The INCREASE project: Intelligent Collections of food-legume genetic resources for European agrofood systems.

Food legumes are crucial for all agriculture-related societal challenges, including climate change mitigation, agrobiodiversity conservation, sustainable agriculture, food security and human health. The transition to plant-based diets, largely based on food legumes, could present major opportunities for adaptation and mitigation, generating significant co-benefits for human health. The characterization, maintenance and exploitation of food-legume genetic resources, to date largely unexploited, form the core development of both sustainable agriculture and a healthy food system. INCREASE will implement, on chickpea (Cicer arietinum), common bean (Phaseolus vulgaris), lentil (Lens culinaris) and lupin (Lupinus albus and L. mutabilis), a new approach to conserve, manage and characterize genetic resources. Intelligent Collections, consisting of nested core collections composed of single-seed descent-purified accessions (i.e., inbred lines), will be developed, exploiting germplasm available both from genebanks and on-farm and subjected to different levels of genotypic and phenotypic characterization. Phenotyping and gene discovery activities will meet, via a participatory approach, the needs of various actors, including breeders, scientists, farmers and agri-food and non-food industries, exploiting also the power of massive metabolomics and transcriptomics and of artificial intelligence and smart tools. Moreover, INCREASE will test, with a citizen science experiment, an innovative system of conservation and use of genetic resources based on a decentralized approach for data management and dynamic conservation. By promoting the use of food legumes, improving their quality, adaptation and yield and boosting the competitiveness of the agriculture and food sector, the INCREASE strategy will have a major impact on economy and society and represents a case study of integrative and participatory approaches towards conservation and exploitation of crop genetic resources.

Defence-related pathways, phytohormones and primary metabolism are key players in kiwifruit plant tolerance to Pseudomonas syringae pv. actinidiae.

The reasons underlying the differential tolerance of Actinidia spp. to the pandemic pathogen Pseudomonas syringae pv. actinidiae (Psa) have not yet been elucidated. We hypothesized that differential plant-defence strategies linked to transcriptome regulation, phytohormones and primary metabolism might be key and that Actinidia chinensis susceptibility results from an inefficient activation of defensive mechanisms and metabolic impairments shortly following infection. Here, 48 h postinoculation bacterial density was 10-fold higher in A. chinensis var. deliciosa than in Actinidia arguta, accompanied by significant increases in glutamine, ornithine, jasmonic acid (JA) and salicylic acid (SA) (up to 3.2-fold). Actinidia arguta showed decreased abscisic acid (ABA) (0.7-fold), no changes in primary metabolites, and 20 defence-related genes that were only differentially expressed in this species. These include GLOX1, FOX1, SN2 and RBOHA, which may contribute to its higher tolerance. Results suggest that A. chinensis' higher susceptibility to Psa is due to an inefficient activation of plant defences, with the involvement of ABA, JA and SA, leading to impairments in primary metabolism, particularly the ammonia assimilation cycle. A schematic overview on the interaction between Psa and genotypes with distinct tolerance is provided, highlighting the key transcriptomic and metabolomic aspects contributing to the different plant phenotypes after infection.

Effect of the Interaction between Elevated Carbon Dioxide and Iron Limitation on Proteomic Profiling of Soybean.

Elevated atmospheric CO2 (eCO2) and iron (Fe) availability are important factors affecting plant growth that may impact the proteomic profile of crop plants. In this study, soybean plants treated under Fe-limited (0.5 mM) and Fe-sufficient (20 mM) conditions were grown at ambient (400 µmol mol-1) and eCO2 (800 µmol mol-1) in hydroponic solutions. Elevated CO2 increased biomass from 2.14 to 3.14 g plant-1 and from 1.18 to 2.91 g plant-1 under Fe-sufficient and Fe-limited conditions, respectively, but did not affect leaf photosynthesis. Sugar concentration increased from 10.92 to 26.17 µmol g FW-1 in roots of Fe-sufficient plants and from 8.75 to 19.89 µmol g FW-1 of Fe-limited plants after exposure to eCO2. In leaves, sugar concentration increased from 33.62 to 52.22 µmol g FW-1 and from 34.80 to 46.70 µmol g FW-1 in Fe-sufficient and Fe-limited conditions, respectively, under eCO2. However, Fe-limitation decreases photosynthesis and biomass. Pathway enrichment analysis showed that cell wall organization, glutathione metabolism, photosynthesis, stress-related proteins, and biosynthesis of secondary compounds changed in root tissues to cope with Fe-stress. Moreover, under eCO2, at sufficient or limited Fe supply, it was shown an increase in the abundance of proteins involved in glycolysis, starch and sucrose metabolism, biosynthesis of plant hormones gibberellins, and decreased levels of protein biosynthesis. Our results revealed that proteins and metabolic pathways related to Fe-limitation changed the effects of eCO2 and negatively impacted soybean production.

Systematic review on lignin valorization in the agro-food system: From sources to applications.

Lignocellulosic biomass is the most abundant renewable resource on earth and currently most of this biomass is considered a low-value waste. Specifically, lignin is an underrated bioresource that is mostly burned for energy production and few value-added products have been created. Since the agro-food industry produces large amounts of wastes that can be potential sources of high-quality lignin, scientific efforts should be directed to this industry. Thus, this review provides a systematic overview of the trends and evolution of research on agro-food system-derived lignin (from 2010 to 2020), including the extraction of lignin from various agro-food sources and emergent applications of lignin in the agro-food chain. Crops with the highest average production/year (n = 26) were selected as potential lignin sources. The extraction process efficiency (yield) and lignin purity were used as indicators of the raw material potential. Overall, it is notable that research interest on agro-food lignin has increased exponentially over the years, both as source (567%) and application (128%). Wheat, sugarcane, and maize are the most studied sources and are the ones that render the highest lignin yields. As for the extraction methods used, alkaline and organosolv methods are the most employed (∼50%). The main reported applications are related to lignin incorporation in polymers (∼55%) and as antioxidant (∼24%). Studies on agro-food system-derived lignin is of most importance since there are numerous possible sources that are yet to be fully valorized and many promising applications that need to be further developed.

Foliar application of 3-hydroxy-4-pyridinone Fe-chelate [Fe(mpp)3 ] induces responses at the root level amending iron deficiency chlorosis in soybean.

Iron (Fe) deficiency chlorosis (IDC) affects the growth of several crops, especially when growing in alkaline soils. The application of synthetic Fe-chelates is one of the most commonly used strategies in IDC amendment, despite their associated negative environmental impacts. In a previous work, the Fe-chelate tris(3-hydroxy-1-(H)-2-methyl-4-pyridinonate) iron(III) [Fe(mpp)3 ] has shown great potential for alleviating IDC in soybean (Glycine max) in the early stages of plant development under hydroponic conditions. Herein, its efficacy was verified under soil conditions in soybean grown from seed to full maturity. Chlorophyll levels, plant growth, root and shoot mineral accumulation (K, Mg, Ca, Na, P, Mn, Zn, Ni, and Co) and FERRITIN expression were accessed at V5 phenological stage. Compared to a commonly used Fe chelate, FeEDDHA, supplementation with [Fe(mpp)3 ] led to a 29% higher relative chlorophyll content, 32% higher root biomass, 36% higher trifoliate Fe concentration, and a twofold increase in leaf FERRITIN gene expression. [Fe(mpp)3 ] supplementation also resulted in increased accumulation of P, K, Zn, and Co. At full maturity, the remaining plants were harvested and [Fe(mpp)3 ] application led to a 32% seed yield increase when compared to FeEDDHA. This is the first report on the use of [Fe(mpp)3 ] under alkaline soil conditions for IDC correction, and we show that its foliar application has a longer-lasting effect than FeEDDHA, induces efficient root responses, and promotes the uptake of other nutrients.

Sargassum muticum and Osmundea pinnatifida Enzymatic Extracts: Chemical, Structural, and Cytotoxic Characterization.

Seaweeds, which have been widely used for human consumption, are considered a potential source of biological compounds, where enzyme-assisted extraction can be an efficient method to obtain multifunctional extracts. Chemical characterization of Sargassum muticum and Osmundea pinnatifida extracts obtained by Alcalase and Viscozyme assisted extraction, respectively, showed an increment of macro/micro elements in comparison to the corresponding dry seaweeds, while the ratio of Na/K decreased in both extracts. Galactose, mannose, xylose, fucose, and glucuronic acid were the main monosaccharides (3.2-27.3 mg/glyophilized extract) present in variable molar ratios, whereas low free amino acids content and diversity (1.4-2.7 g/100gprotein) characterized both extracts. FTIR-ATR and 1H NMR spectra confirmed the presence of important polysaccharide structures in the extracts, namely fucoidans from S. muticum or agarans as sulfated polysaccharides from O. pinnatifida. No cytotoxicity against normal mammalian cells was observed from 0 to 4 mglyophilized extract/mL for both extracts. The comprehensive characterization of the composition and safety of these two extracts fulfils an important step towards their authorized application for nutritional and/or nutraceutical purposes.

Biochemical markers to predict internal browning disorders in 'Rocha' pear during storage under high CO2.

BACKGROUND: The aim of this study was to identify biochemical markers to predict internal browning disorders (IBD) in 'Rocha' pear. Fruits from five orchards were stored for 45 days under cold air followed by 100 days in browning-inducing controlled atmosphere (CA) conditions (1 kPa O2 + 10 kPa CO2 ). RESULTS: Relationships between concentrations of ethanol (EtOH), acetaldehyde (AcDH) and ascorbic acid (AA), activities of peroxidase (POX) and polyphenoloxidase (PPO) and IBD incidence were established. The partial least square (PLS) model using the most promising markers, EtOH and AcDH, explained 89% of the variance in IBD incidence, whereas the univariate models based on the same markers explained between 89 and 94%. In contrast, the models based on AA levels and AA depletion rate only explained 57 and 82% of the variance in IBD incidence respectively. Model validation confirmed the robustness of EtOH for the prediction (R2 = 0.91, RMSE = 11.1) and allowed proposing a threshold level of 30 µL EtOH L-1 above which IBD may occur. Using this threshold value, the storage time limit associated with the occurrence of the first IBD symptoms was predicted with an acceptable RMSE of 9 days. CONCLUSION: This work clearly identifies biochemical IBD markers for 'Rocha' pear and shows that dynamic changes in ethanol concentration during the beginning of storage may be used to predict IBD development. Therefore the results presented herein represent a major step forward in the prediction of IBD in 'Rocha' pear. © 2017 Society of Chemical Industry.

Nuclear magnetic resonance metabolomics of iron deficiency in soybean leaves.

Iron (Fe) deficiency is an important agricultural concern that leads to lower yields and crop quality. A better understanding of the condition at the metabolome level could contribute to the design of strategies to ameliorate Fe-deficiency problems. Fe-sufficient and Fe-deficient soybean leaf extracts and whole leaves were analyzed by liquid (1)H nuclear magnetic resonance (NMR) and high-resolution magic-angle spinning NMR spectroscopy, respectively. Overall, 30 compounds were measurable and identifiable (comprising amino and organic acids, fatty acids, carbohydrates, alcohols, polyphenols, and others), along with 22 additional spin systems (still unassigned). Thus, metabolite differences between treatment conditions could be evaluated for different compound families simultaneously. Statistically relevant metabolite changes upon Fe deficiency included higher levels of alanine, asparagine/aspartate, threonine, valine, GABA, acetate, choline, ethanolamine, hypoxanthine, trigonelline, and polyphenols and lower levels of citrate, malate, ethanol, methanol, chlorogenate, and 3-methyl-2-oxovalerate. The data indicate that the main metabolic impacts of Fe deficiency in soybean include enhanced tricarboxylic acid cycle activity, enhanced activation of oxidative stress protection mechanisms and enhanced amino acid accumulation. Metabolites showing accumulation differences in Fe-starved but visually asymptomatic leaves could serve as biomarkers for early detection of Fe-deficiency stress.

More than a meat- or synthetic nitrogen fertiliser-substitute: a review of legume phytochemicals as drivers of 'One Health' via their influence on the functional diversity of soil- and gut-microbes.

Legumes are essential to healthy agroecosystems, with a rich phytochemical content that impacts overall human and animal well-being and environmental sustainability. While these phytochemicals can have both positive and negative effects, legumes have traditionally been bred to produce genotypes with lower levels of certain plant phytochemicals, specifically those commonly termed as 'antifeedants' including phenolic compounds, saponins, alkaloids, tannins, and raffinose family oligosaccharides (RFOs). However, when incorporated into a balanced diet, such legume phytochemicals can offer health benefits for both humans and animals. They can positively influence the human gut microbiome by promoting the growth of beneficial bacteria, contributing to gut health, and demonstrating anti-inflammatory and antioxidant properties. Beyond their nutritional value, legume phytochemicals also play a vital role in soil health. The phytochemical containing residues from their shoots and roots usually remain in-field to positively affect soil nutrient status and microbiome diversity, so enhancing soil functions and benefiting performance and yield of following crops. This review explores the role of legume phytochemicals from a 'one health' perspective, examining their on soil- and gut-microbial ecology, bridging the gap between human nutrition and agroecological science.

Metabolic Evaluation of Lupin-Enriched Yogurt by Nuclear Magnetic Resonance Metabolomics.

Untargeted nuclear magnetic resonance (NMR) metabolomics was used to evaluate compositional changes during yogurt fermentation upon lupin enrichment compared to traditional conditions. Lupin significantly changed the sample metabolic profile and its time course dynamics, seemingly delaying microbial action. The levels of organic and amino acids were significantly altered, along with those of some sugars, nucleotides, and choline compounds. Lupin seemed to favor acetate and formate synthesis, compared to that of citrate and fumarate; a higher formate levels may suggest increased levels of Streptococcus thermophilus action, compared toLactobacillus bulgaricus. Lupin-yogurt was poorer in hippurate, lactose (and hence lactate), galactose, glucose-1-phosphate, and galactose-1-phosphate, containing higher orotate levels (possibly related to increased uridine derivatives), among other differences. Trigonelline was confirmed as a lupin marker, possibly together with glutamate and histidine. Other metabolite trajectories remained unchanged upon lupin addition, unveiling unaffected underlying processes. These results demonstrate the usefulness of untargeted NMR metabolomics to understand/develop new foodstuffs and their production processes, highlighting the identity of a variety of bioactive metabolites with importance for human health.

Enzymatic and Non-Enzymatic Antioxidant Responses of Young Tomato Plants (cv. Micro-Tom) to Single and Combined Mild Nitrogen and Water Deficit: Not the Sum of the Parts.

This study aims to perform a broad analysis of the antioxidant (AOX) responses of young tomato plants exposed to single and combined mild nitrogen (N) and water deficits through the evaluation of oxidative biomarkers, non-enzymatic and enzymatic AOX components. 'Micro-Tom' seedlings were subjected to four treatments: control (CTR; 100%N + 100%W), N deficit (N; 50%N), water deficit (W; 50%W), and combined deficits (N + W; 50%N + 50%W). An enhancement of several non-enzymatic and enzymatic components was found in plants subjected to N + W deficit, which presented higher anthocyanins accumulation (up to 103%) as well as higher levels of superoxide dismutase (SOD) transcripts at root level and of ascorbate peroxidase (APX) and catalase (CAT) transcripts at shoot level. This increase in the gene expression was also translated in augmented SOD (up to 202%), APX (up to 155%) and CAT (up to 108%) activity compared to CTR plants and the single deficits. Overall, tomato plants were able to employ defense strategies to cope with this combined deficit, as demonstrated by the higher total AOX capacity (up to 87%) compared to the single deficits, which contributed to the maintenance of their redox homeostasis, with unchanged values of lipid peroxidation and hydrogen peroxide compared with CTR plants.

Young Tomato Plants Respond Differently under Single or Combined Mild Nitrogen and Water Deficit: An Insight into Morphophysiological Responses and Primary Metabolism.

This study aimed to understand the morphophysiological responses and primary metabolism of tomato seedlings subjected to mild levels of nitrogen and/or water deficit (50% N and/or 50% W). After 16 days of exposure, plants grown under the combined deficit showed similar behavior to the one found upon exposure to single N deficit. Both N deficit treatments resulted in a significantly lower dry weight, leaf area, chlorophyll content, and N accumulation but in a higher N use efficiency when compared to control (CTR) plants. Moreover, concerning plant metabolism, at the shoot level, these two treatments also responded in a similar way, inducing higher C/N ratio, nitrate reductase (NR) and glutamine synthetase (GS) activity, expression of RuBisCO encoding genes as well as a downregulation of GS2.1 and GS2.2 transcripts. Interestingly, plant metabolic responses at the root level did not follow the same pattern, with plants under combined deficit behaving similarly to W deficit plants, resulting in enhanced nitrate and proline concentrations, NR activity, and an upregulation of GS1 and NR genes than in CTR plants. Overall, our data suggest that the N remobilization and osmoregulation strategies play a relevant role in plant acclimation to these abiotic stresses and highlight the complexity of plant responses under a combined N+W deficit.

Scientific and technological advances in the development of sustainable disease management tools: a case study on kiwifruit bacterial canker.

Plant disease outbreaks are increasing in a world facing climate change and globalized markets, representing a serious threat to food security. Kiwifruit Bacterial Canker (KBC), caused by the bacterium Pseudomonas syringae pv. actinidiae (Psa), was selected as a case study for being an example of a pandemic disease that severely impacted crop production, leading to huge economic losses, and for the effort that has been made to control this disease. This review provides an in-depth and critical analysis on the scientific progress made for developing alternative tools for sustainable KBC management. Their status in terms of technological maturity is discussed and a set of opportunities and threats are also presented. The gradual replacement of susceptible kiwifruit cultivars, with more tolerant ones, significantly reduced KBC incidence and was a major milestone for Psa containment - which highlights the importance of plant breeding. Nonetheless, this is a very laborious process. Moreover, the potential threat of Psa evolving to more virulent biovars, or resistant lineages to existing control methods, strengthens the need of keep on exploring effective and more environmentally friendly tools for KBC management. Currently, plant elicitors and beneficial fungi and bacteria are already being used in the field with some degree of success. Precision agriculture technologies, for improving early disease detection and preventing pathogen dispersal, are also being developed and optimized. These include hyperspectral technologies and forecast models for Psa risk assessment, with the latter being slightly more advanced in terms of technological maturity. Additionally, plant protection products based on innovative formulations with molecules with antibacterial activity against Psa (e.g., essential oils, phages and antimicrobial peptides) have been validated primarily in laboratory trials and with few compounds already reaching field application. The lessons learned with this pandemic disease, and the acquired scientific and technological knowledge, can be of importance for sustainably managing other plant diseases and handling future pandemic outbreaks.

Impact of soybean-associated plant growth-promoting bacteria on plant growth modulation under alkaline soil conditions.

Conventional strategies to manage iron (Fe) deficiency still present drawbacks, and more eco-sustainable solutions are needed. Knowledge on soybean-specific diversity and functional traits of their plant growth-promoting bacteria (PGPB) potentiates their applicability as bioinoculants to foster soybean performance under calcareous soil conditions. This work aimed to assess the efficacy of PGPB, retrieved from soybean tissues/rhizosphere, in enhancing plant growth and development as well as crop yield under alkaline soil conditions. Seventy-six bacterial strains were isolated from shoots (18%), roots (53%), and rhizosphere (29%) of soybean. Twenty-nine genera were identified, with Bacillus and Microbacterium being the most predominant. Based on distinct plant growth-promoting traits, the endophyte Bacillus licheniformis P2.3 and the rhizobacteria Bacillus aerius S2.14 were selected as bioinoculants. In vivo tests showed that soybean photosynthetic parameters, chlorophyll content, total fresh weight, and Fe concentrations were not significantly affected by bioinoculation. However, inoculation with B. licheniformis P2.3 increased pod number (33%) and the expression of Fe-related genes (FRO2, IRT1, F6'H1, bHLH38, and FER4), and decreased FC-R activity (45%). Moreover, bioinoculation significantly affected Mn, Zn, and Ca accumulation in plant tissues. Soybean harbors several bacterial strains in their tissues and in the rhizosphere with capacities related to Fe nutrition and plant growth promotion. The strain B. licheniformis P2.3 showed the best potential to be incorporated in bioinoculant formulations for enhancing soybean performance under alkaline soil conditions.

Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening.

BACKGROUND: Pine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant's molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN). RESULTS: Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species. CONCLUSIONS: Defense-related genes triggered by nematode infestation were detected in both P. pinaster and P. pinea transcriptomes utilizing 454 pyrosequencing technology. P. pinaster showed higher abundance of genes related to transcriptional regulation, terpenoid secondary metabolism (including some with nematicidal activity) and pathogen attack. P. pinea showed higher abundance of genes related to oxidative stress and higher levels of expression in general of stress responsive genes. This study provides essential information about the molecular defense mechanisms utilized by P. pinaster and P. pinea against PWN infestation and contributes to a better understanding of PWD.

Evaluation of plant elicitation with methyl-jasmonate, salicylic acid and benzo (1,2,3)-thiadiazole-7-carbothioic acid-S-methyl ester for the sustainable management of the pine wilt disease.

Treatment with plant elicitors can be a promising method to induce Pinus pinaster tolerance against the pinewood nematode (PWN), Bursaphelenchus xylophilus, by promoting plant antioxidant system, micronutrient accumulation and by modulating plant-associated bacterial populations. To test this hypothesis, plants were sprayed with methyl jasmonate (MeJA), salicylic acid (SA) or benzo (1,2,3)-thiadiazole-7-carbothioic acid-S-methyl ester (BTH), and evaluated until 35 days after-inoculation (dai) for: i) extent of foliar symptoms; ii) nematode density inside stem tissues; iii) proxies for oxidative damage and antioxidant activity, iv) micronutrient concentration and v) bacterial diversity. Compared with non-elicited plants, plant elicitation, particularly with BTH, significantly decreased nematodes density inside stem tissues (by 0.63-fold). Concordantly, without elicitation plant mortality reached 12.5% while no mortality was observed in elicited plants. BTH-elicited plants had significantly higher concentrations of anthocyanins and carotenoids at the end of the assay than SA-elicited and MeJA-elicited plants, which possibly contributed to the lower PWN colonization and degree of foliar symptoms observed. Accordingly, MeJA and SA led to increased lipid peroxidation at 28 dai (by 2.64- and 2.52-fold, respectively) in comparison with BTH (by 1.10-fold), corroborating its higher potential in increasing plant antioxidative response during infection. Moreover, carotenoids showed a negative correlation with nematode migration, whereas polyphenols showed a positive correlation. Elicitors also induced changes in the bacterial community of infected P. pinaster plants, increasing the diversity of specific populations. Finally, elicitors induced significant changes in micronutrients accumulation in plant tissues, namely a decrease in the concentration of B, Mn and Ni in plants treated with BTH compared to those treated with the other elicitors. Altogether, results suggest that elicitation with MeJA, SA and, particularly, BTH, increases tolerance against B. xylophilus by promoting plant antioxidant system, changing the accumulation of essential micronutrients and modulating plant-associated bacterial diversity.

Widening the Perspectives for Legume Consumption: The Case of Bioactive Non-nutrients.

Legume grains have provided essential nutrients in human diets for centuries, being excellent sources of proteins, carbohydrates, fatty acids, and fibers. They also contain several non-nutrients that historically have been connotated as toxic but that in recent years have been shown to have interesting bioactive properties. The discussion on the role of bioactive non-nutrients is becoming more important due to increasing science-based evidence on their potential antioxidant, hypoglycemic, hypolipidemic, and anticarcinogenic properties. At a time when legume-based products consumption is being strongly promoted by national governments and health authorities, there is a need to clearly define the recommended levels of such non-nutrients in human diets. However, there is insufficient data determining the ideal amount of non-nutrients in legume grains, which will exert the most positive health benefits. This is aligned with insufficient studies that clearly demonstrate if the positive health effects are due to the presence of specific non-nutrients or a result of a dietary balance. In fact, rather than looking directly at the individual food components, most nutritional epidemiology studies relate disease risk with the food and dietary patterns. The purpose of this perspective paper is to explore different types of non-nutrients present in legume grains, discuss the current evidence on their health benefits, and provide awareness for the need for more studies to define a recommended amount of each compound to identify the best approaches, either to enhance or reduce their levels.

Effect of alkaline and deep eutectic solvents pretreatments on the recovery of lignin with antioxidant activity from grape stalks.

Grape stalks are lignocellulosic residues that can be valorized through the extraction of lignin - an underutilized biopolymer with high potential. Two lignin extraction methods, alkaline and deep eutectic solvents (DES), were studied, and experimental designs were carried out to obtain the best extraction conditions. The defined parameters for alkaline extraction allowed the recovery of ~48 % of lignin with low purity that was further improved with an autohydrolysis pretreatment (~79 % purity; ~32 % yield). Optimum parameters of DES method rendered high purity lignin (~90 %) without the need of a pretreatment and with a better yield (50.2 % (±2.3)) than the alkaline method. Both lignin fractions presented high antioxidant activities, being close to the antioxidant capacity of BHT for DPPH scavenging. Structural analysis proved the presence of lignin in both alkaline and DES samples with similar morphology. Overall, DES method was more efficient in the extraction of lignin from grape stalks besides its greener and sustainable nature. This work uses DES to extract lignin from this biomass while comparing it with a commonly classical method, proving that grape stalks can be used to extract lignin with a sustainable and efficient method rendering a final ingredient with value-added properties.

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster).

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera. The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA. The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines. In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate.

Chitosan increases Pinus pinaster tolerance to the pinewood nematode (Bursaphelenchus xylophilus) by promoting plant antioxidative metabolism.

The pine wilt disease (PWD), for which no effective treatment is available at the moment, is a constant threat to Pinus spp. plantations worldwide, being responsible for significant economic and environmental losses every year. It has been demonstrated that elicitation with chitosan increases plant tolerance to the pinewood nematode (PWN) Bursaphelenchus xylophilus, the causal agent of the PWD, but the biochemical and genetic aspects underlying this response have not been explored. To understand the influence of chitosan in Pinus pinaster tolerance against PWN, a low-molecular-weight (327 kDa) chitosan was applied to mock- and PWN-inoculated plants. Nematode population, malondialdehyde (MDA), catalase, carotenoids, anthocyanins, phenolic compounds, lignin and gene expression related to oxidative stress (thioredoxin 1, TRX) and plant defence (defensin, DEF, and a-farnesene synthase, AFS), were analysed at 1, 7, 14, 21 and 28 days post-inoculation (dpi). At 28 dpi, PWN-infected plants elicited with chitosan showed a sixfold lower nematode population when compared to non-elicited plants. Higher levels of MDA, catalase, carotenoids, anthocyanins, phenolic compounds, and lignin were detected in chitosan-elicited plants following infection. The expression levels of DEF gene were higher in elicited plants, while TRX and AFS expression was lower, possibly due to the disease containment-effect of chitosan. Combined, we conclude that chitosan induces pine defences against PWD via modulation of metabolic and transcriptomic mechanisms related with plant antioxidant system.