Rapid and direct low micromolar NMR method for the simultaneous detection of hydrogen peroxide and phenolics in plant extracts

A rapid and direct low micromolar 1H NMR method for the simultaneous identification and quantification of hydrogen peroxide and phenolic compounds in plant extracts was developed. The method is based on the highly deshielded 1H NMR signal of H?O? at ～10.30 ppm in DMSO-d? and the combined use of picric acid and low temperature, near the freezing point of the solution, in order to achieve the minimum proton exchange rate. Line widths of H?O? below 3.8 Hz were obtained for several Greek oregano extracts which resulted in a detection limit of 0.7 μmol L?1. Application of an array of NMR experiments, including 2D 1H-13C HMBC, spiking of the samples with H?O?, and variable temperature experiments, resulted in the unequivocal assignment of H?O? precluding any confusion with interferences from intrinsic phenolics in the extract.     

The impact of biological pesticides on arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi have a key role for plant nutrition in organic farming systems where crop protection relies on biopesticides. Although these are considered safe, their effects on non-target organisms, such as AM fungi, are not known and should be evaluated. A pot and a field experiment were employed to investigate the impact of biological pesticides (azadirachtin, spinosad, pyrethrum and terpens) on exogenous AM fungal inoculum (pots) and on indigenous AM fungi (field). The synthetic fungicide carbendazim and non-pesticide treated controls with or without mycorrhizal inoculation were also included. Plant growth and root colonization were measured 20 and 40 days post inoculation (dpi) in the pot experiment, or 40 and 90 dpi in the field study. Pesticide effects on the structure of the intraradical AM fungal community were determined via DGGE and cloning. Spinosad, pyrethrum and terpenes did not affect the colonization ability and the structure of the AM fungal community. On the contrary, pot application of azadirachtin resulted in a selective inhibition of the Glomus etunicatum strain of the inoculum. DGGE analysis showed that the field application of azadirachtin induced significant and persistent shifts in the AM fungal community. Carbendazim completely hampered mycorrhizal colonization in pots, compared to its field application which had a transitory effect on the colonization ability and the community structure of indigenous AM fungi. Our study provides first evidence for the effects of biological pesticides on the diversity of AM fungi.     

Quantitative bioluminometric method for DNA-based species/varietal identification in food authenticity assessment

A method is reported for species quantification by exploiting single-nucleotide polymorphisms (SNPs). These single-base changes in DNA are particularly useful because they enable discrimination of closely related species and/or varieties. As a model, quantitative authentication studies were performed on coffee. These involved the determination of the percentage of Arabica and Robusta species based on a SNP in the chloroplastic trnL(UAA)-trnF(GAA) intraspacer region. Following polymerase chain reaction (PCR), the Robusta-specific and Arabica-specific fragments were subjected to 15 min extension reactions by DNA polymerase using species-specific primers carrying oligo(dA) tags. Biotin was incorporated into the extended strands. The products were captured in streptavidin-coated microtiter wells and quantified by using oligo(dT)-conjugated photoprotein aequorin. Aequorin was measured within 3 s via its characteristic flash-type bioluminescent reaction that was triggered by the addition of Ca(2+). Because of the close resemblance between the two DNA fragments, during PCR one species serves as an internal standard for the other. The percentage of the total luminescence signal obtained from a certain species was linearly related to the percent content of the sample with respect to this species. The method is accurate and reproducible. The microtiter well-based assay configuration allows high sample throughput and facilitates greatly the automation.     

Bioactive Lipids of Marine Microalga Chlorococcum sp. SABC 012504 with Anti-Inflammatory and Anti-Thrombotic Activities

Microalgae are at the start of the food chain, and many are known producers of a significant amount of lipids with essential fatty acids. However, the bioactivity of microalgal lipids for anti-inflammatory and antithrombotic activities have rarely been investigated. Therefore, for a sustainable source of the above bioactive lipids, the present study was undertaken. The total lipids of microalga Chlorococcum sp., isolated from the Irish coast, were fractionated into neutral-, glyco-, and phospho-lipids, and were tested in vitro for their anti-inflammatory and antithrombotic activities. All tested lipid fractions showed strong anti-platelet-activating factor (PAF) and antithrombin activities in human platelets (half maximal inhibitory concentration (IC₅₀) values ranging ~25–200 μg of lipid) with the highest activities in glyco- and phospho-lipid fractions. The structural analysis of the bioactive lipid fraction-2 revealed the presence of specific sulfoquinovosyl diacylglycerols (SQDG) bioactive molecules and the HexCer-t36:2 (t18:1/18:1 and 18:2/18:0) cerebrosides with a phytosphingosine (4-hydrosphinganine) base, while fraction-3 contained bioactive phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules. These novel bioactive lipids of Chlorococcum sp. with putative health benefits may indicate that marine microalgae can be a sustainable alternative source for bioactive lipids production for food supplements and nutraceutical applications. However, further studies are required towards the commercial technology pathways development and biosafety analysis for the use of the microalga.     

Towards sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.) breeding: phenotyping evaluation of newly developed hybrids

An increasing demand for cherry production (Prunus avium L.) in Greece led to the development of new high quality sweet cherry cultivars. Self-incompatibility in cherry is one of the most challenging issues for the species’ cultivation and top breeding priority. Τhe present study focuses on the development of new hybrids with improved traits such as productivity, fruit size, organoleptic characteristics and self-compatibility. For this purpose, thirty different cultivars were crossed and produced hybrids that were evaluated according to 34 morpho-physiological characteristics. The results were analyzed using the XLSTAT (version 2014.1) software and a dendrogram was constructed using the agglomerative hierarchical clustering method. Optimal hybrid clustering was achieved when characteristics of great economic importance such as fruit shape and size, growth habit and days to blooming were included in the analysis. Based on the results, new sweet cherry hybrids with the special character of self-compatibility were developed. Our findings provide crucial new information for sweet cherry future breeding programs and cultivation.     

Competitive interactions of fifty barley cultivars with Avena sterilis and Asperugo procumbens

A 2-year study was conducted in northern Greece to assess the ability of 29 six-row and 21 two-row barley (Hordeum vulgare L.) cultivars to compete with winter wild oat [Avena sterilis spp. ludoviciana (Durieu) Gill & Magne] and German-madwort (Asperugo procumbens L.). The fresh weight of both weeds grown in competition with six of the most competitive six-row barley cultivars (Bulk Resist. Desc-Sutter, Lignee 640, Paico's, Mantnan's-EH 165, Pistacho and Malebo) was reduced by 61–75% compared with one of the least competitive six-row cultivars [Gloria's-Copal's (30B-4Y-20M)]. The corresponding fresh weight reduction of both weeds grown in competition with three of the most competitive two-row barley cultivars (Goldmarker, Shanon and Helena) ranged from 59 to 76% compared with one of the least competitive two-row cultivars (Guanajuato). The ability to withstand competition (AWC) from winter wild oat and German-madwort of the six-row and two-row cultivars ranged from 33.7 to 78.3% and 26.7 to 69.2%, respectively. Representative yield losses ranged from 24 to 68% and 30 to 75%, respectively. The corresponding ability to compete (AC) indices ranged from 22.5 to 64.8% and 21.1 to 59.1%. The results of this study indicated that the barley cultivars Ligne 640 and Pistacho (2B-1Y-1B) had high AWC from winter wild oat and German-madwort and provided high grain yield in both weedy and weed-free conditions. Consequently, they could successfully be used in Mediterranean organic (no herbicide use) and conventional agricultural systems with reduced herbicide use. Barley cultivars with high AC and ability to produce high grain yield under weedy conditions [with Bulk Resist. Desc-Sutter, Arupo's and Gloria's-Copal's (30B-4Y-23M) among the most efficient] could also successfully be used in organic fields infected by the two weeds.     

Nutritional quality of greenhouse lettuce at harvest and after storage in relation to N application and cultivation season

The effect of five levels of nitrogen fertilization on the growth and nutritional quality of Cos lettuce (Lactuca sativa L. cv. Parris Island) at harvest and after storage was studied during autumn and winter in South-West Greece. Plants were cultivated hydroponically in a greenhouse and the nitrate, chlorophyll and ascorbic acid (vitamin C) concentrations within the plant tissues were measured at harvest and following storage at 5 or 10 °C for 10 days. Nitrate accumulated in the leaves with increasing amounts of N within the nutrient solution and was higher in the winter than in the autumn. At the lowest N level (20 mg L⁻¹), the inner leaves accumulated more nitrate than the outer leaves, whereas at higher N levels (140, 200 or 260 mg L⁻¹) nitrate accumulation was higher in the outer leaves. Overall, the highest nitrate concentrations were detected in the petiole and the proximal end of the leaf, but at the lowest N application rate (20 mg L⁻¹) nitrate accumulated in the distal region of the leaf too. Although the nitrate concentrations within the leaves did not change significantly during 10 days storage at 5 or 10 °C, the chlorophyll and vitamin C concentrations decreased. Chlorophyll loss was higher in lettuce that was grown under low N levels and was higher at 10 °C than at 5 °C, but was reduced by enclosure of the lettuce in polyethylene film. It is concluded that the optimum N application rate for Cos lettuce grown hydroponically under cover during autumn and winter in South-West Greece, and in other areas with a similar climate, is 200 mg N L⁻¹ because at this N rate yield is satisfactory and leaf nitrate concentrations are below the maximum acceptable level for human consumption. Nutritional value (vitamin C concentration) and market quality (chlorophyll content) are highest at harvest and decrease during storage, but quality in terms of nitrate concentration does not change.     

Evaluation of zinc supplementation in European sea bass (Dicentrarchus labrax) juvenile diets

This study aimed to investigate the effect of increased zinc dietary levels on the growth performance, feed utilization, immune status and induced wound healing in European sea bass (Dicentrarchus labrax). Fish weighing 10 g were fed five diets containing organic zinc at 30, 70, 110 and 150 mg kg^?1 diet and one inorganic zinc source (zinc oxide) at 150 mg kg^?1 diet for a period of 12 weeks. No significant (P>0.05) differences were found in the growth performance parameters (final weight, specific growth rate), immunological indices tested (respiratory burst activity in whole blood) and the wound‐healing process. Zinc accumulation in the skin, vertebrae and liver increased significantly (P<0.01) with increased dietary zinc levels but not in muscle. The highest zinc concentrations were obtained in skin tissue, followed by vertebrae, liver and muscle tissue. Using the concentration of zinc in skin as a response criterion, broken‐line analysis showed that the supplementation of 148 mg organic Zn kg^?1 diet seemed to be the optimum dietary zinc supplementation level for sea bass juveniles.