 10.5 24.7 ± 5.1 Se60 1.69 ± 0.44 0.16 ± 0.05 1.53 ± 0.39 a 35.9 ± 10.9 24.2 ± 5.7 CONT: wheat fertilized with dry blood meal in a single application at seeding; CONT + N: CONT plus N foliar application at the beginning of heading (BBCH stage 51); CONT + S: CONT plus foliar S application at flag leaf sheath opening stage (BBCH stage 47); CONT + NS: CONT plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively; Se0: control without selenium; Se60: application of sodium selenate (Na 2 SeO4), at rate of 60 g ha − 1 at booting stage (BBCH stage 41). Grain [N], [S], [Se], [Fe] and [Zn]: nitrogen, sulphur, selenium, iron and zinc grain concentrations. According to Tukey's test, for each factor, values in each column followed by different letters are significantly different ( p -value ≤ 0.05). Data are reported as means ± standard deviation (3 replicates). 3.3 Principal Component Analysis (PCA) Correlation analysis revealed a complex network of relationships among protein fractions, gluten quality traits, and grain mineral concentrations (Fig. 1 ). ILP showed positive associations with polymeric protein fraction, UPP, GI, and grain [Fe], while SLP was positively related to polymeric protein fraction and grain [S] and [Zn]. Polymeric protein fraction correlated positively with UPP, and grain [S] and [Zn]. Grain [N] was positively associated with monomeric protein fraction and grain [Fe], and grain [S] correlated positively with grain [Zn]. Grain [Fe] also showed positive associations with UPP, GI, and grain [N] (Fig. 1 ). Negative correlations were observed between ILP and SLP, monomeric protein fraction, and grain [N]; between SLP and monomeric protein fraction, GI, and grain [N] and [Fe]; and between polymeric protein fraction and grain [N]. Grain [N] was negatively associated with grain [S] and [Zn], while grain [S] showed negative relationships with monomeric protein fraction, UPP, and GI. Finally, grain [Fe] was negatively related to monomeric protein fraction, grain [Zn], and GI (Fig. 1 ). Due to the high number of correlations observed among the different parameters evaluated, these were jointly considered in a multivariate approach and were processed statistically for principal component analysis (PCA). The PCA analysis showed that PC1 explained 45.2% of the total variance and was positively correlated with polymeric protein fraction, SLP, grain [S] and [Zn], and negatively correlated with monomeric protein fraction, mon/pol ratio, and grain [N] (Table 4 ). Thus, PC1 is linked to the “degree of polymerization”, mainly related to the capacity to form covalent bonds. On the other hand, PC2 explained 26.9% of the total variance and had strong positive correlations with ILP, UPP, GI, and grain [Fe] and could be described as a factor associated with "protein assembly degree" (Table 4 ). Table 4 Correlation coefficients between active quantitative variables, supplementary qualitative variables, and the first two Principal Components (PC), with the indication of the explained variance. Variable PC1 PC2 Quantitative active variables Polymeric protein fraction 0.94 *** 0.27 *** Monomeric protein fraction -0.94 *** -0.27 *** mon/pol -0.95 *** -0.23 *** SLP 0.64 *** -0.59 *** ILP 0.41 *** 0.88 *** UPP 0.15 * 0.95 *** GI -0.31 *** 0.75 *** Grain [N] -0.82 *** 0.08 ns Grain [S] 0.79 *** -0.35 *** Grain [Se] -0.03 ns 0.12 ns Grain [Fe] -0.26 *** 0.52 *** Grain [Zn] 0.83 *** -0.15 * Qualitative supplementary variables Year 0.76 *** 0.04 ** Cultivar 0.11 *** 0.65 *** Foliar Fertilization 0.00007 ns 0.03 ns Se application 0.002 ns 0.0008 ns Explained variance 45.2% 26.9% SLP: Soluble large polymers; ILP: insoluble large polymers; mon/pol: monomeric/polymeric ratio; UPP: unextractable polymeric protein; GI: gluten index; Grain [N], [S], [Se], [Fe] and [Zn]: nitrogen, sulphur, selenium, iron and zinc grain concentrations. As for the qualitative supplementary variables, the “year” variable had a stronger correlation with PC1, while the qualitative supplementary variable “cultivar” had a stronger correlation with PC2. No correlation was found for organic foliar fertilization and Se application with PC1 and PC2 (Table 4 ). 3.4 Cluster analysis Three clusters emerged from the hierarchical clustering performed on the extracted PCs (Figs. 2 and 3 ). Category frequency distributions within clusters for the qualitative variables highlighted that the year and the cultivar were significantly different from the overall frequency distribution according to χ 2 test at p-value ≤ 0.001, whereas organic foliar fertilization and Se application were not significant. As for the year, Cluster 1 (C1) was entirely composed of experimental data collected in 2018, Cluster 2 (C2) grouped 76% of the data from the 2019 growing season, and Cluster 3 (C3) grouped 95.5% of the data from the 2019 growing season. As for the cultivars, in C1 Marco Aurelio, Nadif and old Saragolla were equally represented, while Cappelli contributed to the cluster only for 6.4%. In C2, 57.7% of the data belonged to Cappelli, 33.8% to old Saragolla, and only 8.5% to Nadif, while Marco Aurelio was absent. Finally, for C3, 54.5% of the data belonged to Marco Aurelio, 40.9% to Nadif, and only 4.6% to Cappelli, while old Saragolla was absent. In all of the three clusters the four organic foliar fertilization and the Se application were equally represented (Fig. 2 ). C1 was positioned on the negative side of the PC1 factor; it was characterized by the lowest proportion of polymeric protein (Fig. 4 A) and SLP (Fig. 4 D), the highest proportion of the monomeric protein and mon/pol ratio (Fig. 4 B and C), the highest value of grain [N] and [Fe] (Fig. 5 A and D) and the lowest grain [S] and [Zn] (Fig. 5 B and E). C2 was mainly positioned on the negative side of the PC2 factor and it was characterized by the highest proportion of SLP (Fig. 4 D) and the lowest proportion of ILP, UPP, GI (Fig. 4 E, F, G) and grain [Fe] (Fig. 5 D). Cluster 3 was positioned on the positive side of both PC1 and PC2 (Fig. 3 ) and it was characterized by the highest proportion of polymeric protein fraction, ILP and UPP (Fig. 4 A, E and F), and the lowest proportion of monomeric protein fraction, mon/pol ratio (Fig. 4 B and C), and grain [N] (Fig. 5 A). 3.5 Mycotoxin contamination Each of the compared growing seasons and agronomic factors led to a significant effect on the grain contamination (Table S4; additional file). The 2018 growing season resulted in a higher content of all the detected mycotoxins compared to 2019, with an increase comprise between + 13% (DON) and + 46% (DON-3-G) (Table 5 ). According to the type of mycotoxins the cultivar resulted in a different effect: Nadif resulted in a content of DON and DON-3-G that is less than half of that measured in old Saragolla, otherwise this old genotype showed a lower contamination of both MON (­8%) and ENNtot (­82%). The application of S foliar fertilizer led to a significant reduction of all the mycotoxins, between 14% for DON and 50% for MON. Se application led to a stronger effect, with a reduction of 30% for DON, 50% for MON and 141% for ENNtot. Table 5 Effect of year, cultivar, organic foliar fertilization and Se biofortification on the mycotoxin contamination in durum wheat grain resulting from analysis of variance (ANOVA). DONtot DON DON-3-G MON ENNtot (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) Year 2018 359 ± 173 a 279 ± 135 a 57 ± 28 a 26 ± 7 a 133 ± 133 a 2019 302 ± 132 b 246 ± 108 b 35 ± 16 b 23 ± 11 b 91 ± 74 b Cultivar old Saragolla 463 ± 102 a 368 ± 75 a 63 ± 23 a 24 ± 7 b 35 ± 24 b Nadif 198 ± 44 b 156 ± 35 b 28 ± 9 b 26 ± 11 a 190 ± 104 a Foliar fertilization CONT + N 353 ± 155 a 278 ± 119 a 49 ± 26 a 30 ± 10 a 134 ± 124 a CONT + NS 309 ± 154 b 246 ± 125 b 43 ± 24 b 20 ± 5 b 90 ± 86 b Selenium application Se0 386 ± 168 a 304 ± 131 a 55 ± 28 a 30 ± 11 a 159 ± 129 a Se60 275 ± 119 b 220 ± 97 b 36 ± 17 b 20 ± 3 b 66 ± 51 b CONT + N: wheat fertilized with dry blood meal in a single application at seeding plus N foliar application at the beginning of heading (BBCH stage 51); CONT + NS: wheat fertilized with dry blood meal in a single application at seeding plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively; Se0: control without selenium; Se60: application of sodium selenate (Na 2 SeO 4 ), at rate of 60 g ha − 1 at booting stage (BBCH stage 41). DONtot: total deoxynivalenol forms, sum of DON, DON-3-G and 3-ADON; DON: deoxynivalenol; DON-3-G, deoxynivalenol-3-glucoside; MON: moniliformin; ENNtot: total enniatin forms, sum of ENN A, A 1 , B and B 1 . According to Tukey's test, for each factor, values in each column followed by different letters are significantly different ( p -value ≤ 0.05). Data are reported as means ± standard deviation (3 replicates). The interaction of year with cultivar and with organic foliar fertilization was significant. The combination of cultivar and year did not lead to a significant difference for DON-3-G and ENNtot, while only in 2019, old Saragolla showed a lower MON contamination compared to Nadif (Table 6 ). As far as the DONtot is concerned, in both growing seasons Nadif showed a lower content than old Saragolla, although the difference was higher in 2018. In a single year, the organic foliar fertilization did not significantly affect the contamination of DON forms; otherwise, the application of S significantly minimized the MON and ENNtot content in both growing years, with a higher percentage reduction in the 2019 growing season. Table 6 Effect of combination of the durum wheat cultivar or organic foliar fertilization with year on the mycotoxin contamination resulting from analysis of variance (ANOVA). DONtot DON DON-3-G MON ENNtot (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) Year Cultivar 2018 old Saragolla 514 ± 89 a 401 ± 63 a 79 ± 20 a 28 ± 6 a 37 ± 21 a Nadif 203 ± 44 c 156 ± 34 c 34 ± 8 a 25 ± 8 a 230 ± 127 a 2019 old Saragolla 412 ± 90 b 336 ± 73 b 48 ± 12 a 20 ± 5 b 34 ± 28 a Nadif 193 ± 45 c 156 ± 38 c 22 ± 5 a 27 ± 14 a 149 ± 57 a Year Foliar fertilization 2018 CONT + N 380 ± 179 a 293 ± 137 a 60 ± 29 a 30 ± 7 a 154 ± 158 a CONT + NS 338 ± 172 a 264 ± 137 a 53 ± 27 a 23 ± 5 b 112 ± 104 b 2019 CONT + N 325 ± 129 a 263 ± 103 a 37 ± 17 a 29 ± 13 a 153 ± 79 a CONT + NS 280 ± 136 a 229 ± 114 a 32 ± 15 a 18 ± 3 c 68 ± 62 c CONT + N: wheat fertilized with dry blood meal in a single application at seeding plus N foliar application at the beginning of heading (BBCH stage 51); CONT + NS: wheat fertilized with dry blood meal in a single application at seeding plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively. DONtot: total deoxynivalenol forms, sum of DON, DON-3-G and 3-ADON; DON: deoxynivalenol; DON-3-G: deoxynivalenol-3-glucoside; MON: moniliformin; ENNtot: total enniatin forms, sum of ENN A, A 1 , B and B 1 . According to Tukey's test, for each combination of agronomic factor and year, values in each column followed by different letters are significantly different ( p -value ≤ 0.05). Data are reported as means ± standard deviation (3 replicates). The effect of S application in term of mycotoxin contamination showed a significant interaction with the cultivar: this fertilization strategy was able to reduce the DONtot and DON contamination only for Nadif cultivar (Table 7 ). Otherwise, CONT + NS treatment reduced mycotoxins produced by F. avenaceum for both cultivars, with a higher effect for MON in Nadif (­39%) and for ENNtot in old Saragolla (­51%). Table 7 Effect of combination of the durum wheat cultivar and organic foliar fertilization on the mycotoxin contamination resulting from analysis of variance (ANOVA) performed. DONtot DON DON-3-G MON ENNtot (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) (µg kg − 1 ) Cultivar Foliar fertilizatio old Saragolla CONT + N 482 ± 109 a 379 ± 80 a 67 ± 24 a 26 ± 6 b 47 ± 27 c CONT + NS 444 ± 95 a 358 ± 71 a 60 ± 22 a 21 ± 6 c 23 ± 14 d Nadif CONT + N 223 ± 43 b 177 ± 34 b 31 ± 9 a 33 ± 12 a 222 ± 121 a CONT + NS 173 ± 29 c 135 ± 22 c 26 ± 8 a 20 ± 3 c 157 ± 76 b CONT + N: wheat fertilized with dry blood meal in a single application at seeding plus N foliar application at the beginning of heading (BBCH stage 51); CONT + NS: wheat fertilized with dry blood meal in a single application at seeding plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively. DONtot: total deoxynivalenol forms, sum of DON, DON-3-G and 3-ADON; DON: deoxynivalenol; DON-3-G: deoxynivalenol-3-glucoside; MON: moniliformin; ENNtot: total enniatin forms, sum of ENN A, A 1 , B and B 1 . According to Tukey's test, values in each column followed by different letters are significantly different ( p -value ≤ 0.05). Data are reported as means ± standard deviation (3 replicates). Although the Se application was always able to reduce significantly the content of the compared mycotoxins, the positive impact of this practice was more effective within the highest level of contamination: in Nadif cultivar for ENNtot (Fig. 6 ) or the CONT + N treatment for MON and ENNtot (Fig. 7 ). Finally, a network of relationships among grain mycotoxin contamination and protein fractions and grain mineral concentrations were considered (Fig. 8 ). In particular, significant positive correlations were observed between DONtot, DON, DON-3-G and monomeric protein fraction, mon/pol, [N] grain and [S] grain, and between ENNtot and UPP and GI. Significant negative correlations were observed between DONtot, DON, DON-3-G and ILP, polymeric protein fraction, UPP, GI and [Se] grain and between ENNtot and [S] grain and [Zn] grain. 4. Discussion Our two-year field trial provides novel evidence that targeted agronomic practices, including organic foliar fertilization and selenium biofortification, enhance both the technological and nutritional traits of durum wheat, and at the same time support food safety and sustainable production under Mediterranean conditions. Gluten proteins are among the major determinants of wheat quality and consist of monomeric (gliadins) and polymeric subunits high (HMW-GS) and low (LMW-GS) molecular weight, which interact each other, leading to the formation of polymers with different sizes and structures [43]. The glutenin polymers can further polymerize by intermolecular disulphide bonds, forming SLP which can also assemble through hydrogen bonds, leading to the formation of UPP [43]. In bread wheat, the proportion of UPP has often been positively correlated with dough strength [44–46]. As for durum wheat, the relation between UPP and GI, which is the main gluten strength indicator, has been less investigated, and the results are contradictory [32]; however, in our study, the two parameters resulted strongly and positively correlated. Environmental conditions during grain filling, particularly those affecting plant water status, can significantly influence both the polymerization and assembly of glutenin proteins [43], aspect of relevant interest under the ongoing climate change situation. Although this phenomenon is well documented in bread wheat, it remains less explored in durum wheat, despite it is commonly grown in sub-arid environments. In our study, the lower rainfall during the 2019 grain filling period promoted gluten protein polymerization, as evidenced by significantly higher SLP and polymeric protein fractions, and negatively affected protein assembly, as indicated by reduced UPP values. In line with our results, Park et al. [47] reported that water stress can decrease the proportion of UPP in wheat grains, particularly when it shortens the grain filling period [48], as observed in our trial, likely interfering with the assembly process of SLP in UPP. Moreover, drought conditions in 2019 also led to a reduction in grain [N], due to the negative effect of drought stress during grain filling on N assimilation and remobilization in wheat [49, 50], and to a lower grain [Fe] due to lower uptake [51]. In contrast, grain [S] and [Zn] increased, possibly due to drought-induced activation of S assimilation pathways [52, 53] and to the enhanced Zn uptake (Nawaz et al., 2015) [51]. The observed increase in grain [S] favoured protein polymerization through the formation of inter-chain disulphide bonds, particularly among cysteine-rich HMW-GS (Yu et al., 2021) [4], which are abundant in SLPs (Shewry and Lafiandra, 2022) [43]. Similarly, higher grain [Zn] could have contributed to the polymerization, by supporting the synthesis of S-rich proteins [54]. Indeed, both grain [S] and [Zn] were strongly and positively correlated with SLP and polymeric fraction. CONT + S significantly increased SLP content, consisted with Tea et al. [55–56], who reported enhanced polymeric protein proportions following S application in bread wheat, while had a depressing effect on protein assembly (ILP and UPP). Interestingly, we hypothesized that higher grain [S], while promoting disulphide bonding and protein polymerization, may hinder hydrogen bond formation required for protein assembly. On the contrary, the CONT + NS fertilization reduced SLP content and disulphide bond formation potential, likely favouring hydrogen bond-mediated protein assembly. This synergistic effect is supported by Tea et al. [55], who observed increased large polymer aggregates after N + S application. Additionally, CONT + NS enhanced grain [S], [Fe], and [Zn], likely due to both improved root growth and nutrient uptake capacity induced by N [57] and to the involvement of S in metabolic pathways critical to mineral absorption [58]. To our knowledge, this is the first study in durum wheat that demonstrates a clear directional effect of combined organic foliar N and S fertilization on grain S, Fe and Zn accumulation, which may have substantial implications for both nutritional quality and technological performance. Notably, under CONT + NS fertilization, the grain [Fe] and the grain [Zn] consistently exceeded nutritional thresholds to ensure meet human nutritional requirements (30 mg kg − 1 for Fe and 25 mg kg − 1 for Zn; [59–60]). As expected, grain [Se] decreased following CONT + S and CONT + NS fertilization, likely due to competitive inhibition between chemically similar S and Se during uptake [61, 62]. On the contrary Se foliar fertilization did not influence grain [S]. Despite literature suggesting antagonism between Se and Fe/Zn uptake [63], our data indicate that foliar Se application did not compromise grain [Fe] and [Zn] accumulation, confirming the advantage of bypassing soil competition [64, 65]. Thus, Se foliar fertilization in addition to increasing yield [34] offers nutritional benefits by increasing the availability of Se (Se requirements of humans and animals > 50–100 µg Se kg − 1 , [34]) without compromising that of Fe and Zn and without impacting protein quality parameters. Moreover, Se foliar application as sodium selenate had a positive effect on the minimizing mycotoxin grain contamination as reported below. The effect on the absorption of macro and micronutrients in wheat depends not only on the environmental conditions, but also on cultivar [66]. The modern cultivar Marco Aurelio exhibited the highest grain [Fe] and [Zn], as well as the highest yield (3.3 t ha ⁻¹, [34]), suggesting that, contrary to previous assumptions [67], high yield and micronutrient concentration are not necessarily antagonistic. To integrate the numerous parameters investigated and account for their complex interactions, we adopted a multivariate workflow (PCA followed by cluster analysis) to synthesize the experimental evidence and identify the main drivers of wheat technological and nutritional quality. The results of the PCA highlighted two main dimensions of variability: a first component (“polymerization factor”) mainly associated with SLP, total polymeric protein fraction, grain [S] and [Zn], and a second component (“assembly factor”) associated with ILP, UPP, GI and grain [Fe]. This allowed a better understanding of the distinct yet complementary roles of mineral nutrition in gluten protein structure. Indeed, while previous studies reported correlations between grain micronutrients and protein content [68–71], this study provides novel evidence of specific associations between grain [Fe] and gluten protein assembly, and between [Zn] and polymerization in durum wheat. The supplementary qualitative variables “Year” and “Cultivar” showed significant associations with PC1 and PC2, respectively, suggesting that environmental factors mainly influenced protein polymerization, while genotypic traits governed assembly processes. Also the Cluster analysis supported this finding. C1 included only samples from the first wetter year, was defined by the lowest values of SLP, polymeric protein fraction, and grain [S] and [Zn], thus placing it on the negative side of the polymerization factor. However, its moderate position along the assembly factor, linked to relatively higher grain [Fe], suggests that favourable water availability may have promoted Fe uptake and protein aggregation, despite poor polymerization. C2, largely composed of samples from the second drier year, exhibited the highest SLP and polymeric fraction values, but low levels of ILP, UPP, GI, and grain [Fe], placing it on the negative side of the assembly axis. This cluster notably lacked the modern cultivar Marco Aurelio, while the other modern one Nadif contributed to this cluster only with 8.5%, highlighting the limited ability of older cultivars to sustain assembly processes under stress. Conversely, C3, which included mainly the samples from second drier year and from the modern cultivars Marco Aurelio and, stood out as the only group with positive values on both PCA axes. This cluster showed a favourable combination of high SLP, polymeric fraction, ILP, UPP, and grain [Zn], suggesting that this genotype possesses superior capacity to simultaneously support polymerization and assembly processes, potentially due to more efficient nutrient use and stress resilience. Moreover, this study reported a significant effect of the compared agronomic practices also on the mycotoxin occurrence in grain. In general, the DON content was always below the maximum limits for durum wheat (EU Reg. 2024/1022), also when the sum of native form and modified one (DONtot, sum of DON, 3-ADON and DON-3-G) has been considered. The level of contamination of emerging mycotoxins such as MON (on average 25 µg kg − 1 ) and ENNtot (112 µg kg − 1 as a sum of different enniatin forms) was overall low if compared with other cereal growing areas [23], thereby limiting potential health risks for consumers and contributing to safer grain supplies, a key aspect in the broader context of food security. Inorganic Se forms, such as sodium selenite (Na 2 SeO 3 ) or sodium selenate (Na 2 SeO 4, applied in the present experiment), were previously reported to have inhibitory effects on fungi or synergistic effects with fungicides [72, 73]. Both inorganic and organic Se forms were recently reported to inhibit F. graminearum growth and DON production in vitro [74]. Among the different Se tested forms Na 2 SeO 4 resulted the inorganic Se form with the best inhibitory effect on F. graminearum growth and DON accumulation [74]. The authors supposed that Na 2 SeO 4 can be assimilated and transported more efficiently by plants than other inorganic or organic form (Li et al., 2008; 2010) [75, 76]. In the present study the Se foliar application was carried out at booting stage and was able to prevent the DON production during wheat ripening, caused by a fungal infection which occurred at flowering. Moreover, in addition to DON, the data collected highlighted a similar effect of Se foliar application also on ENNtot and MON, emerging mycotoxins produced by other Fusarium species (e.g. F. avenaceum ). No previous studies were instead reported in literature on open field experiments under natural mycotoxin contamination. Kornaś et al. [77] in 2018 investigated whether the application of Se ions directly to the leaf surface can protect plants against infection by the fungal toxin zearalenone (ZEA), applying the Na 2 SeO 4 to the second leaf of seedlings. The presence of Se significantly suppressed changes at the site of ZEA application in all tested plants. Microscopic observations confirmed that foliar administration of ZEA resulted in its penetration to deeper localized cells and that damage induced by ZEA (mainly to chloroplasts) decreased after Se application. Analyses of antioxidant enzymes demonstrated the involvement of Se in antioxidation mechanisms, by activating superoxide dismutases (SOD) and catalases (CAT) under ZEA-induced stress conditions. Thus, Kornaś et al. [77] concluded that the foliar application of Se to seedling leaves may be a non-invasive method of protecting crops against the first steps of ZEA infection. Although with a lower effectiveness compared to Se, the foliar S application at wheat flag leaf sheath opening stage showed a significant effect in minimizing the accumulation of all detected mycotoxins. In addition to the well-known role in preventing several diseases, a positive effect of elemental S application in controlling FHB in field was reported only by Haneklaus et al. [78]. In the experiment carried out on barley artificially inoculated with F. culmorum , the S was applied weekly from the anthesis to the end of ripening, for a total of 5 applications, resulting in a reduction of FHB severity and increasing grain yield. To the best of authors knowledge, the present study is the first one which reported a significant contribution of S foliar application on reducing mycotoxin occurrence in field. In the study of Haneklaus et al. [78] a direct effect of elemental S on fungal infection and development could be hypothesized, due to the timing of application. Conversely, in the present experiment the application of the foliar S was carried out only once and earlier compared to the fungal occurrence; thus, the observed effect could be linked to the formation of substances with antifungal action or to the activation of the plant metabolism pathway able to induce resistant to disease infection. Several S-containing defense compounds, such as sulfur containing amino acids, glucosinolates, phytoalexins and defensins and thionins peptides are reported to enhance plant defense responses to pathogens [79] Transgenic wheat plants overexpressing the gene for the production of S-containing peptides with antimicrobial activity showed an increased resistance to F. graminearum [80]. Finally, in 2018 the development of Fusarium species during ripening could be co-responsible for the lower gluten protein assembly, as suggested by the high negative correlation of the DONtot contamination with ILP (-0.73***) and with UPP (-0.7***). Koga et al. [81] reported that F. graminearum secreted gluten-degrading proteases play an important role in reducing the molecular size of glutenin polymers. Not only proteases from F. graminearum , but also from several other Fusarium spp. have been reported to degrade gluten proteins [82, 83], although in our study the main correlation was reported with DON-producer fungal species. These results highlight the need to better understand the interaction between organic and Se foliar applications and the effects on plant metabolism and the indirect potential role in minimizing fungal diseases. 5. Conclusion This study provides new insights into the complex interactions between genotype, environment, and application of foliar organic fertilizer in shaping both the nutritional and technological and the risk of accumulation of mycotoxin of durum wheat. The results underscore the importance of adopting integrated strategies that combine modern cultivars with targeted organic fertilization approaches and foliar micronutrient fortification to achieve sustainable improvements in grain quality. In particular, the combined foliar application of organic N and S proved effective in enhancing grain concentrations of essential micronutrients such as Fe, Zn, and S. These increases were not only beneficial from a nutritional standpoint, but also contributed to improving technological traits, such as gluten protein polymerization and assembly and could contribute to the reduction of mycotoxin accumulation. Notably, this study offers novel evidence of a specific association between grain [Fe] and gluten protein assembly, and between [Zn] and protein polymerization, highlighting the dual functional role of micronutrients in durum wheat quality. Furthermore, foliar Se application was shown to enrich grain Se content without compromising Fe and Zn accumulation or protein quality, confirming its value as a biofortification strategy that bypasses competitive uptake mechanisms in the soil. In addition, these agronomic practices resulted in a clear and consistent effect in minimizing the DON contamination in wheat grain at harvest. Principal component and cluster analyses revealed that while environmental factors primarily influenced polymerization, genotypic traits governed the protein assembly process. The modern cultivar Marco Aurelio, in particular, demonstrated a remarkable ability to combine elevated concentrations of micronutrients and favorable gluten quality indices, even under water-limited conditions. Overall, our results highlight that the strategic use of organic fertilizers, when combined with modern cultivars, can enhance both the nutritional and technological quality of durum wheat and reduce the DON contamination, although the potential risk of other emerging mycotoxins needs to be carefully verified. This integrated approach supports more sustainable production systems that align with current goals of environmental stewardship, climate resilience, and human health. CRediT authorship contribution statement FC: Writing – original draft, Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation, Visualization, Conceptualization. GG: Writing – review & editing, Methodology, Data curation, Visualization. MB: Writing – original draft, Writing – review & editing, Investigation, Resources. VS: Writing – original draft, Writing – review & editing, Formal analysis. GMM: Writing – review & editing, Investigation, Formal analysis. YM-A: Writing – review & editing, Supervision, Resources. MMG: Writing – original draft, Writing – review & editing, Methodology, Investigation, Supervision, Data curation, Visualization, Conceptualization, Resources. Declarations CRediT authorship contribution statement FC: Writing – original draft, Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation, Visualization, Conceptualization. GG: Writing – review & editing, Methodology, Data curation, Visualization. MB: Writing – original draft, Writing – review & editing, Investigation, Resources. VS: Writing – original draft, Writing – review & editing, Formal analysis. GMM: Writing – review & editing, Investigation, Formal analysis. YM-A: Writing – review & editing, Supervision, Resources. MMG: Writing – original draft, Writing – review & editing, Methodology, Investigation, Supervision, Data curation, Visualization, Conceptualization, Resources. Funding PID2023-148425NB-I00 project funded by MCIU/AEI/10.13039/501100011033 and Feder, UE Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was part of project SOFT (Smart Organic Farming Tecniques) financed under the PSR Puglia 2014–2020 funds, Measure 16—Cooperation, Submeasure 16.2—Support for pilot projects and the development of new products, practices, processes, and technologies (CUP B79J20000080009). Field experiments and wheat cultivation were supported by Dott. Pasquale De Vita, Dott. Ivano Pecorella and the staff of C.R.A. Experimental Institute for Cereal Research, S.S. 16 km 675, 71100 Foggia, Italy. Data availability Data will be made available on request. References Martínez-Moreno F, Ammar K, Solís I.. Global changes in cultivated area and breeding activities of durum wheat from 1800 to date: a historical review. Agronomy. 2022;12(5):1135. https://doi.org/10.3390/agronomy12051135. Sarkar A, Fu BX. Impact of quality improvement and milling innovations on durum wheat and end products. Foods. 2022;11(12):1796. https://doi.org/10.3390/foods11121796. Mefleh M, Conte P, Fadda C, Giunta F, Piga A, Hassoun G, Motzo R. From ancient to old and modern durum wheat varieties: Interaction among cultivar traits, management, and technological quality. J. Sci. Food Agric. 2019;99(5):2059-2067. https://doi.org/doi:10.1002/jsfa.9388. Yu Z, She M, Zheng T, Diepeveen D, Islam S, Zhao Y, Blanchard CL, Ma W. Impact and mechanism of sulphur-deficiency on modern wheat farming nitrogen-related sustainability and gliadin content. Commun. Biol. 2021;4(1):945. https://doi.org/10.1038/s42003-021-02458-7 . Fan M, Shen J, Yuan L, Jiang R, Chen X, Davies WJ, Zhang F. Improving crop productivity and resource use efficiency to ensure food security and environmental quality in China. J. Exp. Bot. 2012;63 (1):13-24. https://doi.org/10.1093/jxb/err248. Zhang Y, Dai X, Jia D, Li H, Wang Y, Li C, Xu H, He M. Effects of plant density on grain yield, protein size distribution, and breadmaking quality of winter wheat grown under two nitrogen fertilisation rates. Europ. J. Agron. 2016;73:1-10. https://doi.org/10.1016/j.eja.2015.11.015. Liu Y, Han M, Zhou X, Li W, Du C, Zhang Y, Zhang Y, Sun Z, Wang Z. Optimizing nitrogen fertilizer application under reduced irrigation strategies for winter wheat of the north China plain. Irrig. Sci. 2022;40(2):255-265. https://doi.org/10.1007/s00271-021-00764-w. Boix-Fayos C, de Vente J. Challenges and potential pathways towards sustainable agriculture within the European Green Deal. Agric. Syst. 2023;207:103634. https://doi.org/10.1016/j.agsy.2023.103634 Tappi M, Carucci F, Gatta G, Giuliani MM, Lamonaca E, Santeramo FG. Temporal and design approaches and yield-weather relationships. Clim. Risk Manag. 2023°;40:100522. https://doi.org/10.1016/j.crm.2023.100522. Tappi M, Carucci F, Gagliardi A, Gatta G, Giuliani MM, Santeramo FG. Earliness, phenological phases and yield-temperature relationships: evidence from durum wheat in Italy. Bio-based Appl. Econ. 2023b;12(2):115-125. https://doi.org/10.36253/bae-13745. Qaswar M, Jing H, Ahmed W, Dongchu L, Shujun L, Lu Z, Cai A, Liu L, Xu Y, Gao J, Huimin Z. Yield sustainability, soil organic carbon sequestration and nutrients balance under long-term combined application of manure and inorganic fertilizers in acidic paddy soil. Soil Tillage Res. 2020;198:104569. https://doi.org/10.1016/j.still.2019.104569. Xu F, Liu Y, Du W, Li C, Xu M, Xie T, Yin Y, Guo H. Response of soil bacterial communities, antibiotic residuals, and crop yields to organic fertilizer substitution in North China under wheat–maize rotation. Sci. Total Environ. 2021;785:147248. https://doi.org/10.1016/j.scitotenv.2021.147248. Geng Y, Cao G, Wang L, Wang S. Effects of equal chemical fertilizer substitutions with organic manure on yield, dry matter, and nitrogen uptake of spring maize and soil nitrogen distribution. PLoS One. 2019;14(7): e0219512. https://doi.org/10.1371/journal.pone.0219512. Guiducci M, Tosti G, Falcinelli B, Benincasa P. Sustainable management of nitrogen nutrition in winter wheat through temporary intercropping with legumes. Agron. Sustain. Dev. 2018;(38)31. https://doi.org/10.1007/s13593-018-0509-3. Konvalina P, Moudry Jr J., Capouchova I, Moudry J. Baking quality of winter wheat varieties in organic farming, Agron. Res. 2009;7:612–617. Lacko-Bartosova M, Lacko-Bartosova L, Konvalina P. Wheat rheological and Mixolab quality in relation to cropping systems and plant nutrition sources, Czech J. Food Sci. 2021;39. https://doi.org/10.17221/189/2020-CJFS. Reynolds LP, Caton JS. Role of the pre-and post-natal environment in developmental programming of health and productivity. Mol. Cell. Endocrinol., 2012;354(1-2):54-59. https://doi.org/10.1016/j.mce.2011.11.013. FAO, IFAD, UNICEF, WFP and WHO. 2019. The State of Food Security and Nutrition in the World 2019. Safeguarding against economic slowdowns and downturns. Rome, FAO. Licence: CC BY-NC-SA 3.0 IGO. Stoffaneller R, Morse NL. A review of dietary selenium intake and selenium status in Europe and the Middle East. Nutrients. 2015;7(3):1494-1537. https://doi.org/10.3390/nu7031494. Jones GD, Droz B, Greve P, Gottschalk P, Poffet D, McGrath SP, Seneviratne SI, Smith P, Winkel LH. Selenium deficiency risk predicted to increase under future climate change. Proceedings of the National Academy of Sciences. 2017;114(11):2848-2853. https://doi.org/10.1073/pnas.1611576114. Poblaciones MJ, Rodrigo S, Santamaría O, Chen Y, McGrath SP. Agronomic selenium biofortification in Triticum durum under Mediterranean conditions: From grain to cooked pasta. Food Chem. 2014;146:378-384. https://doi.org/10.1016/j.foodchem.2013.09.070. Sharma S, Kaur N, Kaur S, Nayyar H. Selenium as a nutrient in biostimulation and biofortification of cereals. Indian J. Plant Physiol. 2017; 22 (1): 1-15. https://doi.org/10.1007/s40502-016-0249-9. Blandino M, Scarpino V, Sulyok M, Krska R, Reyneri A. Effect of agronomic programmes with different susceptibility to deoxynivalenol risk on emerging contamination in winter wheat. Europ. J. of Agronomy. 2017;85:12-24. https://doi.org/10.1016/j.eja.2017.01.001. Scarpino V, Blandino M. Effects of durum wheat cultivars with different degrees of FHB susceptibility grown under different meteorological conditions on the contamination of regulated, modified and emerging mycotoxins. Microorganisms. 2021;9(2):408. https://doi.org/10.3390/microorganisms9020408. Scala V, Aureli G, Cesarano G, Incerti G, Fanelli C, Scala F, Reverberi M, Bonanomi G. Climate, Soil Management, and Cultivar Affect Fusarium Head Blight Incidence and Deoxynivalenol Accumulation in Durum Wheat of Southern Italy. Front. Microbiol. 2016;7:186895. https://doi.org/10.3389/fmicb.2016.01014. Zanini B, Simonetto A, Marconi S, Marullo M, Castellano M, Gilioli G. Whole grain perceptions and consumption attitudes: Results of a survey in Italy. Health Educ. J. 2023;82(7):739-751. https://doi.org/10.1177/00178969231185662. Streit E, Schwab C, Sulyok M, Naehrer K, Krska R, Schatzmayr G. Multi-mycotoxin screening reveals the occurrence of 139 different secondary metabolites in feed and feed ingredients. Toxins. 2013;5(3):504-523. https://doi.org/10.3390/toxins5030504. Scarpino V, Reyneri A, Sulyok M, Krska R, Blandino M. Effect of fungicide application to control Fusarium head blight and 20Fusarium and Alternaria mycotoxins in winter wheat (Triticum aestivum L.). World Mycotoxin J. 2015;8(4):499-510. Carucci F, Gatta G, Gagliardi A, De Vita P, Bregaglio S, Giuliani MM. Agronomic strategies to improve N efficiency indices in organic durum wheat grown in Mediterranean area. Plants. 2021;10 (11):2444. https://doi.org/10.3390/plants10112444. Lancashire PD, Bleiholder H, Boom TVD, Langelüddeke P, Stauss R, Weber E, Witzenberger A. A uniform decimal code for growth stages of crops and weeds. Ann. Appl. Biol. 1991;119(3):561-601. https://doi.org/10.1111/j.1744-7348.1991.tb04895.x. De Vita P, Platani C, Fragasso M, Ficco DBM, Colecchia SA, Del Nobile MA, Padalino L, Di Gennaro S, Petrozza A. Selenium-enriched durum wheat improves the nutritional profile of pasta without altering its organoleptic properties. Food Chem. 2017;214:374-382. https://doi.org/10.1016/j.foodchem.2016.07.015. Gagliardi A, Carucci F, Masci S, Flagella Z, Gatta G, Giuliani MM. Effects of genotype, growing season and nitrogen level on gluten protein assembly of durum wheat grown under Mediterranean conditions. Agronomy. 2020;10(5):755. https://doi.org/10.3390/agronomy10050755. International Association of Cereal Chemistry (ICC). Standard Methods of the ICC; ICC: Vienna, Austria, 1986 Carucci F, Moreno-Martín G, Madrid-Albarrán Y, Gatta G, De Vita P, Giuliani MM. Selenium agronomic biofortification of durum wheat fertilized with organic products: se content and speciation in grain. Agronomy. 2022;12(10):2492. https://doi.org/10.3390/agronomy12102492. Scarpino V, Reyneri A, Blandino M. Development and comparison of two multiresidue methods for the determination of 17 Aspergillus and Fusarium mycotoxins in cereals using HPLC-ESI-TQ-MS/MS. Front. Microbiol. 2019;10,361:1-12. https://doi.org/10.3389/fmicb.2019.00361. Box GE, Cox DR. An analysis of transformations. J R Stat Soc Series B Stat Methodol. 1964;26(2):211-243. Pinheiro JC, Bates DM. Mixed-effects models in S and S-PLUS. 2020.New York, NY: Springer New York. Carucci F, Gatta G, Gagliardi A, Bregaglio S, Giuliani MM. Individuation of the best agronomic practices for organic durum wheat cultivation in the Mediterranean environment: a multivariate approach. Agric. Food Secur. 2023;12:12. https://doi.org/10.1186/s40066-023-00417-5. Mongiano G, Titone P, Tamborini L, Pilu R, Bregaglio S. Evolutionary trends and phylogenetic association of key morphological traits in the Italian rice varietal landscape. Sci Rep. 2018;8(1):1–12. https://doi.org/10.1038/s41598- 018- 31909-1. Lê S, Josse J, Husson F. FactoMineR: an R package for multivariate analysis. J. of Stat. Softw. 2008;25:1-18. https://doi.org/10.18637/jss.v025.i01. Wei T, Simko V. R package “corrplot”: Visualization of a Correlation Matrix (Version 0.84). 2017. https://github.com/taiyun/corrplot Wickham H. ggplot2. Wiley interdisciplinary reviews: computational statistics. 2011; 3 (2):180-185. https://doi.org/10.1002/wics.147. Shewry PR, Lafiandra D. Wheat glutenin polymers 1. Structure, assembly and properties. J. Cereal Sci. 2022;106:103486. https://doi.org/10.1016/j.jcs.2022.103486. Wardlaw IF, Blumenthal CS, Larroqu O, Wrigley CW. Contrasting effects of chronic heat stress and heat shock on kernel weight and flour quality in wheat. Funct. Plant Biol. 2002;29:25–34. https://doi.org/10.1071/PP00147. Don C, Lookhart G, Naeem H, MacRitchie F, Hamer RJ. Heat stress and genotype affect the glutenin particles of the glutenin macropolymer-gel fraction. J. Cereal Sci. 2005;42(1):69-80. https://doi.org/10.1016/j.jcs.2005.01.005. Labuschagne MT, Elago O, Koen E. Influence of extreme temperatures during grain filling on protein fractions, and its relationship to some quality characteristics in bread, biscuit, and durum wheat. Cereal Chem . 2009;86:61–66. https://doi.org/10.1094/CCHEM-86-1-0061. Park H, Clay DE, Hall RG, Rohila JS, Kharel TP, Clay SA, Lee S. Winter wheat quality responses to water, environment, and nitrogen fertilization. Commun. Soil Sci. Plan. 2014;45(14):1894-1905. https://doi.org/10.1080/00103624.2014.909833. Martre P, Jamieson PD, Semenov MA, Zyskowski RF, Portr JR, Triboi E. Modelling protein content and composition in relation to crop nitrogen dynamics for wheat. Europ. J. Agronomy. 2006;25:138-154. https://doi.org/doi:10.1016/j.eja.2006.04.007. Gooding MJ, Pinyosinwat A, Addisu M. Nitrogen fertilizer and grain quality in wheat. In Y. A. El-Shemy (Ed.), Plant Fertilization for Agricultural Improvement. 2012;1–23. Carucci F, Gatta G, Gagliardi A, De Vita P, Giuliani MM. Strobilurin effects on nitrogen use efficiency for the yield and protein in durum wheat grown under rainfed Mediterranean conditions. Agronomy. 2020;10(10):1508. https://doi.org/10.3390/agronomy10101508. Nawaz F, Ahmad R, Ashraf MY, Waraich EA, Khan SZ. 2015 Effect of selenium foliar spray on physiological and biochemical processes and chemical constituents of wheat under drought stress. Ecotoxicol. Environ. Saf., 113, 191-200. https://doi.org/10.1016/j.ecoenv.2014.12.003 Ernst L, Goodger JQD, Alvarez S, Marsh EL, Berla B, Lockhart E, Jung J, Li P, Bohnert HJ, Schachtman D.P. Sulphate as a xylem-borne chemical signal precedes the expression of ABA biosynthetic genes in maize roots. J. Exp Bot. 2010;61(12):3395–3405. https://doi.org/10.1093/jxb/erq160. Chan KX, Wirtz M, Phua SY, Estavillo GM, Pogson BJ. Balancing metabolites in drought: the sulfur assimilation conundrum. Trends Plant Sci. 2013;18(1):18–29. https://doi.org/10.1016/j.tplants.2012.07.005. Liu, H.E., Wang, Q.Y., Rengel, Z., Zhao, P., 2015. Zinc fertilization alters flour protein composition of winter wheat genotypes varying in gluten content. Plant Soil Environ ., 61 (5), 195-200. https://doi.org/10.17221/817/2014-PSE Tea I, Genter T, Naulet N, Boyer V, Lummerzheim M, Kleiber D. Effect of foliar sulfur and nitrogen fertilization on wheat storage protein composition and dough mixing properties. Cereal Chem. 2004;81(6):759-766. http://dx.doi.org/10.1094/CCHEM.2004.81.6.759. Tea I, Genter T, Naulet N, Lummerzheim M, Kleiber D. Interaction between nitrogen and sulfur by foliar application and its effects on flour bread‐making quality. J. Sci. Food Agric. 2007; 87(15):2853-2859. https://doi.org/10.1002/jsfa.3044. Kutman UB, Yildiz B, Cakmak I. Effect of nitrogen on uptake, remobilization and partitioning of zinc and iron throughout the development of durum wheat. Plant Soil. 2011;342(1):149-164. https://doi.org/10.1007/s11104-010-0679-5. Hellemans T, Landschoot S, Dewitte K, Van Bockstaele F, Vermeir P, Eeckhout M, Haesaert G. Impact of crop husbandry practices and environmental conditions on wheat composition and quality: a review. J Agric Food Chem. 2018;66(11):2491-2509. https://doi.org/10.1021/acs.jafc.7b05450. Bouis HE, Saltzman A. Improving nutrition through biofortification: A review of evidence from HarvestPlus, 2003 through 2016. Global Food Secur. 2017;12:49-58. https://doi.org/10.1016/j.gfs.2017.01.009. Miner GL, Delgado JA, Ippolito JA, Johnson JJ, Kluth DL, Stewart CE. Wheat grain micronutrients and relationships with yield and protein in the U.S. Central Great Plains. Field Crops Res. 2022;279:108453. https://doi.org/10.1016/j.fcr.2022.108453. Sors TG, Ellis DR, Salt DE. Selenium uptake, translocation, assimilation and metabolic fate in plants. Photosynth. Res. 2005;86(3):373-389. https://doi.org/10.1007/s11120-005-5222-9. Zhou X, Yang J, Kronzucker HJ, Shi W. Selenium biofortification and interaction with other elements in plants: a review. Front. Plant Sci. 2020;11:586421. https://doi.org/10.3389/fpls.2020.586421. Fargašová A, Pastierová J, Svetkova K. Effect of Se-metal pair combinations (Cd, Zn, Cu, Pb) on photosynthetic pigments production and metal accumulation in Sinapis alba L. seedlings. Plant Soil Environ. 2006;52(1):8. https://doi.org/10.17221/3340-PSE. Zembala M, Filek M, Walas S, Mrowiec H, Hartikainen H, Miszalski Z. The influence of selenium on root growth and oxidative stress in rape seedlings subjected to cadmium stress. Plant and Soil. 2010;337(1–2):451–460. https://doi.org/10.1007/s11104-010-0543-z. Boldrin PF, Faquin V, Ramos SJ, Boldrin KVF, Araujo RA, Guilherme LRG. Selenium biofortification and antioxidant activity in lettuce plants fed with selenate and selenite. Plant Soil Environ. 2013;59(4):171–176. https://doi.org/10.17221/113/2010-PSE. Xia Q, Yang ZP, Xue NW, Dai XJ, Zhang X, Gao, ZQ. Effect of foliar application of selenium on nutrient concentration and yield of colored-grain wheat in China. Appl. Ecol. Environ. Res. 2019;17(2). http://dx.doi.org/10.15666/aeer/1702_21872202 Fan MS, Zhao FJ, Fairweather-Tait SJ, Poulton PR, Dunham SJ, McGrath SP. Evidence of decreasing mineral density in wheat grain over the last 160 years. J. Trace Elem. Med. Biol. 2008; 22 (4):315-324. https://doi.org/10.1016/j.jtemb.2008.07.002. Oury FX, Leenhardt F, Remesy C, Chanliaud E, Duperrier B, Balfourier F, Charmet G. Genetic variability and stability of grain magnesium, zinc and iron concentrations in bread wheat. Europ. J. Agronomy. 2006;25(2):177-185. https://doi.org/10.1016/j.eja.2006.04.011. Zhao FJ, Su YH, Dunham SJ, Rakszegi M, Bedo Z, McGrath SP, Shewry PR. Variation in mineral micronutrient concentrations in grain of wheat lines of diverse origin. J. Cereal Sci. 2009;49(2):290-295. https://doi.org/10.1016/j.jcs.2008.11.007. Guttieri MJ, Seabourn BW, Liu C, Baenziger PS, Waters BM. Distribution of cadmium, iron, and zinc in millstreams of hard winter wheat (Triticum aestivum L.). J. Agric. Food Chem. 2015. https://doi.org/10.1021/acs.jafc.5b04337 Dapkekar A, Deshpande P, Oak MD, Paknikar KM, Rajwade JM. Zinc use efficiency is enhanced in wheat through nanofertilization. Scientific Reports. 2018;8(1):6832. https://doi.org/10.1038/s41598-018-25247-5. Liu K, Cai M, Hu C, Sun X, Cheng Q, Jia W, Yang T, Nie M, Zhao X. Selenium (Se) reduces Sclerotinia stem rot disease incidence of oilseed rape by increasing plant Se concentration and shifting soil microbial community and functional profiles. Environ. Pollut. 2019;254:113051. https://doi.org/10.1016/j.envpol.2019.113051. Xu JY, Jia W, Hu CX, Nie M, Ming JJ, Cheng Q, Cai M, Sun X, Li X, Zheng X, Wang J, Zhao X. Selenium as a potential fungicide could protect oilseed rape leaves from sclerotinia sclerotiorum infection. Environ. pollut. 2020;257:113495. http://dx.doi.org/10.1016/j.envpol.2019.113495 Mao XY, Hua C, Yang L, Zhang YH, Sun ZX, Li L, Li T. The effects of selenium on wheat fusarium head blight and DON accumulation were selenium compound-dependent. Toxins. 2020;12(9):573. https://doi.org/10.3390/toxins12090573. Li HF, McGrath SP, Zhao FJ. Selenium uptake, translocation and speciation in wheat supplied with selenate or selenite. New Phytol. 2008;178(1):92–102. https://doi.org/10.1111/j.1469-8137.2007.02343.x Li HF, Lombi E, Stroud JL, McGrath SP, Zhao FJ. Selenium speciation in soil and rice: influence of water management and Se fertilization. J. Agric. Food Chem.2010;58(22):11837–11843. https://doi.org/10.1021/jf1026185 Kornaś A, Filek M, Sieprawska A, Bednarska-Kozakiewicz E, Gawrońska K, Miszalski Z. Foliar application of selenium for protection against the first stages of mycotoxin infection of crop plant leaves. J. Sci. Food Agric. 2018;99(1):482-485. https://doi.org/10.1002/jsfa.9145 . Haneklaus S, Bloem E, Funder U, Schnug E. Effect of foliar-applied elemental sulphur on Fusarium infections in barley. LBF.2007;57(3):213. Künstler A, Gullner G, Ádám AL, Kolozsváriné Nagy J, Király L. The versatile roles of sulfur-containing biomolecules in plant defense-a road to disease resistance. Plants. 2020;9:1705. https://doi.org/10.3390/plants9121705. Sasaki K; Kuwabara C; Umeki N, Fujioka M, Saburi W, Matsui H, Abe F, Imai R. The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat. J. Biotechnol. 2016;228:3–7. https://doi.org/10.1016/j.jbiotec.2016.04.015. Koga S, Aamot H, Uhlen A, Seehusen T, Veiseth-Kent E, Hofgaard I, Moldestad A, Böcker U. Environmental factors associated with glutenin polymer assembly during grain maturation. J. Cereal Sci. 2020;91:102865. https://doi.org/10.1016/j.jcs.2019.102865. Pekkarinen AI, Mannonen L, Jones BL, Niku-Paavola ML. Production of proteases by Fusarium species grown on barley grains and in media containing cereal proteins. J. Cereal Sci. 2000; 31 (3):253-261. https://doi.org/10.1006/jcrs.2000.0305. Wang J, Wieser H, Pawelzik E, Weinert J, Keutgen AJ, Wolf GA. Impact of the fungal protease produced by Fusarium culmorum on the protein quality and breadmaking properties of winter wheat. Eur. Food Res. Technol. 2005;220(5-6):552-559. https://doi.org/10.1007/s00217-004-1112-1. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7797922","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":531530219,"identity":"27f44e1b-e385-46cf-be15-2ceae0df9c85","order_by":0,"name":"Federica Carucci","email":"","orcid":"","institution":"University of Tuscia","correspondingAuthor":false,"prefix":"","firstName":"Federica","middleName":"","lastName":"Carucci","suffix":""},{"id":531530220,"identity":"6ebda3cb-26d2-4cad-b18e-ed6e80fb8722","order_by":1,"name":"Giuseppe Gatta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDACZlSuDVCE8QEDgwFxWhgbDjCkAUWYgeoN8OlB1XIYZApIOW4t8u48xh8+7rgjx8C/+PnjDzXnE7ezMzO/YCj4g1OL4WEeM8mZZ54ZM0g8M2w4cOx24s5mZjYLfA4zbOYxY+ZtO5zYIHEAqIXtduKGw/zHDAhoMf78t+1wfYPE8Y8NB/6dA2phZsOrRZ6Zx0Case1wAgN/j2HDwbYDIC3MD/BpMWBmK5PsPfPMsE2Cp3DG2b5kY5AtDAkGxrht6T+8+cPPHXfk+fmPb/hQ8c1OdsP5w8wfPvyRw23LAQZIhLBJJMAFkdlYbGmAamHgPwAXZP6AR8coGAWjYBSMPAAAIbpaTLPi6gUAAAAASUVORK5CYII=","orcid":"","institution":"University of Foggia","correspondingAuthor":true,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Gatta","suffix":""},{"id":531530221,"identity":"bb823b21-228a-417a-9486-61104218663e","order_by":2,"name":"Massimo Blandino","email":"","orcid":"","institution":"University of Turin","correspondingAuthor":false,"prefix":"","firstName":"Massimo","middleName":"","lastName":"Blandino","suffix":""},{"id":531530222,"identity":"cfe38ae3-72e7-4f3f-b500-72ee526dfe98","order_by":3,"name":"Valentina Scarpino","email":"","orcid":"","institution":"University of Turin","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Scarpino","suffix":""},{"id":531530223,"identity":"7d1389b3-763b-4555-9b25-2b093bebcced","order_by":4,"name":"Gustavo Moreno-Martín","email":"","orcid":"","institution":"Complutense University of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Moreno-Martín","suffix":""},{"id":531530224,"identity":"70e2b258-3032-4eb7-b75a-0fc65a014a39","order_by":5,"name":"Yolanda Madrid-Albarrán","email":"","orcid":"","institution":"Complutense University of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Yolanda","middleName":"","lastName":"Madrid-Albarrán","suffix":""},{"id":531530225,"identity":"c79743c6-7bd3-4712-9ebb-acc616c4892f","order_by":6,"name":"Marcella Michela Giuliani","email":"","orcid":"","institution":"University of Foggia","correspondingAuthor":false,"prefix":"","firstName":"Marcella","middleName":"Michela","lastName":"Giuliani","suffix":""}],"badges":[],"createdAt":"2025-10-07 09:23:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7797922/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7797922/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94015043,"identity":"9c9d13f7-c5ef-447a-86ff-c5be5ea410d0","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1352585,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptCaruccietal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/749d3d942615ad32897a9bbc.docx"},{"id":94015038,"identity":"6e2f098f-6e60-43a6-bbb9-649a079acf2b","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9546,"visible":true,"origin":"","legend":"","description":"","filename":"b517e4409dc647dea88c779321313daf.json","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/ad842fb061b698e8069a41e0.json"},{"id":94015047,"identity":"dff440c5-0796-4b7a-a7bb-094ddd63007e","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":32282,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/9d13d471f34e53f17d32e432.docx"},{"id":94015054,"identity":"a0aa92f6-f345-45c7-9289-361496967b00","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":187162,"visible":true,"origin":"","legend":"","description":"","filename":"b517e4409dc647dea88c779321313daf1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/3620bce8f92df4fe85c38cf8.xml"},{"id":94015513,"identity":"d1a90632-76e4-4dad-b163-0d61b0ddd567","added_by":"auto","created_at":"2025-10-21 11:08:00","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147777,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/11510cc15607dd6113aaaabb.png"},{"id":94015058,"identity":"82f2f65a-123b-4119-ae94-f6e192d51bcb","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":331371,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/ea11202426f5ad98b1155b47.png"},{"id":94016969,"identity":"b6e078e0-eb46-4036-98ba-99972390829b","added_by":"auto","created_at":"2025-10-21 11:24:01","extension":"emf","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1441596,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.emf","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/742807f049bae137c59ea759.emf"},{"id":94015064,"identity":"c79abe6b-c7a5-4e9c-9911-0432399a822c","added_by":"auto","created_at":"2025-10-21 11:00:01","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":225725,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/5f2df4f891df1cfca87447a6.png"},{"id":94016088,"identity":"08bb8ec4-195d-4bd7-b159-596dc93617f6","added_by":"auto","created_at":"2025-10-21 11:16:00","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168326,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/4a41d2d8a4a6141e64f785a8.png"},{"id":94015062,"identity":"05f5e409-97eb-4184-9581-466a89eaf058","added_by":"auto","created_at":"2025-10-21 11:00:01","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":30909,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/8b148684bb4f24dcf8358aef.png"},{"id":94015049,"identity":"a1c4a136-6acb-4f27-8be2-05c821df63ed","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49874,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/056850ffc9f88c5da7eeb95c.jpeg"},{"id":94015517,"identity":"bfe6c8a2-e765-4ca2-b260-db69a828804b","added_by":"auto","created_at":"2025-10-21 11:08:00","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150164,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/e4efde49f649f7f47e037977.png"},{"id":94016087,"identity":"743552ca-3655-4baf-afc9-e13d4fd4931b","added_by":"auto","created_at":"2025-10-21 11:16:00","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":45984,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/cc0b159c1ea7f729f31ee23c.png"},{"id":94015052,"identity":"ef3edede-0778-46b4-8882-1de8c6ae5b50","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":56783,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/f402f56c29e146a32bc15b89.png"},{"id":94015519,"identity":"1afcba88-4477-4bc4-81c0-9a777824329b","added_by":"auto","created_at":"2025-10-21 11:08:00","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19561,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/8f9dc162a959dec47c13d9c9.png"},{"id":94015516,"identity":"a1f4f3bc-51ea-4bad-b732-28c37afa2f53","added_by":"auto","created_at":"2025-10-21 11:08:00","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":30233,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/5bd8daa0342788a5b2d005e6.png"},{"id":94015056,"identity":"e5862382-a062-46c8-b985-b070000ccef3","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23304,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/6a62f7e47d69eea867d2810e.png"},{"id":94015523,"identity":"0a93d256-17a2-4f63-b741-e1bd08a4185c","added_by":"auto","created_at":"2025-10-21 11:08:01","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25449,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/6db39d28344fd9b41691beab.png"},{"id":94016968,"identity":"00079675-db36-47cf-be88-73cd12f70be4","added_by":"auto","created_at":"2025-10-21 11:24:01","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13498,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/847f79db5e3cfadb86fd8d31.png"},{"id":94015065,"identity":"fb40394f-ff35-4bac-a834-e0a9a27a7fcf","added_by":"auto","created_at":"2025-10-21 11:00:01","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65283,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/e782912a125d151ada40f954.png"},{"id":94015067,"identity":"e6627072-076f-4ac0-8b19-95e745fb3dea","added_by":"auto","created_at":"2025-10-21 11:00:01","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185245,"visible":true,"origin":"","legend":"","description":"","filename":"b517e4409dc647dea88c779321313daf1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/fcedaa4aed99dda18931bf39.xml"},{"id":94015066,"identity":"40b46174-8022-45be-a570-66398a55c0f6","added_by":"auto","created_at":"2025-10-21 11:00:01","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192219,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/b78a6912720ccd0fca1bb6ee.html"},{"id":94015037,"identity":"a917a6af-e623-4770-8244-280e98ad541c","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147777,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation matrix with Pearson’s r values, performed on protein fractions, gluten index (GI), and grain mineral concentrations. SLP: soluble large polymers; ILP: insoluble large polymers; mon/pol: monomeric/polymeric protein fraction ratio, UPP: unextractable polymeric protein; [N] grain: grain nitrogen concentration; [S] grain: grain sulphur concentration; grain [Se]: grain selenium concentration; grain [Fe]: grain iron concentration; grain [Zn]: grain zinc concentrations.\u003c/p\u003e\n\u003cp\u003e*** \u003cem\u003ep\u003c/em\u003e-value ≤ 0.001; ** \u003cem\u003ep\u003c/em\u003e-value ≤ 0.01; * \u003cem\u003ep\u003c/em\u003e-value ≤ 0.05; not significant.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/90ae549713a3b4470f791f1d.png"},{"id":94015509,"identity":"ebe1dfb9-c4e1-40b8-baf5-f02a64694aed","added_by":"auto","created_at":"2025-10-21 11:08:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":295776,"visible":true,"origin":"","legend":"\u003cp\u003eWithin-cluster distributions (Mod.Cla) and the level of significance of supplementary qualitative variables contributing to each cluster. CONT: wheat fertilized with dry blood meal in a single application at seeding; CONT+N: CONT plus N foliar application at the beginning of heading (BBCH stage 51); CONT+S: CONT plus foliar S application at flag leaf sheath opening stage (BBCH stage 47);\u0026nbsp; CONT+NS: CONT plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively; Se0: control without selenium; Se60 = one application of sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e), at rate of 60 g ha\u003csup\u003e-1\u003c/sup\u003e at booting stage (BBCH stage 41).\u003c/p\u003e\n\u003cp\u003e*** \u003cem\u003ep\u003c/em\u003e-value ≤ 0.001; ** \u003cem\u003ep\u003c/em\u003e-value ≤ 0.01; * \u003cem\u003ep\u003c/em\u003e-value ≤ 0.05; ns not significant.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/f16e2f844774039c914e4de8.png"},{"id":94015039,"identity":"1261428a-d99f-4f9b-80c1-d6c3d837b3bd","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108255,"visible":true,"origin":"","legend":"\u003cp\u003ePCA biplot with clusters delimitation; the barycenter of the supplementary variables significantly contributing to cluster variances are highlighted with colours and symbols: the years (2018 and 2019) in red with diamond shape symbol, and the cultivar (Cappelli, old Saragolla, Marco Aurelio, and Nadif) in blue with square shape symbol. The other non-significant supplementary variables are reported in grey. CONT: wheat fertilized with dry blood meal in a single application at seeding; CONT+N: CONT plus N foliar application at the beginning of heading (BBCH stage 51); CONT+S: CONT plus foliar S application at flag leaf sheath opening stage (BBCH stage 47);\u0026nbsp; CONT+NS: CONT plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively; Se0: control without selenium; Se60 = one application of sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e), at rate of 60 g ha\u003csup\u003e-1\u003c/sup\u003e at booting stage (BBCH stage 41).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/e1e0502681a216b4a5c5f817.png"},{"id":94015042,"identity":"a98a3028-889e-4a55-82b8-c4f49a356bfc","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":201290,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplots show the distributions of the proportions of the polymeric protein fraction (A), monomeric protein fraction (B), monomeric and polymeric ratio (mon/pol; C), soluble large polymer (SLP; D), insoluble large polymer (ILP; E), unextractable polymeric protein (UPP; F), and gluten index (GI; G) resulting from the cluster analysis. The colors of the boxplot charts reflect the visuals used in the PCA biplot. Different superscript letters indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e-value≤0.05, Tukey’s test). The asterisk symbol denotes the mean value.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/53fa01601e4a60187799055a.png"},{"id":94015512,"identity":"99878df7-3859-4909-b7e2-6e259ce692d5","added_by":"auto","created_at":"2025-10-21 11:08:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154554,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplots show the distributions of grain nitrogen ([N]; A), grain sulphur ([S]; B), grain selenium ([Se]; C), grain iron ([Fe]; D), and grain zinc ([Zn]; E) concentrations, resulting from the cluster analysis. The colors of the boxplot charts reflect the visuals used in the PCA biplot. Different superscript letters indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e-value≤0.05, Tukey’s test). The asterisk symbol denotes the mean value.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/8c1f75101b327a3b19c37ca5.png"},{"id":94015511,"identity":"35c7e975-b79f-431d-ae83-d010fa866880","added_by":"auto","created_at":"2025-10-21 11:08:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":47037,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the combination of cultivars and Se application on the contamination of enniatins (ENNtot, sum of ENN A, A\u003csub\u003e1\u003c/sub\u003e, B and B\u003csub\u003e1\u003c/sub\u003e). Different superscript letters are significantly different at \u003cem\u003ep\u003c/em\u003e-value ≤ 0.05 according to Tukey’s test. Vertical bars indicate standard errors (3 replicates). Se0: control without selenium; Se60: application of sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e), at rate of 60 g ha\u003csup\u003e-1\u003c/sup\u003e at booting stage (BBCH stage 41).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/0f8e0c65bb7be1337b767771.png"},{"id":94015045,"identity":"b5b3d899-ab5b-4603-8d5d-d87153504e5b","added_by":"auto","created_at":"2025-10-21 11:00:00","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":49874,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the combination of organic foliar fertilization and Se application on the contamination of enniatins (ENN tot = sum of ENN A, A\u003csub\u003e1\u003c/sub\u003e, B and B\u003csub\u003e1\u003c/sub\u003e) and MON. Different superscript letters are significantly different at \u003cem\u003ep\u003c/em\u003e-value ≤ 0.05 according to Tukey’s test. Vertical bars indicate standard errors (3 replicates). CONT+N: wheat fertilized with dry blood meal in a single application at seeding plus N foliar application at the beginning of heading (BBCH stage 51); CONT+NS: wheat fertilized with dry blood meal in a single application at seeding plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively. Se0: control without selenium; Se60: application of sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e), at rate of 60 g ha\u003csup\u003e-1\u003c/sup\u003e at booting stage (BBCH stage 41).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/bfa215f1c40f7cc1808f5416.jpeg"},{"id":94016086,"identity":"f49f0e38-1c60-40f6-9c59-4fb269f31e7b","added_by":"auto","created_at":"2025-10-21 11:16:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":161449,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation matrix with Pearson’s r values, performed on mycotoxin contamination, protein fractions, gluten quality parameters, and grain mineral concentrations. DONtot: total deoxynivalenol forms, sum of DON, DON-3-G and 3-ADON; DON: deoxynivalenol; DON-3-G: deoxynivalenol-3-glucoside; MON: moniliformin; ENNtot: total enniatin forms, sum of ENN A, A\u003csub\u003e1\u003c/sub\u003e, B and B\u003csub\u003e1.\u003c/sub\u003e SLP: soluble large polymers; ILP: insoluble large polymers; mon/pol: monomeric/polymeric protein fraction ratio; UPP: unextractable polymeric protein; GI: gluten index; [N] grain: grain nitrogen concentration; [S] grain: grain sulphur concentration; grain [Se]: grain selenium concentration; grain [Fe]: grain iron concentration; grain [Zn]: grain zinc concentrations.\u003c/p\u003e\n\u003cp\u003e*** \u003cem\u003ep\u003c/em\u003e-value ≤ 0.001; ** \u003cem\u003ep\u003c/em\u003e-value ≤ 0.01; * \u003cem\u003ep\u003c/em\u003e-value ≤ 0.05; not significant.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/baf44b53f9a0710e7d6b2cb6.png"},{"id":94017114,"identity":"d306c98a-f893-4fe3-b520-547eff598bd3","added_by":"auto","created_at":"2025-10-21 11:32:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2495094,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/0151ce3d-01b4-4993-b45d-85592c9bed4d.pdf"},{"id":94016085,"identity":"5b3fb3f7-50ba-401d-9d34-52c5d397dde5","added_by":"auto","created_at":"2025-10-21 11:16:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":32282,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7797922/v1/8947cbd78422ebe7c788f2d8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing Durum Wheat Grain Quality and Safety: The Role of Organic Foliar Fertilization and Selenium Biofortification in Mediterranean Farming Systems","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDurum wheat (\u003cem\u003eTriticum turgidum\u003c/em\u003e spp. \u003cem\u003edurum\u003c/em\u003e L.) represents about 7% of global wheat production [1]. Still, its economic and nutritional importance is considerable due to its wide use in products such as pasta, couscous, bulgur, and specialty breads [2]. The technological suitability of durum wheat semolina for these products is largely determined by its gluten protein composition [3, 4]. In recent years, however, increasing attention has been paid not only to gluten-related processing traits, but also to broader aspects of grain quality, including nutritional value, food safety, and environmental sustainability. This shift reflects the evolving priorities of both consumers and policymakers, moving beyond yield as the sole breeding and agronomic target.\u003c/p\u003e\u003cp\u003eOne major challenge in sustainable wheat production is the widespread use of mineral nitrogen (N) fertilizers, which, despite their productivity benefits, are associated with low uptake efficiency (\u0026lt;\u0026thinsp;40%), high costs, soil degradation, and significant environmental impacts [5, 6, 7]. To mitigate these effects, the European Green Deal is proposing targets to reduce mineral fertilizer use [8]. This goal, however, raises critical issues for high-input crops like wheat, for which fertilization also plays a key role in quality, especially under increasing abiotic stress conditions linked to climate change [9, 10]. Organic fertilizers offer a promising alternative [11, 12], even if their lower nutrient content and slower mineralization can limit durum wheat yield and quality [13, 14]. In organic systems, N is often applied only at sowing, and delayed mineralization during spring can result in insufficient availability during key phenological stages [15, 16]. Foliar application of organic N formulations may represent a viable strategy to improve N use efficiency and guarantee the wheat grain quality target.\u003c/p\u003e\u003cp\u003eAs well as the need for agronomic sustainability, there is a growing awareness of the nutritional limitations of wheat, which, despite providing about 20% of global dietary protein and calories [17, 18], is inherently low in essential micronutrients such as selenium (Se). In Europe, Se intake is often below recommended levels [19], and this deficiency may worsen due to climate-driven declines in soil Se content [20]. Agronomic biofortification has been shown to improve Se accumulation in cereal grains [21, 22], yet its broader effects on plant physiology, including interactions with N, S, Zn, and Fe uptake, remain underexplored.\u003c/p\u003e\u003cp\u003eDurum wheat also faces increasing threats to food safety due to its susceptibility to Fusarium Head Blight (FHB), especially in Mediterranean environments. FHB leads to contamination by deoxynivalenol (DON) and other mycotoxins, posing a major barrier to production and export [23, 24] affecting also the food security. Although DON levels in Southern Italy often remain within regulatory limits [25], recent EU regulations (e.g., EC No. 2024/1022) and the rise in wholegrain consumption [26] have increased the demand for mitigation strategies. Moreover, the presence of emerging mycotoxins such as DON-3-glucoside, enniatins (ENNs), and moniliformin (MON) raises additional concerns [27, 28]. With a planned reduction in pesticide use requested by the legislation and supply chain (Boix-Fayos and de Vente, 2023) [8], agronomic tools with dual physiological and protective effects, such as S and Se, may offer novel solutions.\u003c/p\u003e\u003cp\u003eTo date, however, to the best of our knowledge, no evidence is available on the combined effects of organic foliar fertilization and selenium biofortification in durum wheat, particularly regarding their impact on gluten protein structure, nutrient enrichment, and mycotoxin contamination.\u003c/p\u003e\u003cp\u003eTherefore, this study aims to provide new insights into the role of organic foliar fertilization and Se biofortification in durum wheat. Specifically, we evaluate their effects on gluten protein polymerization and assembly, the accumulation of key nutrients (S, Fe, Zn, Se), and the occurrence of major and emerging mycotoxins under different seasonal conditions and genetic backgrounds. By integrating nutritional, technological, and safety traits, the main goal is to identify sustainable agronomic strategies that simultaneously enhanced durum wheat technological, nutritional and safety quality.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Field Experiments\u003c/h2\u003e\u003cp\u003eThe field experiments conducted in two consecutive growing seasons, 2017-18 and 2018-19 (namely 2018 and 2019, respectively), have already been described in Carucci et al. [29]. Briefly, two old (Cappelli and old Saragolla) and two moderns (Marco Aurelio and Nadif) Italian durum wheat cultivars were grown in southern Italy (Foggia, 41\u0026deg;46\u0026prime; N, 16\u0026deg;54\u0026prime; E) under four different organic foliar fertilization: 1) control (CONT) fertilized with dry blood meal (ORGAZOT\u0026reg;, AGM) in a single application at seeding; 2) CONT plus foliar S (water solution of bio sulfur Fytofert\u0026reg;S, De Sangosse (10% \u003cem\u003ew\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e)) application at flag leaf sheath opening stage (BBCH stage 47) [30] (CONT\u0026thinsp;+\u0026thinsp;S); 3) CONT plus N foliar application (water solution of blood meal EUTROFIT\u0026reg;, AGM (5% \u003cem\u003ew\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e)) at the beginning of heading (BBCH stage 51) (CONT\u0026thinsp;+\u0026thinsp;N); 4) CONT plus the combination of N and S foliar applications at flag leaf sheath opening stage and at the beginning of the heading, respectively (CONT\u0026thinsp;+\u0026thinsp;NS).\u003c/p\u003e\u003cp\u003eFurthermore, two different Se applications were also considered: 1) Se0, control without Se; 2) Se60, with one application of sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e), at rate of 60 g ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [31] at the booting stage (BBCH stage 41). A split-split-plot design with three replicates was adopted: the cultivar was the main plot, the organic foliar fertilization was the plot, and the Se application was the sub-plot (10.2 m\u003csup\u003e2\u003c/sup\u003e) for a total of 96 experimental units. Except for the compared agronomic factor, the standard agronomical management for the growing area and the organic wheat was adopted in all the plots (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; additional file).\u003c/p\u003e\u003cp\u003eThe precipitations and daily temperatures were measured at meteorological stations located near the experimental field. The main meteorological data together with the crop length in the two years are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Durum wheat grain was machine-harvested at full maturity using a Wintersteiger Nursery Master Elite plot combine (Wintersteiger Inc., Ried im Innkreis, Austria). The harvested grains were mixed thoroughly, and 1 kg grain samples were taken from each plot to obtain a representative sample for the qualitative analysis. For mycotoxin analysis and determination of the macro and microelement grain concentration, representative sub-samples (300 g) were ground to whole-meal using a laboratory centrifugal mill Cyclotec Sample Mill 1093 (Foss Tecator, Hiller\u0026oslash;d, Denmark) and then manually sieved through a 1-mm sieve. The semolina flour used for gluten protein polymers and gluten index analysis has been obtained from kernels milled by Bona mill 4 cylinders (sieve 180 \u0026micro;m).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMeteorological data and crop length related to the two experimental years.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMeasurement unit\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2018\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2019\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrop cycle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003edays\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e207\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrain filling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003edays\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrop cycle rainfall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e299\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrain filling rainfall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrop cycle Avg. T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e12.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAvg. T\u003csub\u003emax\u003c/sub\u003e during grain filling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e24.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u0026deg; C\u0026thinsp;\u0026lt;\u0026thinsp;T\u003csub\u003emax\u003c/sub\u003e \u0026lt;35\u0026deg; C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003edays\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e5.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e \u0026gt;35\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003edays\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Analysis of gluten protein parameters and gluten index\u003c/h2\u003e\u003cp\u003eSize exclusion high performance liquid chromatography (SEC-HPLC) analysis of Sodium Dodecyl Sulfate (SDS) extractable and unextractable proteins was performed according to the method reported in Gagliardi et al. [32]. The SDS-unextractable fraction profile showed one peak containing the largest glutenin polymers, insoluble in SDS solution alone, but rendered soluble by sonication called insoluble large protein (ILP). The SDS soluble fraction profiles were divided into four areas. The first two areas correspond to soluble large (SLP) and medium-sized polymers, respectively, which, together with ILP determine the polymeric protein fraction. The third and the fourth areas represent the amounts of the monomeric protein fraction together. The proportions of each area were calculated as percentages of the total areas of the two chromatograms (SDS-insoluble and SDS-soluble fractions). Finally, the amount of monomeric over polymeric proteins (mon/pol) and the proportion of unextractable polymeric protein (UPP), as the ratio between ILP and the sum of SLP and ILP areas (*100), were calculated.\u003c/p\u003e\u003cp\u003eFurthermore, the gluten index (GI), an indicator of gluten strength, was determined on semolina samples using the Glutomatic system according to ICC standard 155 [33].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Determination of N, S, Se, Fe and Zn grain concentration\u003c/h2\u003e\u003cp\u003eN and S grain concentrations (hereinafter called [N] and [S], respectively) of all whole-meal samples were determined in triplicate using a Leco CHNS 628 Analyzer (Leco corporation, St. Joseph, Michigan). The grain [N] was determined on 0.0500 g of whole grain flour, while the grain [S] was determined on 0.1000 g of material.\u003c/p\u003e\u003cp\u003eThe Se grain concentration (hereinafter called [Se]) was determined using an inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700x, Agilent Technologies Inc., USA) after acid digestion in a microwave oven (MSP 1000, CEM, Matheus, NC), as reported in Carucci et al. [34]. Briefly, 0.5000 g of whole grain flour was digested with nitric acid and hydrogen peroxide. The experimental conditions for ICP-MS analyses were as follows: RF power of 1550 W, sample depth of 8 mm, plasma gas flow rate 15.0 L\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, auxiliary gas flow rate 0.90 L\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and conical nebulizer with a flow rate 1.01 L\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Se quantification was performed by external calibration with the isotope \u003csup\u003e78\u003c/sup\u003eSe.\u003c/p\u003e\u003cp\u003eRegarding the analysis of Fe and Zn grain concentration (hereinafter called [Fe] and [Zn], respectively), around 0.5000 g of wheat grains were accurately weight in an Xpress tube and then digested in a microwave oven (MSP 1000, CEM, Matheus, NC) with 6.0 mL of a mixture of concentrated nitric acid (Scharlab, Spain) and 33% (v/v) hydrogen peroxide (Dismadel, Spain) in a 3:1 ratio. The program of the microwave over for the mineralization of the samples is detailed in previous work (Moreno-Mart\u0026iacute;n et al. 2020). The digested extracts were transferred to 25.0 mL with ultrapure water, filtered through a 0.22 \u0026micro;m syringe Nylon filters (Dismadel, Spain) and analyzed by flame atomic absorption spectrometry (FAAS).\u003c/p\u003e\u003cp\u003eThe operating conditions of the FAAS were a flame of air-acetylene mixture with a flow rate of 2.5/ 4.0 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Fe and Zn were measured in continuous mode with a band pass of 0.2 mm with lamp currents of 30 and 15 mA, and at wavelengths of 248.3 and 213.9 nm, respectively. External calibration using dissolved Fe and Zn ionic standards (Merck, Spain), ranging from 0 to 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Zn and 0 to 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Fe, with an acid content equivalent to that of the samples resulting from the digestion procedure, was utilized to quantify both elements. Analyses were performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Determination of Mycotoxin\u003c/h2\u003e\u003cp\u003eMycotoxin analysis was performed by a multi-mycotoxin liquid chromatography with tandem mass spectrometry (LC-MS/MS) method as described by Scarpino et al. [35]. The analysis was carried out on a sub-set of the considered treatment for both growing seasons, considering the combination of two cultivars (old Saragolla \u003cem\u003evs\u003c/em\u003e Nadif), two organic foliar fertilizers (CONT\u0026thinsp;+\u0026thinsp;N \u003cem\u003evs\u003c/em\u003e CONT\u0026thinsp;+\u0026thinsp;NS), and two Se application (Se0 \u003cem\u003evs\u003c/em\u003e Se60). Briefly, 5.0000 g of each wholegrain flour was extracted with 20 mL of CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO/CH\u003csub\u003e3\u003c/sub\u003eCOOH (79/20/1, v/v/v), according to the dilute-and-shoot method reported by Scarpino et al. [35], and 20 \u0026micro;L of the diluted filtered extracts was analyzed without any further pretreatment. LC-MS/MS analysis was carried out on a Varian 310 triple quadrupole (TQ) mass spectrometer (Varian, Milan, Italy), equipped with an electrospray ionization (ESI) source, a 212 LC pump, a ProStar 410 AutoSampler and dedicated software. LC separations were performed on a Gemini-NX C18 100 x 2.0 mm i.d., 3 \u0026micro;m particle size, 110 \u0026Aring;, equipped with a C18 4 x 2 mm security guard cartridge column (Phenomenex, Torrance, CA, United States), using water (eluent A) and methanol (eluent B), both acidified with 0.1% v/v CH\u003csub\u003e3\u003c/sub\u003eCOOH, as eluents that were delivered at 200 \u0026micro;L min\u003csup\u003e\u0026shy;1\u003c/sup\u003e. The chromatographic and MS conditions and the results pertaining to the linearity range, the limit of detection (LOD), the limit of quantification (LOQ), the apparent recovery RA (%), the matrix effects obtained through the evaluation of the signal suppression/enhancement SSE (%), and the recovery of the extraction RE (%) were described in detail by Scarpino et al. [35]. The results of the mycotoxin concentrations were corrected for the recovery rates and expressed as \u0026micro;g kg\u003csup\u003e\u0026shy;-1\u003c/sup\u003e on dry matter basis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were conducted by using the R statistical environment (R Core Team, 2018). The dataset was tested according to the basic assumptions of analysis of variance (ANOVA). The normal distribution of the experimental error and the common variance of the experimental error were verified through Shapiro\u0026ndash;Wilk and Levene\u0026rsquo;s tests, respectively. When required, Box\u0026ndash;Cox transformations [36] were applied before analysis. Gluten protein polymers, gluten index, and macro and microelements concentration in grain were analyzed using the split-split-plot linear mixed model ANOVA approach, using the package \u0026ldquo;nlme\u0026rdquo; [37]. Cultivar (Cappelli, old Saragolla, Marco Aurelio, and Nadif), organic foliar fertilization (CONT, CONT\u0026thinsp;+\u0026thinsp;S, CONT\u0026thinsp;+\u0026thinsp;N, and CONT\u0026thinsp;+\u0026thinsp;NS), and Se application (Se0 \u003cem\u003evs\u003c/em\u003e Se60) were included in the model as fixed factors, together with their interaction. The growing season was considered as a fixed factor, while the \u0026ldquo;blocks within growing season\u0026rdquo;, the \u0026ldquo;main-plots within blocks within growing season\u0026rdquo; and the \u0026ldquo;plots within main-plots within blocks within growing season\u0026rdquo; were added as random effects to account for blocking units. The statistical significance of the difference among the means was determined using post-hoc Tukey\u0026rsquo;s test (function \u0026ldquo;means\u0026rdquo;) at the 5% probability level. Moreover, to analyze the relationships between gluten protein polymers, GI, and the grain [N], [S], [Se], [Fe] and [Zn], a multivariate analysis was applied to reduce dataset complexity and enhance correlation visualization [38]. Four cultivars, four organic foliar fertilization treatments, and two Se applications were incorporated across two growing seasons as descriptors according to the procedure outlined by Mongiano et al. [39]. Thus, Principal Component Analysis (PCA) was performed after first conducting a correlation analysis. The following experimental traits, proportion of ILP, SLP, monomeric protein, mon/pol ratio, UPP, GI, and grain [N], [S], [Se], [Fe] and [Zn] were used as active quantitative variables, which were centered and scaled prior to PCA, through a procedure which involved diagonalizing the correlation matrix and extracting the associated eigenvectors and eigenvalues. The supplementary qualitative variables, which included factors such as year, cultivar, organic foliar fertilization, and Se application, did not influence the calculation of the principal components. Instead, their coordinates were determined as the barycenter of individuals plotted in the PCA space. Next, we implemented Hierarchical Clustering on Principal Components (HCPC) using Euclidean distance and Ward\u0026rsquo;s method to identify data clusters with similar behavior. For each experimental factor, the average value within each cluster was tested against the null hypothesis that its distribution remained consistent across all clusters:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:u=\\frac{{x̄}_{q}-\\:x̄}{\\sqrt{\\frac{{s}^{2}}{{n}_{q}}\\:\\left(\\frac{N-{n}_{q}}{N-1}\\right)}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere n\u003csub\u003eq\u003c/sub\u003e is the number of data points in cluster q, N is the total number of data points, and s is the global standard deviation. We also assessed each cluster's composition, which was characterized considering the representativeness of the qualitative variables used in the PCA, using an alpha level α\u0026thinsp;=\u0026thinsp;0.05 for all statistical tests. PCA and clustering computations were performed using the FactoMineR package [40]. To assess differences between the resulting clusters, analysis of variance (one-way ANOVA, function \u0026ldquo;aov\u0026rdquo;) and the post-hoc Tukey\u0026rsquo;s test (function \u0026ldquo;means\u0026rdquo;) were performed at the 5% probability level.\u003c/p\u003e\u003cp\u003eThe mycotoxin concentrations in grain were modelled in a linear mixed model using the package \u0026ldquo;nlme\u0026rdquo; [37] for data analysis. Cultivar (Nadif, old Saragolla), organic foliar fertilization (CONT\u0026thinsp;+\u0026thinsp;N and CONT\u0026thinsp;+\u0026thinsp;NS), and Se application (Se0 and Se60) were included in the model as fixed factors, together with their interaction. The growing season was considered as a fixed factor, while the \u0026ldquo;blocks within growing seasons\u0026rdquo;, the \u0026ldquo;main-plots within blocks within growing seasons\u0026rdquo;, and the \u0026ldquo;plots within main-plots within blocks within growing seasons\u0026rdquo; were added as random effects to account for blocking units. The ANOVA was performed, and the statistical significance of the difference among the means was determined using post-hoc Tukey\u0026rsquo;s test (function \u0026ldquo;emmeans\u0026rdquo;) at the 5% probability level. Finally, Pearson\u0026rsquo;s correlations were calculated between the mycotoxin concentration in grain and all the durum wheat traits investigated (gluten protein parameters, gluten index, macro- and micro-elements concentration in grain) on old Saragolla and Nadif cultivars, on CONT\u0026thinsp;+\u0026thinsp;N and CONT\u0026thinsp;+\u0026thinsp;NS organic foliar fertilization, and on the Se application under the two growing seasons, using the \u0026ldquo;corrplot\u0026rdquo; package [41].\u003c/p\u003e\u003cp\u003eThe package ggplot2 was used [42] for all the visual representation of the data.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Gluten protein parameters and gluten index\u003c/h2\u003e\n \u003cp\u003eANOVA generally showed a significant effect of year, cultivar, foliar fertilization, Se biofortification, and their interactions on gluten protein parameters and gluten index (Table S2, additional file) due to the high number and complexity of the interactions, these will be better explored through multivariate analysis.\u003c/p\u003e\n \u003cp\u003eThe two experimental years significantly affected the proportion of gluten polymers and monomers and GI (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In the second year, there was the highest proportion of both SLP and ILP with a consequent significant increase of the polymeric fraction, and the lowest proportion of monomeric proteins, mon/pol ratio, UPP, and GI. The effect of the durum wheat cultivars was also significant (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Among the two old cultivars, Cappelli exhibited the highest proportion of SLP and the lowest GI values, while old Saragolla had the highest proportion of monomeric protein and mon/pol ratio and the lowest proportion of polymeric protein. For the modern cultivars, Marco Aurelio showed the highest proportions of ILP, UPP, and polymeric protein, along with the highest GI value, and the lowest proportions of monomeric protein and mon/pol ratio, while Nadif showed intermediate values for all the parameters considered.\u003c/p\u003e\n \u003cp\u003eRegarding organic foliar fertilization, CONT\u0026thinsp;+\u0026thinsp;S significantly increased the proportion of SLP and GI value. Conversely, CONT\u0026thinsp;+\u0026thinsp;NS significantly decreased the proportion of SLP and significantly increased the proportion of UPP. CONT\u0026thinsp;+\u0026thinsp;N resulted in the lowest GI value. The organic foliar fertilization adopted did not affect the proportion of polymeric and monomeric proteins or their mon/pol ratio (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFinally, Se application did not significantly impact gluten protein parameters or the GI (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of year, cultivar, foliar fertilization, and selenium application on protein fraction resulting from analysis of variance (ANOVA).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFactor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSLP\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eILP\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePolymeric protein fraction (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMonomeric protein fraction (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emon/pol\u003c/p\u003e\n \u003cp\u003e(-)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUPP\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGI\u003c/p\u003e\n \u003cp\u003e(-)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.2\u0026thinsp;\u0026plusmn;\u0026thinsp;31.6 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.9\u0026thinsp;\u0026plusmn;\u0026thinsp;31.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCultivar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCappelli\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.1 d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eold Saragolla\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMarco Aurelio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12.8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNadif\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.5\u0026thinsp;\u0026plusmn;\u0026thinsp;15.3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFoliar fertilization\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.6\u0026thinsp;\u0026plusmn;\u0026thinsp;34.5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.7\u0026thinsp;\u0026plusmn;\u0026thinsp;32.6 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.5\u0026thinsp;\u0026plusmn;\u0026thinsp;31.2 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.26 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.4\u0026thinsp;\u0026plusmn;\u0026thinsp;34.3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSelenium application\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSe0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.2\u0026thinsp;\u0026plusmn;\u0026thinsp;34.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSe60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.9\u0026thinsp;\u0026plusmn;\u0026thinsp;31.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eCONT: wheat fertilized with dry blood meal in a single application at seeding; CONT\u0026thinsp;+\u0026thinsp;N: CONT plus N foliar application at the beginning of heading (BBCH stage 51); CONT\u0026thinsp;+\u0026thinsp;S: CONT plus foliar S application at flag leaf sheath opening stage (BBCH stage 47); CONT\u0026thinsp;+\u0026thinsp;NS: CONT plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively; Se0: control without selenium; Se60: application of sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO4), at rate of 60 g ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at booting stage (BBCH stage 41).\u003c/p\u003e\n \u003cp\u003eSLP: Soluble large polymers; ILP: insoluble large polymers; mon/pol: monomeric/polymeric ratio; UPP: unextractable polymeric protein; GI: gluten index According to Tukey\u0026apos;s test, for each factor, values in each column followed by different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05). Data are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (3 replicates).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Macro and microelements concentration in the grain\u003c/h2\u003e\n \u003cp\u003eThe analysis of variance (ANOVA) generally showed a significant effect of year, cultivar, foliar fertilization, Se biofortification, and their interactions on grain macro and microelements concentration (Table S3, additional file). Due to the high number and complexity of the interactions, these will be better explored through multivariate analysis.\u003c/p\u003e\n \u003cp\u003eThe two years significantly affected the concentration of macro and microelements in the grain. In the first wetter year, the highest values of grain [N], [Se], and [Fe] were observed. On the contrary, the grain [S] and [Zn] were higher in the second drier year (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eAmong the cultivars, Cappelli showed a significantly higher grain [N] and [S], the latter together with the cultivar old Saragolla. Old Saragolla differed from the other cultivars for its lowest grain [Se]. As for the two modern cultivars, Marco Aurelio differed for the highest grain [Fe] and Nadif for the highest grain [Se] and the lowest grain [S] and [Zn] (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eAs for the organic foliar fertilization strategies under study, the highest grain [S] and [Fe] were obtained by CONT\u0026thinsp;+\u0026thinsp;NS fertilization, while grain [Se] significantly decreased using CONT\u0026thinsp;+\u0026thinsp;S or CONT\u0026thinsp;+\u0026thinsp;NS strategies. Finally, the highest grain [Zn] was observed utilizing CONT\u0026thinsp;+\u0026thinsp;NS and the lowest utilizing CONT\u0026thinsp;+\u0026thinsp;N strategies (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The foliar Se application significantly increased the grain [Se] with no effect on the other detected micronutrient grain concentration (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of year, cultivar, foliar fertilization, and selenium application on macro and micro nutrient grain concentration resulting from analysis of variance (ANOVA).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrain [N]\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrain [S]\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrain [Se]\u003c/p\u003e\n \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrain [Fe]\u003c/p\u003e\n \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrain [Zn]\u003c/p\u003e\n \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCultivar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCappelli\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eold Saragolla\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMarco Aurelio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.2\u0026thinsp;\u0026plusmn;\u0026thinsp;12.8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNadif\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eFoliar fertilization\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSelenium application\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSe0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSe60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eCONT: wheat fertilized with dry blood meal in a single application at seeding; CONT\u0026thinsp;+\u0026thinsp;N: CONT plus N foliar application at the beginning of heading (BBCH stage 51); CONT\u0026thinsp;+\u0026thinsp;S: CONT plus foliar S application at flag leaf sheath opening stage (BBCH stage 47); CONT\u0026thinsp;+\u0026thinsp;NS: CONT plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively; Se0: control without selenium; Se60: application of sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO4), at rate of 60 g ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at booting stage (BBCH stage 41).\u003c/p\u003e\n \u003cp\u003eGrain [N], [S], [Se], [Fe] and [Zn]: nitrogen, sulphur, selenium, iron and zinc grain concentrations. According to Tukey\u0026apos;s test, for each factor, values in each column followed by different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05). Data are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (3 replicates).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Principal Component Analysis (PCA)\u003c/h2\u003e\n \u003cp\u003eCorrelation analysis revealed a complex network of relationships among protein fractions, gluten quality traits, and grain mineral concentrations (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). ILP showed positive associations with polymeric protein fraction, UPP, GI, and grain [Fe], while SLP was positively related to polymeric protein fraction and grain [S] and [Zn]. Polymeric protein fraction correlated positively with UPP, and grain [S] and [Zn]. Grain [N] was positively associated with monomeric protein fraction and grain [Fe], and grain [S] correlated positively with grain [Zn]. Grain [Fe] also showed positive associations with UPP, GI, and grain [N] (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eNegative correlations were observed between ILP and SLP, monomeric protein fraction, and grain [N]; between SLP and monomeric protein fraction, GI, and grain [N] and [Fe]; and between polymeric protein fraction and grain [N]. Grain [N] was negatively associated with grain [S] and [Zn], while grain [S] showed negative relationships with monomeric protein fraction, UPP, and GI. Finally, grain [Fe] was negatively related to monomeric protein fraction, grain [Zn], and GI (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Due to the high number of correlations observed among the different parameters evaluated, these were jointly considered in a multivariate approach and were processed statistically for principal component analysis (PCA).\u003c/p\u003e\n \u003cp\u003eThe PCA analysis showed that PC1 explained 45.2% of the total variance and was positively correlated with polymeric protein fraction, SLP, grain [S] and [Zn], and negatively correlated with monomeric protein fraction, mon/pol ratio, and grain [N] (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Thus, PC1 is linked to the \u0026ldquo;degree of polymerization\u0026rdquo;, mainly related to the capacity to form covalent bonds. On the other hand, PC2 explained 26.9% of the total variance and had strong positive correlations with ILP, UPP, GI, and grain [Fe] and could be described as a factor associated with \u0026quot;protein assembly degree\u0026quot; (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCorrelation coefficients between active quantitative variables, supplementary qualitative variables, and the first two Principal Components (PC), with the indication of the explained variance.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eQuantitative active variables\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolymeric protein fraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.94\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.27\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMonomeric protein fraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.94\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.27\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emon/pol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.95\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.23\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.64\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.59\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eILP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.41\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.88\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.95\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.31\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrain [N]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.82\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrain [S]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.35\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrain [Se]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.03\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrain [Fe]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.26\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.52\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrain [Zn]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.83\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.15\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eQualitative supplementary variables\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.76\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCultivar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.65\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFoliar Fertilization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00007\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSe application\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0008\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExplained variance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eSLP: Soluble large polymers; ILP: insoluble large polymers; mon/pol: monomeric/polymeric ratio; UPP: unextractable polymeric protein; GI: gluten index; Grain [N], [S], [Se], [Fe] and [Zn]: nitrogen, sulphur, selenium, iron and zinc grain concentrations.\u003c/p\u003e\n \u003cp\u003eAs for the qualitative supplementary variables, the \u0026ldquo;year\u0026rdquo; variable had a stronger correlation with PC1, while the qualitative supplementary variable \u0026ldquo;cultivar\u0026rdquo; had a stronger correlation with PC2. No correlation was found for organic foliar fertilization and Se application with PC1 and PC2 (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Cluster analysis\u003c/h2\u003e\n \u003cp\u003eThree clusters emerged from the hierarchical clustering performed on the extracted PCs (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Category frequency distributions within clusters for the qualitative variables highlighted that the year and the cultivar were significantly different from the overall frequency distribution according to \u0026chi;\u003csup\u003e2\u003c/sup\u003e test at p-value\u0026thinsp;\u0026le;\u0026thinsp;0.001, whereas organic foliar fertilization and Se application were not significant.\u003c/p\u003e\n \u003cp\u003eAs for the year, Cluster 1 (C1) was entirely composed of experimental data collected in 2018, Cluster 2 (C2) grouped 76% of the data from the 2019 growing season, and Cluster 3 (C3) grouped 95.5% of the data from the 2019 growing season.\u003c/p\u003e\n \u003cp\u003eAs for the cultivars, in C1 Marco Aurelio, Nadif and old Saragolla were equally represented, while Cappelli contributed to the cluster only for 6.4%. In C2, 57.7% of the data belonged to Cappelli, 33.8% to old Saragolla, and only 8.5% to Nadif, while Marco Aurelio was absent. Finally, for C3, 54.5% of the data belonged to Marco Aurelio, 40.9% to Nadif, and only 4.6% to Cappelli, while old Saragolla was absent. In all of the three clusters the four organic foliar fertilization and the Se application were equally represented (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). C1 was positioned on the negative side of the PC1 factor; it was characterized by the lowest proportion of polymeric protein (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) and SLP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD), the highest proportion of the monomeric protein and mon/pol ratio (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and C), the highest value of grain [N] and [Fe] (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA and D) and the lowest grain [S] and [Zn] (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB and E). C2 was mainly positioned on the negative side of the PC2 factor and it was characterized by the highest proportion of SLP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD) and the lowest proportion of ILP, UPP, GI (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE, F, G) and grain [Fe] (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Cluster 3 was positioned on the positive side of both PC1 and PC2 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) and it was characterized by the highest proportion of polymeric protein fraction, ILP and UPP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, E and F), and the lowest proportion of monomeric protein fraction, mon/pol ratio (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and C), and grain [N] (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Mycotoxin contamination\u003c/h2\u003e\n \u003cp\u003eEach of the compared growing seasons and agronomic factors led to a significant effect on the grain contamination (Table S4; additional file). The 2018 growing season resulted in a higher content of all the detected mycotoxins compared to 2019, with an increase comprise between +\u0026thinsp;13% (DON) and +\u0026thinsp;46% (DON-3-G) (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). According to the type of mycotoxins the cultivar resulted in a different effect: Nadif resulted in a content of DON and DON-3-G that is less than half of that measured in old Saragolla, otherwise this old genotype showed a lower contamination of both MON (\u0026shy;8%) and ENNtot (\u0026shy;82%). The application of S foliar fertilizer led to a significant reduction of all the mycotoxins, between 14% for DON and 50% for MON. Se application led to a stronger effect, with a reduction of 30% for DON, 50% for MON and 141% for ENNtot.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of year, cultivar, organic foliar fertilization and Se biofortification on the mycotoxin contamination in durum wheat grain resulting from analysis of variance (ANOVA).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDONtot\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDON-3-G\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eENNtot\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e359\u0026thinsp;\u0026plusmn;\u0026thinsp;173 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e279\u0026thinsp;\u0026plusmn;\u0026thinsp;135 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57\u0026thinsp;\u0026plusmn;\u0026thinsp;28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;7 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e133\u0026thinsp;\u0026plusmn;\u0026thinsp;133 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e302\u0026thinsp;\u0026plusmn;\u0026thinsp;132 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e246\u0026thinsp;\u0026plusmn;\u0026thinsp;108 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u0026thinsp;\u0026plusmn;\u0026thinsp;16 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;11 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91\u0026thinsp;\u0026plusmn;\u0026thinsp;74 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCultivar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eold Saragolla\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e463\u0026thinsp;\u0026plusmn;\u0026thinsp;102 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e368\u0026thinsp;\u0026plusmn;\u0026thinsp;75 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63\u0026thinsp;\u0026plusmn;\u0026thinsp;23 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u0026thinsp;\u0026plusmn;\u0026thinsp;7 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u0026thinsp;\u0026plusmn;\u0026thinsp;24 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNadif\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e198\u0026thinsp;\u0026plusmn;\u0026thinsp;44 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156\u0026thinsp;\u0026plusmn;\u0026thinsp;35 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;9 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;11 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e190\u0026thinsp;\u0026plusmn;\u0026thinsp;104 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFoliar fertilization\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e353\u0026thinsp;\u0026plusmn;\u0026thinsp;155 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e278\u0026thinsp;\u0026plusmn;\u0026thinsp;119 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49\u0026thinsp;\u0026plusmn;\u0026thinsp;26 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;10 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134\u0026thinsp;\u0026plusmn;\u0026thinsp;124 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e309\u0026thinsp;\u0026plusmn;\u0026thinsp;154 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e246\u0026thinsp;\u0026plusmn;\u0026thinsp;125 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43\u0026thinsp;\u0026plusmn;\u0026thinsp;24 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u0026thinsp;\u0026plusmn;\u0026thinsp;86 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSelenium application\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSe0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e386\u0026thinsp;\u0026plusmn;\u0026thinsp;168 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e304\u0026thinsp;\u0026plusmn;\u0026thinsp;131 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55\u0026thinsp;\u0026plusmn;\u0026thinsp;28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;11 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e159\u0026thinsp;\u0026plusmn;\u0026thinsp;129 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSe60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e275\u0026thinsp;\u0026plusmn;\u0026thinsp;119 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e220\u0026thinsp;\u0026plusmn;\u0026thinsp;97 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u0026thinsp;\u0026plusmn;\u0026thinsp;17 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66\u0026thinsp;\u0026plusmn;\u0026thinsp;51 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N: wheat fertilized with dry blood meal in a single application at seeding plus N foliar application at the beginning of heading (BBCH stage 51); CONT\u0026thinsp;+\u0026thinsp;NS: wheat fertilized with dry blood meal in a single application at seeding plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively; Se0: control without selenium; Se60: application of sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e), at rate of 60 g ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at booting stage (BBCH stage 41).\u003c/p\u003e\n \u003cp\u003eDONtot: total deoxynivalenol forms, sum of DON, DON-3-G and 3-ADON; DON: deoxynivalenol; DON-3-G, deoxynivalenol-3-glucoside; MON: moniliformin; ENNtot: total enniatin forms, sum of ENN A, A\u003csub\u003e1\u003c/sub\u003e, B and B\u003csub\u003e1\u003c/sub\u003e. According to Tukey\u0026apos;s test, for each factor, values in each column followed by different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05). Data are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (3 replicates).\u003c/p\u003e\n \u003cp\u003eThe interaction of year with cultivar and with organic foliar fertilization was significant. The combination of cultivar and year did not lead to a significant difference for DON-3-G and ENNtot, while only in 2019, old Saragolla showed a lower MON contamination compared to Nadif (Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). As far as the DONtot is concerned, in both growing seasons Nadif showed a lower content than old Saragolla, although the difference was higher in 2018.\u003c/p\u003e\n \u003cp\u003eIn a single year, the organic foliar fertilization did not significantly affect the contamination of DON forms; otherwise, the application of S significantly minimized the MON and ENNtot content in both growing years, with a higher percentage reduction in the 2019 growing season.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of combination of the durum wheat cultivar or organic foliar fertilization with year on the mycotoxin contamination resulting from analysis of variance (ANOVA).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDONtot\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDON-3-G\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eENNtot\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCultivar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eold Saragolla\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e514\u0026thinsp;\u0026plusmn;\u0026thinsp;89 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e401\u0026thinsp;\u0026plusmn;\u0026thinsp;63 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79\u0026thinsp;\u0026plusmn;\u0026thinsp;20 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;6 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;21 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNadif\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e203\u0026thinsp;\u0026plusmn;\u0026thinsp;44 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156\u0026thinsp;\u0026plusmn;\u0026thinsp;34 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u0026thinsp;\u0026plusmn;\u0026thinsp;8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e230\u0026thinsp;\u0026plusmn;\u0026thinsp;127 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eold Saragolla\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e412\u0026thinsp;\u0026plusmn;\u0026thinsp;90 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336\u0026thinsp;\u0026plusmn;\u0026thinsp;73 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u0026thinsp;\u0026plusmn;\u0026thinsp;12 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u0026thinsp;\u0026plusmn;\u0026thinsp;28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNadif\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e193\u0026thinsp;\u0026plusmn;\u0026thinsp;45 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156\u0026thinsp;\u0026plusmn;\u0026thinsp;38 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;5 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u0026thinsp;\u0026plusmn;\u0026thinsp;14 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e149\u0026thinsp;\u0026plusmn;\u0026thinsp;57 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFoliar fertilization\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e380\u0026thinsp;\u0026plusmn;\u0026thinsp;179 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e293\u0026thinsp;\u0026plusmn;\u0026thinsp;137 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u0026thinsp;\u0026plusmn;\u0026thinsp;29 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;7 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e154\u0026thinsp;\u0026plusmn;\u0026thinsp;158 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e338\u0026thinsp;\u0026plusmn;\u0026thinsp;172 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e264\u0026thinsp;\u0026plusmn;\u0026thinsp;137 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;27 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112\u0026thinsp;\u0026plusmn;\u0026thinsp;104 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e325\u0026thinsp;\u0026plusmn;\u0026thinsp;129 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e263\u0026thinsp;\u0026plusmn;\u0026thinsp;103 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;17 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;13 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e153\u0026thinsp;\u0026plusmn;\u0026thinsp;79 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e280\u0026thinsp;\u0026plusmn;\u0026thinsp;136 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e229\u0026thinsp;\u0026plusmn;\u0026thinsp;114 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u0026thinsp;\u0026plusmn;\u0026thinsp;15 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;3 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68\u0026thinsp;\u0026plusmn;\u0026thinsp;62 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N: wheat fertilized with dry blood meal in a single application at seeding plus N foliar application at the beginning of heading (BBCH stage 51); CONT\u0026thinsp;+\u0026thinsp;NS: wheat fertilized with dry blood meal in a single application at seeding plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively.\u003c/p\u003e\n \u003cp\u003eDONtot: total deoxynivalenol forms, sum of DON, DON-3-G and 3-ADON; DON: deoxynivalenol; DON-3-G: deoxynivalenol-3-glucoside; MON: moniliformin; ENNtot: total enniatin forms, sum of ENN A, A\u003csub\u003e1\u003c/sub\u003e, B and B\u003csub\u003e1\u003c/sub\u003e. According to Tukey\u0026apos;s test, for each combination of agronomic factor and year, values in each column followed by different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05). Data are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (3 replicates).\u003c/p\u003e\n \u003cp\u003eThe effect of S application in term of mycotoxin contamination showed a significant interaction with the cultivar: this fertilization strategy was able to reduce the DONtot and DON contamination only for Nadif cultivar (Table \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Otherwise, CONT\u0026thinsp;+\u0026thinsp;NS treatment reduced mycotoxins produced by \u003cem\u003eF. avenaceum\u003c/em\u003e for both cultivars, with a higher effect for MON in Nadif (\u0026shy;39%) and for ENNtot in old Saragolla (\u0026shy;51%).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of combination of the durum wheat cultivar and organic foliar fertilization on the mycotoxin contamination resulting from analysis of variance (ANOVA) performed.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDONtot\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDON-3-G\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMON\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eENNtot\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(\u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCultivar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFoliar fertilizatio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eold Saragolla\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e482\u0026thinsp;\u0026plusmn;\u0026thinsp;109 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e379\u0026thinsp;\u0026plusmn;\u0026thinsp;80 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67\u0026thinsp;\u0026plusmn;\u0026thinsp;24 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;6 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47\u0026thinsp;\u0026plusmn;\u0026thinsp;27 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e444\u0026thinsp;\u0026plusmn;\u0026thinsp;95 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e358\u0026thinsp;\u0026plusmn;\u0026thinsp;71 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u0026thinsp;\u0026plusmn;\u0026thinsp;22 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;6 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;14 d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNadif\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e223\u0026thinsp;\u0026plusmn;\u0026thinsp;43 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e177\u0026thinsp;\u0026plusmn;\u0026thinsp;34 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u0026thinsp;\u0026plusmn;\u0026thinsp;9 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u0026thinsp;\u0026plusmn;\u0026thinsp;12 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e222\u0026thinsp;\u0026plusmn;\u0026thinsp;121 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e173\u0026thinsp;\u0026plusmn;\u0026thinsp;29 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e135\u0026thinsp;\u0026plusmn;\u0026thinsp;22 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;3 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e157\u0026thinsp;\u0026plusmn;\u0026thinsp;76 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eCONT\u0026thinsp;+\u0026thinsp;N: wheat fertilized with dry blood meal in a single application at seeding plus N foliar application at the beginning of heading (BBCH stage 51); CONT\u0026thinsp;+\u0026thinsp;NS: wheat fertilized with dry blood meal in a single application at seeding plus the combination of S and N foliar applications at flag leaf sheath opening stage (BBCH stage 47) and at the beginning of the heading (BBCH stage 51), respectively. DONtot: total deoxynivalenol forms, sum of DON, DON-3-G and 3-ADON; DON: deoxynivalenol; DON-3-G: deoxynivalenol-3-glucoside; MON: moniliformin; ENNtot: total enniatin forms, sum of ENN A, A\u003csub\u003e1\u003c/sub\u003e, B and B\u003csub\u003e1\u003c/sub\u003e. According to Tukey\u0026apos;s test, values in each column followed by different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05). Data are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (3 replicates).\u003c/p\u003e\n \u003cp\u003eAlthough the Se application was always able to reduce significantly the content of the compared mycotoxins, the positive impact of this practice was more effective within the highest level of contamination: in Nadif cultivar for ENNtot (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) or the CONT\u0026thinsp;+\u0026thinsp;N treatment for MON and ENNtot (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFinally, a network of relationships among grain mycotoxin contamination and protein fractions and grain mineral concentrations were considered (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). In particular, significant positive correlations were observed between DONtot, DON, DON-3-G and monomeric protein fraction, mon/pol, [N] grain and [S] grain, and between ENNtot and UPP and GI. Significant negative correlations were observed between DONtot, DON, DON-3-G and ILP, polymeric protein fraction, UPP, GI and [Se] grain and between ENNtot and [S] grain and [Zn] grain.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur two-year field trial provides novel evidence that targeted agronomic practices, including organic foliar fertilization and selenium biofortification, enhance both the technological and nutritional traits of durum wheat, and at the same time support food safety and sustainable production under Mediterranean conditions. Gluten proteins are among the major determinants of wheat quality and consist of monomeric (gliadins) and polymeric subunits high (HMW-GS) and low (LMW-GS) molecular weight, which interact each other, leading to the formation of polymers with different sizes and structures [43]. The glutenin polymers can further polymerize by intermolecular disulphide bonds, forming SLP which can also assemble through hydrogen bonds, leading to the formation of UPP [43]. In bread wheat, the proportion of UPP has often been positively correlated with dough strength [44\u0026ndash;46]. As for durum wheat, the relation between UPP and GI, which is the main gluten strength indicator, has been less investigated, and the results are contradictory [32]; however, in our study, the two parameters resulted strongly and positively correlated.\u003c/p\u003e\u003cp\u003eEnvironmental conditions during grain filling, particularly those affecting plant water status, can significantly influence both the polymerization and assembly of glutenin proteins [43], aspect of relevant interest under the ongoing climate change situation. Although this phenomenon is well documented in bread wheat, it remains less explored in durum wheat, despite it is commonly grown in sub-arid environments.\u003c/p\u003e\u003cp\u003eIn our study, the lower rainfall during the 2019 grain filling period promoted gluten protein polymerization, as evidenced by significantly higher SLP and polymeric protein fractions, and negatively affected protein assembly, as indicated by reduced UPP values. In line with our results, Park et al. [47] reported that water stress can decrease the proportion of UPP in wheat grains, particularly when it shortens the grain filling period [48], as observed in our trial, likely interfering with the assembly process of SLP in UPP. Moreover, drought conditions in 2019 also led to a reduction in grain [N], due to the negative effect of drought stress during grain filling on N assimilation and remobilization in wheat [49, 50], and to a lower grain [Fe] due to lower uptake [51]. In contrast, grain [S] and [Zn] increased, possibly due to drought-induced activation of S assimilation pathways [52, 53] and to the enhanced Zn uptake (Nawaz et al., 2015) [51]. The observed increase in grain [S] favoured protein polymerization through the formation of inter-chain disulphide bonds, particularly among cysteine-rich HMW-GS (Yu et al., 2021) [4], which are abundant in SLPs (Shewry and Lafiandra, 2022) [43]. Similarly, higher grain [Zn] could have contributed to the polymerization, by supporting the synthesis of S-rich proteins [54]. Indeed, both grain [S] and [Zn] were strongly and positively correlated with SLP and polymeric fraction. CONT\u0026thinsp;+\u0026thinsp;S significantly increased SLP content, consisted with Tea et al. [55\u0026ndash;56], who reported enhanced polymeric protein proportions following S application in bread wheat, while had a depressing effect on protein assembly (ILP and UPP). Interestingly, we hypothesized that higher grain [S], while promoting disulphide bonding and protein polymerization, may hinder hydrogen bond formation required for protein assembly. On the contrary, the CONT\u0026thinsp;+\u0026thinsp;NS fertilization reduced SLP content and disulphide bond formation potential, likely favouring hydrogen bond-mediated protein assembly. This synergistic effect is supported by Tea et al. [55], who observed increased large polymer aggregates after N\u0026thinsp;+\u0026thinsp;S application. Additionally, CONT\u0026thinsp;+\u0026thinsp;NS enhanced grain [S], [Fe], and [Zn], likely due to both improved root growth and nutrient uptake capacity induced by N [57] and to the involvement of S in metabolic pathways critical to mineral absorption [58]. To our knowledge, this is the first study in durum wheat that demonstrates a clear directional effect of combined organic foliar N and S fertilization on grain S, Fe and Zn accumulation, which may have substantial implications for both nutritional quality and technological performance. Notably, under CONT\u0026thinsp;+\u0026thinsp;NS fertilization, the grain [Fe] and the grain [Zn] consistently exceeded nutritional thresholds to ensure meet human nutritional requirements (30 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Fe and 25 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Zn; [59\u0026ndash;60]).\u003c/p\u003e\u003cp\u003eAs expected, grain [Se] decreased following CONT\u0026thinsp;+\u0026thinsp;S and CONT\u0026thinsp;+\u0026thinsp;NS fertilization, likely due to competitive inhibition between chemically similar S and Se during uptake [61, 62]. On the contrary Se foliar fertilization did not influence grain [S]. Despite literature suggesting antagonism between Se and Fe/Zn uptake [63], our data indicate that foliar Se application did not compromise grain [Fe] and [Zn] accumulation, confirming the advantage of bypassing soil competition [64, 65]. Thus, Se foliar fertilization in addition to increasing yield [34] offers nutritional benefits by increasing the availability of Se (Se requirements of humans and animals\u0026thinsp;\u0026gt;\u0026thinsp;50\u0026ndash;100 \u0026micro;g Se kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, [34]) without compromising that of Fe and Zn and without impacting protein quality parameters. Moreover, Se foliar application as sodium selenate had a positive effect on the minimizing mycotoxin grain contamination as reported below.\u003c/p\u003e\u003cp\u003eThe effect on the absorption of macro and micronutrients in wheat depends not only on the environmental conditions, but also on cultivar [66]. The modern cultivar Marco Aurelio exhibited the highest grain [Fe] and [Zn], as well as the highest yield (3.3 t ha ⁻\u0026sup1;, [34]), suggesting that, contrary to previous assumptions [67], high yield and micronutrient concentration are not necessarily antagonistic.\u003c/p\u003e\u003cp\u003eTo integrate the numerous parameters investigated and account for their complex interactions, we adopted a multivariate workflow (PCA followed by cluster analysis) to synthesize the experimental evidence and identify the main drivers of wheat technological and nutritional quality. The results of the PCA highlighted two main dimensions of variability: a first component (\u0026ldquo;polymerization factor\u0026rdquo;) mainly associated with SLP, total polymeric protein fraction, grain [S] and [Zn], and a second component (\u0026ldquo;assembly factor\u0026rdquo;) associated with ILP, UPP, GI and grain [Fe]. This allowed a better understanding of the distinct yet complementary roles of mineral nutrition in gluten protein structure. Indeed, while previous studies reported correlations between grain micronutrients and protein content [68\u0026ndash;71], this study provides novel evidence of specific associations between grain [Fe] and gluten protein assembly, and between [Zn] and polymerization in durum wheat.\u003c/p\u003e\u003cp\u003eThe supplementary qualitative variables \u0026ldquo;Year\u0026rdquo; and \u0026ldquo;Cultivar\u0026rdquo; showed significant associations with PC1 and PC2, respectively, suggesting that environmental factors mainly influenced protein polymerization, while genotypic traits governed assembly processes.\u003c/p\u003e\u003cp\u003eAlso the Cluster analysis supported this finding. C1 included only samples from the first wetter year, was defined by the lowest values of SLP, polymeric protein fraction, and grain [S] and [Zn], thus placing it on the negative side of the polymerization factor. However, its moderate position along the assembly factor, linked to relatively higher grain [Fe], suggests that favourable water availability may have promoted Fe uptake and protein aggregation, despite poor polymerization. C2, largely composed of samples from the second drier year, exhibited the highest SLP and polymeric fraction values, but low levels of ILP, UPP, GI, and grain [Fe], placing it on the negative side of the assembly axis. This cluster notably lacked the modern cultivar Marco Aurelio, while the other modern one Nadif contributed to this cluster only with 8.5%, highlighting the limited ability of older cultivars to sustain assembly processes under stress. Conversely, C3, which included mainly the samples from second drier year and from the modern cultivars Marco Aurelio and, stood out as the only group with positive values on both PCA axes. This cluster showed a favourable combination of high SLP, polymeric fraction, ILP, UPP, and grain [Zn], suggesting that this genotype possesses superior capacity to simultaneously support polymerization and assembly processes, potentially due to more efficient nutrient use and stress resilience.\u003c/p\u003e\u003cp\u003eMoreover, this study reported a significant effect of the compared agronomic practices also on the mycotoxin occurrence in grain. In general, the DON content was always below the maximum limits for durum wheat (EU Reg. 2024/1022), also when the sum of native form and modified one (DONtot, sum of DON, 3-ADON and DON-3-G) has been considered. The level of contamination of emerging mycotoxins such as MON (on average 25 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and ENNtot (112 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as a sum of different enniatin forms) was overall low if compared with other cereal growing areas [23], thereby limiting potential health risks for consumers and contributing to safer grain supplies, a key aspect in the broader context of food security.\u003c/p\u003e\u003cp\u003eInorganic Se forms, such as sodium selenite (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e) or sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4,\u003c/sub\u003e applied in the present experiment), were previously reported to have inhibitory effects on fungi or synergistic effects with fungicides [72, 73]. Both inorganic and organic Se forms were recently reported to inhibit \u003cem\u003eF. graminearum\u003c/em\u003e growth and DON production in vitro [74]. Among the different Se tested forms Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e resulted the inorganic Se form with the best inhibitory effect on \u003cem\u003eF. graminearum\u003c/em\u003e growth and DON accumulation [74]. The authors supposed that Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e can be assimilated and transported more efficiently by plants than other inorganic or organic form (Li et al., 2008; 2010) [75, 76]. In the present study the Se foliar application was carried out at booting stage and was able to prevent the DON production during wheat ripening, caused by a fungal infection which occurred at flowering. Moreover, in addition to DON, the data collected highlighted a similar effect of Se foliar application also on ENNtot and MON, emerging mycotoxins produced by other \u003cem\u003eFusarium\u003c/em\u003e species (e.g. \u003cem\u003eF. avenaceum\u003c/em\u003e). No previous studies were instead reported in literature on open field experiments under natural mycotoxin contamination. Kornaś et al. [77] in 2018 investigated whether the application of Se ions directly to the leaf surface can protect plants against infection by the fungal toxin zearalenone (ZEA), applying the Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e to the second leaf of seedlings. The presence of Se significantly suppressed changes at the site of ZEA application in all tested plants. Microscopic observations confirmed that foliar administration of ZEA resulted in its penetration to deeper localized cells and that damage induced by ZEA (mainly to chloroplasts) decreased after Se application. Analyses of antioxidant enzymes demonstrated the involvement of Se in antioxidation mechanisms, by activating superoxide dismutases (SOD) and catalases (CAT) under ZEA-induced stress conditions. Thus, Kornaś et al. [77] concluded that the foliar application of Se to seedling leaves may be a non-invasive method of protecting crops against the first steps of ZEA infection.\u003c/p\u003e\u003cp\u003eAlthough with a lower effectiveness compared to Se, the foliar S application at wheat flag leaf sheath opening stage showed a significant effect in minimizing the accumulation of all detected mycotoxins. In addition to the well-known role in preventing several diseases, a positive effect of elemental S application in controlling FHB in field was reported only by Haneklaus et al. [78]. In the experiment carried out on barley artificially inoculated with \u003cem\u003eF. culmorum\u003c/em\u003e, the S was applied weekly from the anthesis to the end of ripening, for a total of 5 applications, resulting in a reduction of FHB severity and increasing grain yield. To the best of authors knowledge, the present study is the first one which reported a significant contribution of S foliar application on reducing mycotoxin occurrence in field. In the study of Haneklaus et al. [78] a direct effect of elemental S on fungal infection and development could be hypothesized, due to the timing of application. Conversely, in the present experiment the application of the foliar S was carried out only once and earlier compared to the fungal occurrence; thus, the observed effect could be linked to the formation of substances with antifungal action or to the activation of the plant metabolism pathway able to induce resistant to disease infection. Several S-containing defense compounds, such as sulfur containing amino acids, glucosinolates, phytoalexins and defensins and thionins peptides are reported to enhance plant defense responses to pathogens [79]\u003c/p\u003e\u003cp\u003eTransgenic wheat plants overexpressing the gene for the production of S-containing peptides with antimicrobial activity showed an increased resistance to \u003cem\u003eF. graminearum\u003c/em\u003e [80].\u003c/p\u003e\u003cp\u003eFinally, in 2018 the development of \u003cem\u003eFusarium\u003c/em\u003e species during ripening could be co-responsible for the lower gluten protein assembly, as suggested by the high negative correlation of the DONtot contamination with ILP (-0.73***) and with UPP (-0.7***). Koga et al. [81] reported that \u003cem\u003eF. graminearum\u003c/em\u003e secreted gluten-degrading proteases play an important role in reducing the molecular size of glutenin polymers. Not only proteases from \u003cem\u003eF. graminearum\u003c/em\u003e, but also from several other \u003cem\u003eFusarium\u003c/em\u003e spp. have been reported to degrade gluten proteins [82, 83], although in our study the main correlation was reported with DON-producer fungal species.\u003c/p\u003e\u003cp\u003eThese results highlight the need to better understand the interaction between organic and Se foliar applications and the effects on plant metabolism and the indirect potential role in minimizing fungal diseases.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study provides new insights into the complex interactions between genotype, environment, and application of foliar organic fertilizer in shaping both the nutritional and technological and the risk of accumulation of mycotoxin of durum wheat. The results underscore the importance of adopting integrated strategies that combine modern cultivars with targeted organic fertilization approaches and foliar micronutrient fortification to achieve sustainable improvements in grain quality.\u003c/p\u003e\u003cp\u003eIn particular, the combined foliar application of organic N and S proved effective in enhancing grain concentrations of essential micronutrients such as Fe, Zn, and S. These increases were not only beneficial from a nutritional standpoint, but also contributed to improving technological traits, such as gluten protein polymerization and assembly and could contribute to the reduction of mycotoxin accumulation. Notably, this study offers novel evidence of a specific association between grain [Fe] and gluten protein assembly, and between [Zn] and protein polymerization, highlighting the dual functional role of micronutrients in durum wheat quality. Furthermore, foliar Se application was shown to enrich grain Se content without compromising Fe and Zn accumulation or protein quality, confirming its value as a biofortification strategy that bypasses competitive uptake mechanisms in the soil. In addition, these agronomic practices resulted in a clear and consistent effect in minimizing the DON contamination in wheat grain at harvest.\u003c/p\u003e\u003cp\u003ePrincipal component and cluster analyses revealed that while environmental factors primarily influenced polymerization, genotypic traits governed the protein assembly process. The modern cultivar Marco Aurelio, in particular, demonstrated a remarkable ability to combine elevated concentrations of micronutrients and favorable gluten quality indices, even under water-limited conditions. Overall, our results highlight that the strategic use of organic fertilizers, when combined with modern cultivars, can enhance both the nutritional and technological quality of durum wheat and reduce the DON contamination, although the potential risk of other emerging mycotoxins needs to be carefully verified. This integrated approach supports more sustainable production systems that align with current goals of environmental stewardship, climate resilience, and human health.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCRediT authorship contribution statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFC: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Methodology, Investigation, Formal analysis, Data curation, Visualization, Conceptualization. GG: Writing \u0026ndash; review \u0026amp; editing, Methodology, Data curation, Visualization. MB: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Investigation, Resources. VS: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Formal analysis. GMM: Writing \u0026ndash; review \u0026amp; editing, Investigation, Formal analysis. YM-A: Writing \u0026ndash; review \u0026amp; editing, Supervision, Resources. MMG: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Methodology, Investigation, Supervision, Data curation, Visualization, Conceptualization, Resources.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFC: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Methodology, Investigation, Formal analysis, Data curation, Visualization, Conceptualization. GG: Writing \u0026ndash; review \u0026amp; editing, Methodology, Data curation, Visualization. MB:\u0026nbsp;Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Investigation, Resources. VS: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Formal analysis. GMM: Writing \u0026ndash; review \u0026amp; editing, Investigation, Formal analysis. YM-A:\u003csup\u003e\u0026nbsp;\u003c/sup\u003eWriting \u0026ndash; review \u0026amp; editing, Supervision, Resources. MMG: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Methodology, Investigation, Supervision, Data curation, Visualization, Conceptualization, Resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePID2023-148425NB-I00 project funded by MCIU/AEI/10.13039/501100011033 and Feder, UE\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was part of project SOFT (Smart Organic Farming Tecniques) financed under the PSR Puglia 2014\u0026ndash;2020 funds, Measure 16\u0026mdash;Cooperation, Submeasure 16.2\u0026mdash;Support for pilot projects and the development of new products, practices, processes, and technologies (CUP B79J20000080009).\u003c/p\u003e\n\u003cp\u003eField experiments and wheat cultivation were supported by Dott. Pasquale De Vita, Dott. Ivano Pecorella and the staff of C.R.A. Experimental Institute for Cereal Research, S.S. 16 km 675, 71100 Foggia, Italy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMart\u0026iacute;nez-Moreno F, Ammar K, Sol\u0026iacute;s I.. Global changes in cultivated area and breeding activities of durum wheat from 1800 to date: a historical review. Agronomy. 2022;12(5):1135. https://doi.org/10.3390/agronomy12051135. \u003c/li\u003e\n\u003cli\u003eSarkar A, Fu BX. Impact of quality improvement and milling innovations on durum wheat and end products. Foods. 2022;11(12):1796. https://doi.org/10.3390/foods11121796. \u003c/li\u003e\n\u003cli\u003eMefleh M, Conte P, Fadda C, Giunta F, Piga A, Hassoun G, Motzo R. From ancient to old and modern durum wheat varieties: Interaction among cultivar traits, management, and technological quality. J. Sci. Food Agric. 2019;99(5):2059-2067. https://doi.org/doi:10.1002/jsfa.9388.\u003c/li\u003e\n\u003cli\u003eYu Z, She M, Zheng T, Diepeveen D, Islam S, Zhao Y, Blanchard CL, Ma W. Impact and mechanism of sulphur-deficiency on modern wheat farming nitrogen-related sustainability and gliadin content. Commun. Biol. 2021;4(1):945. https://doi.org/10.1038/s42003-021-02458-7 .\u003c/li\u003e\n\u003cli\u003eFan M, Shen J, Yuan L, Jiang R, Chen X, Davies WJ, Zhang F. Improving crop productivity and resource use efficiency to ensure food security and environmental quality in China. J. Exp. Bot. 2012;63 (1):13-24. https://doi.org/10.1093/jxb/err248.\u003c/li\u003e\n\u003cli\u003eZhang Y, Dai X, Jia D, Li H, Wang Y, Li C, Xu H, He M. Effects of plant density on grain yield, protein size distribution, and breadmaking quality of winter wheat grown under two nitrogen fertilisation rates. Europ. J. Agron. 2016;73:1-10. https://doi.org/10.1016/j.eja.2015.11.015.\u003c/li\u003e\n\u003cli\u003eLiu Y, Han M, Zhou X, Li W, Du C, Zhang Y, Zhang Y, Sun Z, Wang Z. Optimizing nitrogen fertilizer application under reduced irrigation strategies for winter wheat of the north China plain. Irrig. Sci. 2022;40(2):255-265. https://doi.org/10.1007/s00271-021-00764-w. \u003c/li\u003e\n\u003cli\u003eBoix-Fayos C, de Vente J. Challenges and potential pathways towards sustainable agriculture within the European Green Deal. Agric. Syst. 2023;207:103634. https://doi.org/10.1016/j.agsy.2023.103634 \u003c/li\u003e\n\u003cli\u003eTappi M, Carucci F, Gatta G, Giuliani MM, Lamonaca E, Santeramo FG. Temporal and design approaches and yield-weather relationships. Clim. Risk Manag. 2023\u0026deg;;40:100522. https://doi.org/10.1016/j.crm.2023.100522.\u003c/li\u003e\n\u003cli\u003eTappi M, Carucci F, Gagliardi A, Gatta G, Giuliani MM, Santeramo FG. Earliness, phenological phases and yield-temperature relationships: evidence from durum wheat in Italy. Bio-based Appl. Econ. 2023b;12(2):115-125. https://doi.org/10.36253/bae-13745.\u003c/li\u003e\n\u003cli\u003eQaswar M, Jing H, Ahmed W, Dongchu L, Shujun L, Lu Z, Cai A, Liu L, Xu Y, Gao J, Huimin Z. Yield sustainability, soil organic carbon sequestration and nutrients balance under long-term combined application of manure and inorganic fertilizers in acidic paddy soil. Soil Tillage Res. 2020;198:104569. https://doi.org/10.1016/j.still.2019.104569. \u003c/li\u003e\n\u003cli\u003eXu F, Liu Y, Du W, Li C, Xu M, Xie T, Yin Y, Guo H. Response of soil bacterial communities, antibiotic residuals, and crop yields to organic fertilizer substitution in North China under wheat\u0026ndash;maize rotation. Sci. Total Environ. 2021;785:147248. https://doi.org/10.1016/j.scitotenv.2021.147248. \u003c/li\u003e\n\u003cli\u003eGeng Y, Cao G, Wang L, Wang S. Effects of equal chemical fertilizer substitutions with organic manure on yield, dry matter, and nitrogen uptake of spring maize and soil nitrogen distribution. PLoS One. 2019;14(7): e0219512. https://doi.org/10.1371/journal.pone.0219512.\u003c/li\u003e\n\u003cli\u003eGuiducci M, Tosti G, Falcinelli B, Benincasa P. Sustainable management of nitrogen nutrition in winter wheat through temporary intercropping with legumes. Agron. Sustain. Dev. 2018;(38)31. https://doi.org/10.1007/s13593-018-0509-3. \u003c/li\u003e\n\u003cli\u003eKonvalina P, Moudry Jr J., Capouchova I, Moudry J. Baking quality of winter wheat varieties in organic farming, Agron. Res. 2009;7:612\u0026ndash;617.\u003c/li\u003e\n\u003cli\u003eLacko-Bartosova M, Lacko-Bartosova L, Konvalina P. Wheat rheological and Mixolab quality in relation to cropping systems and plant nutrition sources, Czech J. Food Sci. 2021;39. https://doi.org/10.17221/189/2020-CJFS.\u003c/li\u003e\n\u003cli\u003eReynolds LP, Caton JS. Role of the pre-and post-natal environment in developmental programming of health and productivity. Mol. Cell. Endocrinol., 2012;354(1-2):54-59. https://doi.org/10.1016/j.mce.2011.11.013.\u003c/li\u003e\n\u003cli\u003eFAO, IFAD, UNICEF, WFP and WHO. 2019. The State of Food Security and Nutrition in the World 2019. Safeguarding against economic slowdowns and downturns. Rome, FAO. Licence: CC BY-NC-SA 3.0 IGO.\u003c/li\u003e\n\u003cli\u003eStoffaneller R, Morse NL. A review of dietary selenium intake and selenium status in Europe and the Middle East. Nutrients. 2015;7(3):1494-1537. https://doi.org/10.3390/nu7031494.\u003c/li\u003e\n\u003cli\u003eJones GD, Droz B, Greve P, Gottschalk P, Poffet D, McGrath SP, Seneviratne SI, Smith P, Winkel LH. Selenium deficiency risk predicted to increase under future climate change. Proceedings of the National Academy of Sciences. 2017;114(11):2848-2853. https://doi.org/10.1073/pnas.1611576114. \u003c/li\u003e\n\u003cli\u003ePoblaciones MJ, Rodrigo S, Santamar\u0026iacute;a O, Chen Y, McGrath SP. Agronomic selenium biofortification in Triticum durum under Mediterranean conditions: From grain to cooked pasta. Food Chem. 2014;146:378-384. https://doi.org/10.1016/j.foodchem.2013.09.070. \u003c/li\u003e\n\u003cli\u003eSharma S, Kaur N, Kaur S, Nayyar H. Selenium as a nutrient in biostimulation and biofortification of cereals. Indian J. Plant Physiol. 2017;\u003cem\u003e22\u003c/em\u003e(1): 1-15. https://doi.org/10.1007/s40502-016-0249-9.\u003c/li\u003e\n\u003cli\u003eBlandino M, Scarpino V, Sulyok M, Krska R, Reyneri A. Effect of agronomic programmes with different susceptibility to deoxynivalenol risk on emerging contamination in winter wheat. Europ. J. of Agronomy. 2017;85:12-24. https://doi.org/10.1016/j.eja.2017.01.001.\u003c/li\u003e\n\u003cli\u003eScarpino V, Blandino M. Effects of durum wheat cultivars with different degrees of FHB susceptibility grown under different meteorological conditions on the contamination of regulated, modified and emerging mycotoxins. Microorganisms. 2021;9(2):408. https://doi.org/10.3390/microorganisms9020408. \u003c/li\u003e\n\u003cli\u003eScala V, Aureli G, Cesarano G, Incerti G, Fanelli C, Scala F, Reverberi M, Bonanomi G. Climate, Soil Management, and Cultivar Affect Fusarium Head Blight Incidence and Deoxynivalenol Accumulation in Durum Wheat of Southern Italy. Front. Microbiol. 2016;7:186895. https://doi.org/10.3389/fmicb.2016.01014. \u003c/li\u003e\n\u003cli\u003eZanini B, Simonetto A, Marconi S, Marullo M, Castellano M, Gilioli G. Whole grain perceptions and consumption attitudes: Results of a survey in Italy. Health Educ. J. 2023;82(7):739-751. https://doi.org/10.1177/00178969231185662.\u003c/li\u003e\n\u003cli\u003eStreit E, Schwab C, Sulyok M, Naehrer K, Krska R, Schatzmayr G. Multi-mycotoxin screening reveals the occurrence of 139 different secondary metabolites in feed and feed ingredients. Toxins. 2013;5(3):504-523. https://doi.org/10.3390/toxins5030504.\u003c/li\u003e\n\u003cli\u003eScarpino V, Reyneri A, Sulyok M, Krska R, Blandino M. Effect of fungicide application to control Fusarium head blight and 20Fusarium and Alternaria mycotoxins in winter wheat (Triticum aestivum L.). World Mycotoxin J. 2015;8(4):499-510.\u003c/li\u003e\n\u003cli\u003eCarucci F, Gatta G, Gagliardi A, De Vita P, Bregaglio S, Giuliani MM. Agronomic strategies to improve N efficiency indices in organic durum wheat grown in Mediterranean area. Plants. 2021;10\u003cem\u003e \u003c/em\u003e(11):2444. https://doi.org/10.3390/plants10112444. \u003c/li\u003e\n\u003cli\u003eLancashire PD, Bleiholder H, Boom TVD, Langel\u0026uuml;ddeke P, Stauss R, Weber E, Witzenberger A. A uniform decimal code for growth stages of crops and weeds. Ann. Appl. Biol. 1991;119(3):561-601. https://doi.org/10.1111/j.1744-7348.1991.tb04895.x.\u003c/li\u003e\n\u003cli\u003eDe Vita P, Platani C, Fragasso M, Ficco DBM, Colecchia SA, Del Nobile MA, Padalino L, Di Gennaro S, Petrozza A. Selenium-enriched durum wheat improves the nutritional profile of pasta without altering its organoleptic properties. Food Chem. 2017;214:374-382. https://doi.org/10.1016/j.foodchem.2016.07.015. \u003c/li\u003e\n\u003cli\u003eGagliardi A, Carucci F, Masci S, Flagella Z, Gatta G, Giuliani MM. Effects of genotype, growing season and nitrogen level on gluten protein assembly of durum wheat grown under Mediterranean conditions. Agronomy. 2020;10(5):755. https://doi.org/10.3390/agronomy10050755. \u003c/li\u003e\n\u003cli\u003eInternational Association of Cereal Chemistry (ICC). Standard Methods of the ICC; ICC: Vienna, Austria, 1986\u003c/li\u003e\n\u003cli\u003eCarucci F, Moreno-Mart\u0026iacute;n G, Madrid-Albarr\u0026aacute;n Y, Gatta G, De Vita P, Giuliani MM. Selenium agronomic biofortification of durum wheat fertilized with organic products: se content and speciation in grain. Agronomy. 2022;12(10):2492. https://doi.org/10.3390/agronomy12102492. \u003c/li\u003e\n\u003cli\u003eScarpino V, Reyneri A, Blandino M. Development and comparison of two multiresidue methods for the determination of 17 Aspergillus and Fusarium mycotoxins in cereals using HPLC-ESI-TQ-MS/MS. Front. Microbiol. 2019;10,361:1-12. https://doi.org/10.3389/fmicb.2019.00361. \u003c/li\u003e\n\u003cli\u003eBox GE, Cox DR. An analysis of transformations. J R Stat Soc Series B Stat Methodol. 1964;26(2):211-243.\u003c/li\u003e\n\u003cli\u003ePinheiro JC, Bates DM. Mixed-effects models in S and S-PLUS. 2020.New York, NY: Springer New York.\u003c/li\u003e\n\u003cli\u003eCarucci F, Gatta G, Gagliardi A, Bregaglio S, Giuliani MM. Individuation of the best agronomic practices for organic durum wheat cultivation in the Mediterranean environment: a multivariate approach. Agric. Food Secur. 2023;12:12. https://doi.org/10.1186/s40066-023-00417-5. \u003c/li\u003e\n\u003cli\u003eMongiano G, Titone P, Tamborini L, Pilu R, Bregaglio S. Evolutionary trends and phylogenetic association of key morphological traits in the Italian rice varietal landscape. Sci Rep. 2018;8(1):1\u0026ndash;12. https://doi.org/10.1038/s41598- 018- 31909-1.\u003c/li\u003e\n\u003cli\u003eL\u0026ecirc; S, Josse J, Husson F. FactoMineR: an R package for multivariate analysis. J. of Stat. Softw. 2008;25:1-18. https://doi.org/10.18637/jss.v025.i01. \u003c/li\u003e\n\u003cli\u003eWei T, Simko V. R package \u0026ldquo;corrplot\u0026rdquo;: Visualization of a Correlation Matrix (Version 0.84). 2017. https://github.com/taiyun/corrplot \u003c/li\u003e\n\u003cli\u003eWickham H. ggplot2. Wiley interdisciplinary reviews: computational statistics. 2011;\u003cem\u003e3\u003c/em\u003e(2):180-185. https://doi.org/10.1002/wics.147. \u003c/li\u003e\n\u003cli\u003eShewry PR, Lafiandra D. Wheat glutenin polymers 1. Structure, assembly and properties. J. Cereal Sci. 2022;106:103486. https://doi.org/10.1016/j.jcs.2022.103486. \u003c/li\u003e\n\u003cli\u003eWardlaw IF, Blumenthal CS, Larroqu O, Wrigley CW. Contrasting effects of chronic heat stress and heat shock on kernel weight and flour quality in wheat. Funct. Plant Biol. 2002;29:25\u0026ndash;34. https://doi.org/10.1071/PP00147. \u003c/li\u003e\n\u003cli\u003eDon C, Lookhart G, Naeem H, MacRitchie F, Hamer RJ. Heat stress and genotype affect the glutenin particles of the glutenin macropolymer-gel fraction. J. Cereal Sci. 2005;42(1):69-80. https://doi.org/10.1016/j.jcs.2005.01.005. \u003c/li\u003e\n\u003cli\u003eLabuschagne MT, Elago O, Koen E. Influence of extreme temperatures during grain filling on protein fractions, and its relationship to some quality characteristics in bread, biscuit, and durum wheat. Cereal Chem\u003cem\u003e.\u003c/em\u003e 2009;86:61\u0026ndash;66. https://doi.org/10.1094/CCHEM-86-1-0061. \u003c/li\u003e\n\u003cli\u003ePark H, Clay DE, Hall RG, Rohila JS, Kharel TP, Clay SA, Lee S. Winter wheat quality responses to water, environment, and nitrogen fertilization. Commun. Soil Sci. Plan. 2014;45(14):1894-1905. https://doi.org/10.1080/00103624.2014.909833.\u003c/li\u003e\n\u003cli\u003eMartre P, Jamieson PD, Semenov MA, Zyskowski RF, Portr JR, Triboi E. Modelling protein content and composition in relation to crop nitrogen dynamics for wheat. Europ. J. Agronomy. 2006;25:138-154. https://doi.org/doi:10.1016/j.eja.2006.04.007. \u003c/li\u003e\n\u003cli\u003eGooding MJ, Pinyosinwat A, Addisu M. Nitrogen fertilizer and grain quality in wheat. In Y. A. El-Shemy (Ed.), Plant Fertilization for Agricultural Improvement. 2012;1\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eCarucci F, Gatta G, Gagliardi A, De Vita P, Giuliani MM. Strobilurin effects on nitrogen use efficiency for the yield and protein in durum wheat grown under rainfed Mediterranean conditions. Agronomy. 2020;10(10):1508. https://doi.org/10.3390/agronomy10101508. \u003c/li\u003e\n\u003cli\u003eNawaz F, Ahmad R, Ashraf MY, Waraich EA, Khan SZ. 2015 Effect of selenium foliar spray on physiological and biochemical processes and chemical constituents of wheat under drought stress. Ecotoxicol. Environ. Saf., 113, 191-200. https://doi.org/10.1016/j.ecoenv.2014.12.003 \u003c/li\u003e\n\u003cli\u003eErnst L, Goodger JQD, Alvarez S, Marsh EL, Berla B, Lockhart E, Jung J, Li P, Bohnert HJ, Schachtman D.P. Sulphate as a xylem-borne chemical signal precedes the expression of ABA biosynthetic genes in maize roots. J. Exp Bot. 2010;61(12):3395\u0026ndash;3405. https://doi.org/10.1093/jxb/erq160. \u003c/li\u003e\n\u003cli\u003eChan KX, Wirtz M, Phua SY, Estavillo GM, Pogson BJ. Balancing metabolites in drought: the sulfur assimilation conundrum. Trends Plant Sci. 2013;18(1):18\u0026ndash;29. https://doi.org/10.1016/j.tplants.2012.07.005.\u003c/li\u003e\n\u003cli\u003eLiu, H.E., Wang, Q.Y., Rengel, Z., Zhao, P., 2015. Zinc fertilization alters flour protein composition of winter wheat genotypes varying in gluten content. Plant Soil Environ\u003cem\u003e., \u003c/em\u003e61 (5), 195-200. https://doi.org/10.17221/817/2014-PSE \u003c/li\u003e\n\u003cli\u003eTea I, Genter T, Naulet N, Boyer V, Lummerzheim M, Kleiber D. Effect of foliar sulfur and nitrogen fertilization on wheat storage protein composition and dough mixing properties. Cereal Chem. 2004;81(6):759-766. http://dx.doi.org/10.1094/CCHEM.2004.81.6.759. \u003c/li\u003e\n\u003cli\u003eTea I, Genter T, Naulet N, Lummerzheim M, Kleiber D. Interaction between nitrogen and sulfur by foliar application and its effects on flour bread‐making quality. J. Sci. Food Agric. 2007; 87(15):2853-2859. https://doi.org/10.1002/jsfa.3044. \u003c/li\u003e\n\u003cli\u003eKutman UB, Yildiz B, Cakmak I. Effect of nitrogen on uptake, remobilization and partitioning of zinc and iron throughout the development of durum wheat. Plant Soil. 2011;342(1):149-164. https://doi.org/10.1007/s11104-010-0679-5. \u003c/li\u003e\n\u003cli\u003eHellemans T, Landschoot S, Dewitte K, Van Bockstaele F, Vermeir P, Eeckhout M, Haesaert G. Impact of crop husbandry practices and environmental conditions on wheat composition and quality: a review. J Agric Food Chem. 2018;66(11):2491-2509. https://doi.org/10.1021/acs.jafc.7b05450.\u003c/li\u003e\n\u003cli\u003eBouis HE, Saltzman A. Improving nutrition through biofortification: A review of evidence from HarvestPlus, 2003 through 2016. Global Food Secur. 2017;12:49-58. https://doi.org/10.1016/j.gfs.2017.01.009. \u003c/li\u003e\n\u003cli\u003eMiner GL, Delgado JA, Ippolito JA, Johnson JJ, Kluth DL, Stewart CE. Wheat grain micronutrients and relationships with yield and protein in the U.S. Central Great Plains. Field Crops Res. 2022;279:108453. https://doi.org/10.1016/j.fcr.2022.108453. \u003c/li\u003e\n\u003cli\u003eSors TG, Ellis DR, Salt DE. Selenium uptake, translocation, assimilation and metabolic fate in plants. Photosynth. Res. 2005;86(3):373-389. https://doi.org/10.1007/s11120-005-5222-9. \u003c/li\u003e\n\u003cli\u003eZhou X, Yang J, Kronzucker HJ, Shi W. Selenium biofortification and interaction with other elements in plants: a review. Front. Plant Sci. 2020;11:586421. https://doi.org/10.3389/fpls.2020.586421. \u003c/li\u003e\n\u003cli\u003eFarga\u0026scaron;ov\u0026aacute; A, Pastierov\u0026aacute; J, Svetkova K. Effect of Se-metal pair combinations (Cd, Zn, Cu, Pb) on photosynthetic pigments production and metal accumulation in Sinapis alba L. seedlings. Plant Soil Environ. 2006;52(1):8. https://doi.org/10.17221/3340-PSE. \u003c/li\u003e\n\u003cli\u003eZembala M, Filek M, Walas S, Mrowiec H, Hartikainen H, Miszalski Z. The influence of selenium on root growth and oxidative stress in rape seedlings subjected to cadmium stress. Plant and Soil. 2010;337(1\u0026ndash;2):451\u0026ndash;460. https://doi.org/10.1007/s11104-010-0543-z.\u003c/li\u003e\n\u003cli\u003eBoldrin PF, Faquin V, Ramos SJ, Boldrin KVF, Araujo RA, Guilherme LRG. Selenium biofortification and antioxidant activity in lettuce plants fed with selenate and selenite. Plant Soil Environ. 2013;59(4):171\u0026ndash;176. https://doi.org/10.17221/113/2010-PSE.\u003c/li\u003e\n\u003cli\u003eXia Q, Yang ZP, Xue NW, Dai XJ, Zhang X, Gao, ZQ. Effect of foliar application of selenium on nutrient concentration and yield of colored-grain wheat in China. Appl. Ecol. Environ. Res. 2019;17(2). http://dx.doi.org/10.15666/aeer/1702_21872202 \u003c/li\u003e\n\u003cli\u003eFan MS, Zhao FJ, Fairweather-Tait SJ, Poulton PR, Dunham SJ, McGrath SP. Evidence of decreasing mineral density in wheat grain over the last 160 years. J. Trace Elem. Med. Biol. 2008;\u003cem\u003e22\u003c/em\u003e(4):315-324. https://doi.org/10.1016/j.jtemb.2008.07.002. \u003c/li\u003e\n\u003cli\u003eOury FX, Leenhardt F, Remesy C, Chanliaud E, Duperrier B, Balfourier F, Charmet G. Genetic variability and stability of grain magnesium, zinc and iron concentrations in bread wheat. Europ. J. Agronomy. 2006;25(2):177-185. https://doi.org/10.1016/j.eja.2006.04.011. \u003c/li\u003e\n\u003cli\u003eZhao FJ, Su YH, Dunham SJ, Rakszegi M, Bedo Z, McGrath SP, Shewry PR. Variation in mineral micronutrient concentrations in grain of wheat lines of diverse origin. J. Cereal Sci. 2009;49(2):290-295. https://doi.org/10.1016/j.jcs.2008.11.007. \u003c/li\u003e\n\u003cli\u003eGuttieri MJ, Seabourn BW, Liu C, Baenziger PS, Waters BM. Distribution of cadmium, iron, and zinc in millstreams of hard winter wheat (Triticum aestivum L.). J. Agric. Food Chem. 2015. https://doi.org/10.1021/acs.jafc.5b04337 \u003c/li\u003e\n\u003cli\u003eDapkekar A, Deshpande P, Oak MD, Paknikar KM, Rajwade JM. Zinc use efficiency is enhanced in wheat through nanofertilization. Scientific Reports. 2018;8(1):6832. https://doi.org/10.1038/s41598-018-25247-5. \u003c/li\u003e\n\u003cli\u003eLiu K, Cai M, Hu C, Sun X, Cheng Q, Jia W, Yang T, Nie M, Zhao X. Selenium (Se) reduces Sclerotinia stem rot disease incidence of oilseed rape by increasing plant Se concentration and shifting soil microbial community and functional profiles. Environ. Pollut. 2019;254:113051. https://doi.org/10.1016/j.envpol.2019.113051.\u003c/li\u003e\n\u003cli\u003eXu JY, Jia W, Hu CX, Nie M, Ming JJ, Cheng Q, Cai M, Sun X, Li X, Zheng X, Wang J, Zhao X. Selenium as a potential fungicide could protect oilseed rape leaves from sclerotinia sclerotiorum infection. Environ. pollut. 2020;257:113495. http://dx.doi.org/10.1016/j.envpol.2019.113495\u003c/li\u003e\n\u003cli\u003eMao XY, Hua C, Yang L, Zhang YH, Sun ZX, Li L, Li T. The effects of selenium on wheat fusarium head blight and DON accumulation were selenium compound-dependent. Toxins. 2020;12(9):573. https://doi.org/10.3390/toxins12090573. \u003c/li\u003e\n\u003cli\u003eLi HF, McGrath SP, Zhao FJ. Selenium uptake, translocation and speciation in wheat supplied with selenate or selenite. New Phytol. 2008;178(1):92\u0026ndash;102. https://doi.org/10.1111/j.1469-8137.2007.02343.x\u003c/li\u003e\n\u003cli\u003eLi HF, Lombi E, Stroud JL, McGrath SP, Zhao FJ. Selenium speciation in soil and rice: influence of water management and Se fertilization. J. Agric. Food Chem.2010;58(22):11837\u0026ndash;11843. https://doi.org/10.1021/jf1026185\u003c/li\u003e\n\u003cli\u003eKornaś A, Filek M, Sieprawska A, Bednarska-Kozakiewicz E, Gawrońska K, Miszalski Z. Foliar application of selenium for protection against the first stages of mycotoxin infection of crop plant leaves. J. Sci. Food Agric. 2018;99(1):482-485. https://doi.org/10.1002/jsfa.9145 .\u003c/li\u003e\n\u003cli\u003eHaneklaus S, Bloem E, Funder U, Schnug E. Effect of foliar-applied elemental sulphur on Fusarium infections in barley. LBF.2007;57(3):213.\u003c/li\u003e\n\u003cli\u003eK\u0026uuml;nstler A, Gullner G, \u0026Aacute;d\u0026aacute;m AL, Kolozsv\u0026aacute;rin\u0026eacute; Nagy J, Kir\u0026aacute;ly L. The versatile roles of sulfur-containing biomolecules in plant defense-a road to disease resistance. Plants.\u003cem\u003e \u003c/em\u003e2020;9:1705. https://doi.org/10.3390/plants9121705.\u003c/li\u003e\n\u003cli\u003eSasaki K; Kuwabara C; Umeki N, Fujioka M, Saburi W, Matsui H, Abe F, Imai R. The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat. J. Biotechnol. 2016;228:3\u0026ndash;7. https://doi.org/10.1016/j.jbiotec.2016.04.015. \u003c/li\u003e\n\u003cli\u003eKoga S, Aamot H, Uhlen A, Seehusen T, Veiseth-Kent E, Hofgaard I, Moldestad A, B\u0026ouml;cker U. Environmental factors associated with glutenin polymer assembly during grain maturation. J. Cereal Sci. 2020;91:102865. https://doi.org/10.1016/j.jcs.2019.102865. \u003c/li\u003e\n\u003cli\u003ePekkarinen AI, Mannonen L, Jones BL, Niku-Paavola ML. Production of proteases by \u003cem\u003eFusarium\u003c/em\u003e species grown on barley grains and in media containing cereal proteins. J. Cereal Sci.\u003cem\u003e \u003c/em\u003e2000;\u003cstrong\u003e31\u003c/strong\u003e(3):253-261. https://doi.org/10.1006/jcrs.2000.0305.\u003c/li\u003e\n\u003cli\u003eWang J, Wieser H, Pawelzik E, Weinert J, Keutgen AJ, Wolf GA. Impact of the fungal protease produced by \u003cem\u003eFusarium culmorum\u003c/em\u003e on the protein quality and breadmaking properties of winter wheat. Eur. Food Res. Technol. 2005;220(5-6):552-559. https://doi.org/10.1007/s00217-004-1112-1. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Agronomic Biofortification, Nitrogen, Sulphur, Selenium, Gluten Protein Assembly, Mycotoxins, Micronutrient","lastPublishedDoi":"10.21203/rs.3.rs-7797922/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7797922/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjectives\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDurum wheat quality and safety are increasingly shaped by agronomic practices that aimed to reduce reliance on synthetic inputs while enhancing nutritional and technological value. Under Mediterranean conditions, where recurrent abiotic stresses linked to climate change pose significant challenges for agricultural production and food security, novel fertilisation strategies are necessary. This study investigates the combined effects of organic foliar fertilization and selenium (Se) biofortification on gluten protein quality, grain nutrient enrichment, and mycotoxin contamination in durum wheat (\u003cem\u003eTriticum turgidum ssp. durum\u003c/em\u003e L.).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eField experiments were carried out over two growing seasons (2018 and 2019), using modern (Marco Aurelio, Nadif) and old (Cappelli, old Saragolla) cultivars. Treatments included foliar applications of organic nitrogen (N), sulphur (S), combined N\u0026thinsp;+\u0026thinsp;S, and Se biofortification. Analyses focused on gluten protein polymerization and assembly, grain concentrations of [N], [S], [Se], [Fe], and [Zn], and the profile of major and emerging mycotoxins.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOrganic foliar N and S application enhanced grain [S], [Fe], and [Zn] and improved gluten protein assembly and gluten index. Se biofortification effectively increased grain Se content without compromising other nutrient concentrations and significantly reduced contamination by both deoxynivalenol and emerging mycotoxins (enniatins, moniliformin). The modern cultivar Marco Aurelio combined high concentrations of micronutrients and gluten quality indices, even under water-limited conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study provides novel evidence that integrating organic foliar fertilization with Se biofortification can simultaneously improve the nutritional, technological, and safety traits of durum wheat. The findings point to agronomic strategies capable of improving grain quality and reducing mycotoxin risks in Mediterranean environments, thereby supporting more sustainable and resilient wheat production systems with direct relevance for food security.\u003c/p\u003e","manuscriptTitle":"Enhancing Durum Wheat Grain Quality and Safety: The Role of Organic Foliar Fertilization and Selenium Biofortification in Mediterranean Farming Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-21 10:59:56","doi":"10.21203/rs.3.rs-7797922/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ccfcbd23-a964-433f-8864-857262e5e9a4","owner":[],"postedDate":"October 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T15:09:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-21 10:59:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7797922","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7797922","identity":"rs-7797922","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}