As a nematotoxics screening biotechnological system, Solanum tuberosum hairy roots (StHR) and S. tuberosum hairy roots with Meloidogyne chitwoodi co-cultures (StHR/CRKN) were evaluated, with and without the addition of the essential oils (EOs) of Satureja montana and Ruta graveolens. EOs nematotoxic and phytotoxic effects were followed weekly by evaluating nematode population density in the co-cultures as well as growth and volatile profiles of both in vitro cultures types. Growth, measured by the dissimilation method and by fresh and dry weight determination, was inhibited after EO addition. Nematode population increased in control cultures, while in EO-added cultures numbers were kept stable. In addition to each of the EOs main components, and in vitro cultures constitutive volatiles, new volatiles were detected by gas chromatography and gas chromatography coupled to mass spectrometry in both culture types. StHR with CRKN co-cultures showed to be suitable for preliminary assessment of nematotoxic EOs.

The Columbia root-knot nematode (CRKN), Meloidogyne chitwoodi , is an EPPO A2 type quarantine pest since 1998. This nematode causes severe damage in economically important crops such as potato and tomato, making agricultural products unacceptable for the fresh market and food processing. Commonly used nematicidal synthetic chemicals are often environmentally unsafe. Essential oils (EOs) may constitute safer alternatives against RKN. EOs, isolated from 56 plant samples, were tested against CRKN hatching, in direct contact bioassays. Some of the most successful EOs were fractionated and the hydrocarbon molecules (HM) and oxygen-containing molecules (OCM) fractions tested separately. 24 EOs displayed very strong hatching inhibitions (≥90 %) at 2 µL mL −1 and were further tested at lower concentrations. Dysphania ambrosioides , Filipendula ulmaria , Ruta graveolens , Satureja montana and Thymbra capitata EOs revealed the lowest EC 50 values (<0.15 µL mL −1 ). The main compounds of these EOs, namely 2-undecanone, ascaridol, carvacrol, isoascaridol, methyl salicylate, p -cymene and/or γ-terpinene, were putatively considered responsible for CRKN hatching inhibition. S. montana and T. capitata OCM fractions showed hatching inhibitions higher than HM fractions. The comparison of EO and corresponding fractions EC 50 values suggests interactions between OCM and HM fractions against CRKN hatching. These species EOs showed to be potential environmentally friendly CRKN hatching inhibitors; nonetheless, bioactivity should be considered globally, since its HM and OCM fractions may contribute, diversely, to the full anti-hatching activity.

Satureja montana (winter savory) and Ruta graveolens (rue) nematotoxic essential oils (EOs) (0.5μl EO/ml culture medium) were assessed for the first time in Pinus pinaster in vitro shoot cultures (Ppi) and P. pinaster shoots with Bursaphelenchus xylophilus co-cultures (PpiBx). The EOs nematotoxic effect was evaluated on B. xylophilus population density in PpiBx co-cultures and the phytotoxic activity to the host was assessed by evaluating relative water content and volatile profiles both on Ppi cultures and on PpiBx co-cultures. Carvacrol-rich S. montana EO showed phytotoxicity, by inducing shoot chlorosis and drooping, whereas no major morphological changes were detected on R. graveolens EO-added Ppi and PpiBx in vitro cultures. Both EOs maintained the nematotoxicity during all experimental phases. R. graveolens EO proved to be an effective PWN antagonist to be further evaluated for pine wilt disease control, given its less phytotoxicity while maintaining nematoxicity.

Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system.

Meloidogyne spp., commonly known as root-knot nematodes (RKNs), are economically important plant sedentary endoparasites that cause galls on susceptible hosts. The Columbia root-knot nematode (CRKN), M. chitwoodi , is a quarantine A2 type pest by the European and Mediterranean Plant Protection Organization since 1998. This nematode has been found associated with economically important crops such as potato and tomato, causing severe damage and making the agricultural products unacceptable for the fresh market and food processing. In vitro co-culture of host and parasite offers an advantageous experimental system for studying plant-RKN interactions. The structure, growth and production of volatiles of Solanum tuberosum hairy roots (HR) and of S. tuberosum HR/CRKN co-cultures were compared. HR were induced by inoculation of aseptic potato tuber segments with Rhizobium rhizogenes . Co-cultures were initiated by inoculating HR with sterilized CRKN eggs. Infection with CRKN induced the RKN symptomatology in the HR and several nematode life stages were observed by light and scanning electron microscopy. Potato HR and HR/CRKN co-cultures exhibited similar growth patterns, evaluated by measuring fresh and dry weight and by the dissimilation method. Volatiles, isolated by distillation–extraction and analyzed by gas chromatography (GC) and gas chromatography coupled to mass spectrometry, revealed that palmitic acid (37–52 %), n –pentadecanal (10–16 %) and linoleic acid (2–16 %) were the main constitutive components of S. tuberosum HR, and of the HR/CRKN co-cultures (24–44, 8–22 and 4–18 %, respectively). S. tuberosum HR/CRKN co-cultures can be considered a suitable biotechnological tool to study RKN infection mechanism by mimicking what occurs under field conditions.

The Portuguese pine forest has become dangerously threatened by pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus. Synthetic chemicals are the most common pesticides used against phytoparasitic nematodes but its use has negative ecological impacts. Phytochemicals may prove to be environmentally friendly alternatives. Essential oils (EOs) and decoction waters, isolated from 84 plant samples, were tested against B. xylophilus, in direct contact assays. Some successful EOs were fractionated and the fractions containing hydrocarbons or oxygen-containing molecules tested separately. Twenty EOs showed corrected mortalities ⩾96% at 2 μL/mL. These were further tested at lower concentrations. Ruta graveolens, Satureja montana and Thymbra capitata EOs showed lethal concentrations (LC100)<0.4μL/mL. Oxygen-containing molecules fractions showing corrected mortality ⩾96% did not always show LC100 values similar to the corresponding EOs, suggesting additive and/or synergistic relationships among fractions. Nine decoction waters (remaining hydrodistillation waters) revealed 100% mortality at a minimum concentration of 12.5μL/mL. R. graveolens, S. montana and T. capitata EOs are potential environmentally friendly alternatives for B. xylophilus control given their high nematotoxic properties. Nematotoxic activity of an EO should be taken in its entirety, as its different components may contribute, in distinct ways, to the overall EO activity.

Wheat germ and corn bran are by-products derived from the bread baking industry. This study aims to development of efficient and low cost processing methods to transform these residues in added value co-products. The basic chemical composition analyses revealed high values of dry matter (87.5–89.8g/100g FW), significant amounts of total ash (13.3–18.0g/100g FW) content and low fat contents (2.2–9.8g/100g FW). The starch values found in the original samples were much lower than after the sequential defatting method revealing a content of starch 3.39, 3.44 and 3.27-fold higher for the samples of wheat germ, maize flour of zootechnical grade and fine, respectively. The performed stabilization studies revealed significant increases up to week 3 in the total phenolics content of the wheat germ and maize cell wall samples. Within the low molecular weight phenolics, quercetin was the major compound (47.3mg/100g FW), in the cell wall of wheat germ samples. The antioxidant activity showed significant differences between liquid and solid fractions, with the highest values (0.38μmol/L Trolox Eq. FW) in the wheat germ liquid fraction. The results show that these by-products can be used as a source of bioactive compounds beneficial for health, while the method used for starch “enrichment” may potentially transform the fine maize flour into an added value co-product.

The main aim of this manuscript is to provide some fundamental concepts related to climate and to its inherent variability, which might be considered an essential tool for a better judgment of the large amounts of information currently available regarding this topic. This is particularly pertinent for agricultural research, as agronomic systems, including horticultural crops, tend to be largely regulated by climate and by atmospheric parameters. Some elementary concepts on the nature of the climate system are first presented. Its components and coupling processes are succinctly described, giving particular emphasis to the spatial and temporal time scales relevant for agro-systems research. Both internal and forced climate variability are discussed from a physically-based perspective with a special focus on the anthropogenic forcing of the climate system. The discussion of these topics is followed by some essential ideas on atmospheric modeling. The need for the development of downscaling strategies is also underlined. A categorization of the different climate system models and their application to agro-system research are thus discussed. The role of greenhouse gases in the radiation and energetic budget of the Earth's system are examined. In this respect, some relevant results of the last IPCC report concerning human-driven forcing mechanisms are also presented. Lastly, the likely impacts of a changing climate on several agronomic sectors are briefly covered.

The aim of this study was to evaluate the effects of various glucosinolate-derived hydrolysis products (HP) as antibacterial compounds against Enterobacteriaceae and Enterococcaceae isolated from intestinal segments of healthy pigs collected directly from slaughter-houses in the North of Portugal. Using a previously described disk-diffusion bioassay, all HP were tested at six different doses (0.015, 0.15, 0.75, 1.5, 3.0, and 15.0 μmoles) in dimethyl-sulfoxide (DMSO), with the exception of sulforaphane (SFN), which was not tested at 15.0 μmoles. Positive (antibiotic standard) and negative controls (DMSO) were included in all experiments. All the experiments were conducted in triplicate. In vitro inhibition of the bacterial growth by the HP was proportional to the concentration used and in many cases was even higher than for the gentamycin, the antibiotic control. The results clearly showed that the glucosinolates-derived HPs were very effective in vitro inhibitors of bacterial growth. The natural products, and specifically the isothiocyanates, should be evaluated as potential alternative control agents for potentially pathogenic bacteria (e.g., dietary amendment of pig foods with glucosinolate-containing plants).

The aim of this study was to evaluate the effects of the level of inclusion of olive leaves and its pre-treatment with Ganoderma resinaceum Baud on growth performance, nutrient digestibilities and caecal fermentation. A total of 48 rabbits were kept in individual cages and divided in 4 groups of 12 animals. Animals were fed ad libitum with one of four diets based on a basal feed for growing rabbits - Control diet (basal feed without olive leaves; OL0), basal diet with 50 g/kg or 100 g/kg of olive leaves (OL5 and OL10), and a basal diet with 50 g/kg of G. resinaceum pre-treated olive leaves (OL5F).The inclusion of olive leaves (OL0 vs. OL5 and OL10) had no effect on growth performance or feed intake. However lower feed intakes (P=0.038) were measured at 100 g/kg of inclusion when compared to 50 g/kg level of inclusion. Organic matter, neutral detergent fibre, crude fat and crude protein digestibilities were lower (P<0.05) for diets with the inclusion of olive leaves (OL0 vs. OL5 and OL10). Although the caecal volatile fatty acid (VFA) concentration was not affected by treatments there was an increase in caecum dry weight contents (P=0.004) in animals fed diets with olive leaves (OL0 vs. OL5 and OL10). Simple phenolics were completely removed by the pre-treatment with G. resinaceum and the concentration of flavonoids and iridoids were reduced by 85%. No effects of the pre-treatment with fungi (OL5F) were observed on the performance of growing rabbits compared to OL5 treatment. The pre-treatment with G. resinaceum increased (P=0.02) crude fat digestibility (0.829 vs. 0.886) and decreased (P=0.006) starch digestibility (0.987 vs. 0.973). Animals fed OL5F diet had lower caecum dry weight contents (-21%; P=0.002), higher caecal valeric acid concentration (+50%; P=0.046) and a trend (P=0.054) for a higher caecal acetic acid concentration (increase of 12%), compared with OL5 treatment.In conclusion, this study showed that increasing amounts of olive leaves in the diet decrease nutrient digestibility. However, fungal treatment of olive leaves seems to alleviate the effect of olive leaves inclusion. (C) 2012 Elsevier B.V. All rights reserved.

The emergence of new diseases and the resurgence of several infections that were controlled in the past, associated with recent increase of bacterial resistance have created the necessity for more studies towards to the development of new antimicrobials and new treatment strategies. The aim of the present study was to evaluate the in vitro synergy between different classes of important glucosinolates hydrolysis products-isothiocyanates with antibiotics (gentamycin and vancomycin), against important pathogenic bacteria: Escherichia coli, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus. A disc diffusion method was used to evaluate the antibacterial activity. The antimicrobial activity of phytochemicals and combinations between gentamycin, vancomycin and phytochemicals were quantitatively assessed by measuring the inhibitory halos. The results showed a selective antimicrobial effect of isothiocyanates, and this effect was strictly related with their chemical structure. In general the benzylisothiocyanate was the most effective compound against both Gram-positive and Gram-negative bacteria. The Listeria monocytogenes and Staphylococcus aureus were the bacteria most affected either by the phytochemicals alone or by the combination phytochemical-antibiotic. The bacteria Pseudomonas aeruginosa was the less affected pathogen. The most important synergism detected occurred between the commercial antibiotics with benzylisothiocyanate and 2-phenylethylisothiocyanate. In conclusion, some isothiocyanates are effective inhibitors of in vitro bacterial growth, and they can act synergistically with antibiotics.

Research on natural and bioactive compounds is increasingly focused on their effects on human health, but there are unexpectedly few studies evaluating the relationship between climate and natural antioxidants. The aim of this study was analyze the biological role of six different Brassica vegetables (Brassica oleracea L. and Brassica rapa L.) as a natural source of antioxidant compounds. The antioxidant activity may be assigned to high levels of L-ascorbic acid, total phenolics and total flavonoids of each sample. The climate seasons affected directly the concentration of bioactive components and the antioxidant activity. Broccoli inflorescences and Portuguese kale showed high antioxidant activity in Spring-Summer whilst turnip leaves did so in Summer-Winter. The Brassica vegetables can provide considerable amounts of bioactive compounds and thus may constitute an important natural source of dietary antioxidants.

This work aims to evaluate the effect of 2-Phenyethyl isothiocyanate (PEITC), a predominant isothiocyanate derived from glucosinolate named glucoranturtiin, particularly important in Brassica rapa (turnip-inflorescences, leaves, stems and roots) and Nasturtion officinalis (watercress), against pathogenic Gram-negative and Gram-positive bacteria, isolated from Human (clinical) and animal gastrointestinal segments. Also standard bacteria strains were sued in the study. Using a disc diffusion method, different doses of PEITC were tested. The compound was dissolved in dimethyl sulfoxide (DMSO) and tested at 5 different concentrations using a standard disk-diffusion assay (0.015, 0.15, 0.75, 1.50, and 3.00 moles). Positive (commercial standards antibiotic) and negative controls (DMSO only) were always included in the tests. All the experiments were conducted in triplicate. The antibacterial activity was also assessed by the application of the percentage of relative inhibition zone (%RIZD) in relation to the antimicrobial effect of commercial standard antibiotics. The results showed that, there were clear concentration differences (P < 0.05) with respect to the in vitro growth inhibition effects as well as differences in the sensitivities of the individual bacteria. The antimicrobial activity of PEITC was always a dose-dependent. The results also showed that antimicrobial activity of PEITC was higher in Gram-positive and lower in Gram-negative. Our results are useful and suggest that PEITC might be useful in controlling Human and animal pathogenic bacteria's growth and therefore could be considered as alternative to, or in combination with, the current antibiotic-based controls used for treating pathogenic bacteria. However, further studies are needed to determine their MIC values, to evaluate their commercial and economical effectiveness, the eventual synergistic effects with others compounds, as found in the consumption of the Brassica foods; toxicity bioassays must as well be performed.

The use of potassium caseinate for wine fining can cause allergic reactions and must be included on wine bottle labels. The focus of this research was to determine if non-allergenic pea protein or polyvinylpolypyrrolidone (PVPP) are possible alternatives for wine fining. Trials were performed to compare the effects of these potential fining agents on phenolic compounds, browning potential, turbidity and sensory attributes. The results indicate that flavonoid and non-flavonoid phenols decreased (p < 0.05) in the wines treated with potassium caseinate, pea protein, and PVPP. All fining agents decreased (p < 0.05) wine colour. Potassium caseinate was the most effective fining agent for reducing browning potential. Pea protein and potassium caseinate resulted in increased clarity of the final wines. No sensorial differences (p < 0.05) were detected among the wines prepared with the different fining agents. This study provides information that could be used to select an appropriate alternative to potassium caseinate. (C) 2011 Elsevier Ltd. All rights reserved.

To access the fermentative response of equine caecal microbial population to nitrogen availability, an in vitro study was conducted using caecal contents provided with adequate energy sources and nitrogen as limiting nutrient. Two nitrogen (N) sources were provided, protein (casein) and non-protein (urea). Caecal fluid, taken from three cannulated horses receiving a hay-concentrate diet, was mixed with a N-free buffer-mineral solution. The influence of four N levels (3.7, 6.3, 12.5 or 25 mg of N in casein or urea) was studied using the gas production technique. Total volatile fatty acids (VFA), NH3-N and gas production were measured after a 24-h incubation period. Microbial biomass was estimated using adenine and guanine bases as internal markers, and ATP production was estimated stoichiometrically. Microbial growth efficiency (YATP) and gas efficiency (Egas) were estimated. Fermentation with casein as the sole N source was generally characterized by lower total VFA, NH3-N, total gas production and higher acetate : propionate (A : P) ratio and YATP than with urea. Results herein presented indicate that, under these in vitro conditions, caecal microbial population does in fact use urea N, but less efficiently than casein in terms of microbial growth.

In this work the total vitamin C contents (ascorbic acid+dehydroascorbic acid) and antioxidant activity of raw and cooked chestnuts was evaluated. The vitamin C contents of raw chestnuts varied significantly between the different cultivars (cv) studied and it varied from 400mg/kg dry weight (cv Lada) to 693mg/kg dry weight (cv Martaínha). The different cultivars behave differently during the cooking process concerning the loss of vitamin C. A significant decrease in the vitamin C content of the chestnuts was observed, 25-54% for the boiling process and 2-77% for the roasting process. Boiled and roasted chestnuts can be good sources of vitamin C since it may represent 22.4%, 16.2%, 26.8% and 19.4%, respectively, of the recommended dietary intake for an adult man and woman. The cooking process significantly changed the antioxidant activity of the chestnuts. A difference was observed between the cultivars during the cooking processes, concerning the antioxidant activity. For the raw chestnuts the variation in vitamin C content of the chestnuts explains 99% of the antioxidant activity variation but for the roasted and boiled chestnuts this percentage significantly decreases to 51% and 88%, respectively. Although a high antioxidant activity is still present in the cooked chestnuts, the cause for this antioxidant activity is less dependent on the vitamin C content of the chestnuts, probably due to the conversion of ascorbic acid to dehydroascorbic acid. The increase in gallic acid during the cooking process, presumably transferred from the peels to the fruit, also contributes to the high antioxidant activity observed for the cooked chestnuts.

Many Brassicaceae species are economically important crops and Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot, is considered one of the most important necrotrophic plant bacterial diseases occurring worldwide on these and many other crops. Therefore identifying resistance mechanisms and genes is crucial. Researchers continue to investigate the role of phytochemicals (plant secondary metabolites) in protecting plants against diseases and pathogens. Glucosinolates (GLS), and more specifically their hydrolysis products, are known to have various biological effects including antimicrobial activity. From the positive results of initial in vitro studies with Xcc and other pathogenic bacteria new experiments were designed to evaluate the possible in planta role of GLS, and also phenolics, in the interaction with Xcc. The in planta studies, with various Brassicaceae seedlings, have shown a correlation between GLS profiles, and therefore the subsequent hydrolysis products, and the inhibition of Xcc growth. There were no significant correlations between Xcc infection and total phenolics. Positive correlations were found between specific and total GLS contents and the severity of disease. Further in vitro and in planta studies need to be performed to evaluate the role of GLS and other defense mechanisms in Xcc and other important bacterial infections of Brassicaceae crops. (C) 2011 Elsevier B.V. All rights reserved.

Banana fruits are important foods, but there have been very few studies evaluating the phenolics associated with their cell walls. In the present study, (+) catechin, gallocatechin, and (-) epicatechin, as well as condensed tannins, were detected in the soluble extract of the fruit pulp; neither soluble anthocyanidins nor anthocyanins were present. In the soluble cell wall fraction, two hydroxycinnamic acid derivatives were predominant, whereas in the insoluble cell wall fraction, the anthocyanidin delphinidin, which is reported in banana cell walls for the first time, was predominant. Cell wall fractions showed remarkable antioxidant capacity, especially after acid and enzymatic hydrolysis, which was correlated with the total phenolic content released after the hydrolysis of the water-insoluble polymer, but not for the posthydrolysis water-soluble polymer. The acid hydrolysis released various monosaccharides, whereas enzymatic hydrolysis released one peak of oligosaccharides. These results indicate that banana cell walls could be a suitable source of natural antioxidants and that they could be bioaccessible in the human gut.