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Mirmajlessi S.M.,Estonian University of Life Sciences | Larena I.,National Institute for Agricultural and Food Research and Technology | Mand M.,Estonian University of Life Sciences | Loit E.,Estonian University of Life Sciences
Zemdirbyste | Year: 2016

The main aim of this investigation was to develop a SYBRGreen real-time polymerase chain reaction (PCR) assay for detection and quantification of Verticillium dahliae directly from affected strawberry roots and soils. The proposed assay utilizes a specifically designed primer pair on the basis of an internal transcribed spacer (ITS) sequence. During 2014-2015, plant and soil samples were randomly collected from different areas including Vasula, Rohu, Unipiha, Utsu and Marjamaa in Estonia and analyzed for V. dahliae. Real-time PCR technique using primers designed to the rDNA ITS1 was highly sensitive and accurate and so allowed reliable quantification of the pathogen DNA at low inoculation in soils (3 × 10-1 pg µl-1) and even in root of symptomless plants (5 × 10-1 pg µl-1). This is the first study of using this technique to quantify the population of V. dahliae in strawberry fields from Estonia. © 2016, Lithuanian Institute of Agriculture. All rights reserved.

Ucles A.,University of Almeria | Ulaszewska M.M.,University of Alcala | Hernando M.D.,National Institute for Agricultural and Food Research and Technology | Ramos M.J.,University of Alcala | And 4 more authors.
Analytical and Bioanalytical Chemistry | Year: 2013

This work introduces a liquid chromatography-electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry (LC-ESI-QTOF-MS)-based method for qualitative and quantitative analysis of poly(amidoamine) (PAMAM) dendrimers of generations 0 to 3 in an aqueous matrix. The multiple charging of PAMAM dendrimers generated by means of ESI has provided key advantages in dendrimer identification by assignation of charge state through high resolution of isotopic clusters. Isotopic distribution in function of abundance of isotopes 12C and 13C yielded valuable and complementarity data for confident characterization. A mass accuracy below 3.8 ppm for the most abundant isotopes (diagnostic ions) provided unambiguous identification of PAMAM dendrimers. Validation of the LC-ESI-QTOF-MS method and matrix effect evaluation enabled reliable and reproducible quantification. The validation parameters, limits of quantification in the range of 0.012 to 1.73 μM, depending on the generation, good linear range (R > 0.996), repeatability (RSD < 13.4 %), and reproducibility (RSD < 10.9 %) demonstrated the suitability of the method for the quantification of dendrimers in aqueous matrices (water and wastewater). The added selectivity, achieved by multicharge phenomena, represents a clear advantage in screening aqueous mixtures due to the fact that the matrix had no significant effect on ionization, with what is evidenced by an absence of sensitivity loss in most generations of PAMAM dendrimers.[Figure not available: see fulltext.] © 2013 Springer-Verlag Berlin Heidelberg.

Guil N.,CSIC - Institute of Refrigeration | Sanchez-Moreno S.,National Institute for Agricultural and Food Research and Technology
Systematics and Biodiversity | Year: 2013

Studies on micrometazoan local distribution patterns are needed to understand microscopic organisms evolution in the light of the related influences of historical and ecological events. Tardigrades encompass a set of rare and unique characteristics to study evolutionary hypotheses. Particularly interesting are leaf litter tardigrades, due to their connection with soil dynamics: although tardigrades have been frequently considered occasional or accidental inhabitants coming from nearby habitats. In this study, leaf litter tardigrade diversity from a central Spain mountain system was determined using four diversity descriptors: species richness, abundance, and two diversity indexes (Shannon and Simpson), and a diversity trophic index (TD). In addition, community composition was analysed using Canonical and Discriminant Analyses. Fifteen types of leaf litter were explored in the present survey, many not previously studied, all of them containing tardigrades. We have found 39 species, 20 are being recorded for the first time in leaf litter, and increasing to 20% the global tardigrade diversity reported for leaf litter worldwide, with the total number of species now being 99. The same genera that were reported being present in high species diversity at global scale were found to be highly diverse at local scale in this study. Three eutardigrade families previously reported as beech litter inhabitants were not found in our survey, probably because beech litter was not included in the present survey, suggesting that further work studying a more diverse range of litter would increase reported tardigrade diversity worldwide. Tardigrade species abundances depended on high level plant categories (plant divisions and type of tree), while tardigrade diversity patterns were related with type of leaves. Broad leaves had higher diversity values (Shannon, Simpson, species richness, trophic groups) than any other leaf, probably due to their association to dense forests presenting humid conditions and high resources availability, associated with this leaf. Xerophilous leaves presented high diversity of species (heterotardigrades) well-adapted to xeric open space in our survey. Type of leaves also changed abundance dynamics of different tardigrade trophic groups. Different biotic and abiotic conditions in different types of leaves promoted varying abundances of carnivore, herbivore and microbivore tardigrades on those different micro-ecosystems. Implications and consequences of these findings in evolutionary hypotheses for microfauna are considered. © 2013 Taylor & Francis Group, LLC.

Herrera Lopez S.,IMDEA Madrid Institute for Advanced Studies | Herrera Lopez S.,University of Almeria | Hernando M.D.,National Institute for Agricultural and Food Research and Technology | Gomez M.J.,IMDEA Madrid Institute for Advanced Studies | And 6 more authors.
Rapid Communications in Mass Spectrometry | Year: 2013

RATIONALE Polycyclic musks have become a concern due to their bioaccumulation potential and ecotoxicological effects. The HHCB transformation product (TP) (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta[γ]- 2-benzopyran; HHCB-lactone) is the most stable intermediate generated and it is frequently detected in river waters. The aim of this work was the identification of relevant TPs generated from UV irradiation and ozone treatments. METHODS Identification of HHCB TPs was carried out by liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (LC/ESI-QTOF-MS) and two-dimensional gas chromatography/electron impact time-of-flight mass spectrometry (GC×GC-EI-TOF-MS). With LC/ESI-QTOF-MS, TPs were characterized by means of mass accuracy in both full-scan and MS/MS modes through information-dependent acquisition (IDA) and direct injection on-column. With stir bar sorptive extraction (SBSE)-GC×GC-EI-TOF-MS, identification was based on the enhanced separation capacity and screening of unknowns through the acquisition of full-range mass spectra. RESULTS The effectiveness of these complementary techniques allowed a detailed evaluation of the main TPs. Eighteen TPs were elucidated based on mass accuracy, in both full-scan and MS/MS modes using LC/ESI-QTOF-MS with mass errors below 5 ppm and 10 ppm (mostly), respectively. Most of the TPs had not been analytically identified in previous studies. Separation of the enantiomeric species (R) and (S) of HHCB-lactone, and the identification of other relevant TPs, was performed using SBSE-GC×GC-EI- TOF-MS. CONCLUSIONS LC/ESI-QTOF-MS and GC×GC-EI-TOF-MS analysis provides the best alternative for TP identification of chemicals of concern, which have a wide range of polarities and isobaric compounds. A prediction of PBT (persistence, bioaccumulation and toxicity) using the PBT Profiler program suggested a classification of 'very persistent' and 'very toxic' for most of the TPs identified. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

Gonzalez-Doncel M.,National Institute for Agricultural and Food Research and Technology | Garcia-Maurino J.E.,Complutense University of Madrid | San Segundo L.,National Institute for Agricultural and Food Research and Technology | Beltran E.M.,National Institute for Agricultural and Food Research and Technology | And 2 more authors.
Environmental Pollution | Year: 2014

Here we proposed a battery of non-invasive biomarkers and a histological survey to examine physiological/anatomical features in embryos, eleutheroembryos (13 days post-fertilization, dpf), and larvae (28-42 dpf) of medaka to investigate the effects of embryonic exposure to propylparaben (PrP). Concentrations <1000 μg PrP/L didn't exert early or late toxic effects. However, survivorship was affected at 4000 μg/L in eleutheroembryos and at ≥1000 μg/L in larvae. Histological alterations were found in 37.5% of eleutheroembryos exposed to 4000 μg PrP/L. Morphometric analysis of the gallbladder revealed significant dilation at ≥400 μg/L throughout embryo development. Ethoxyresorufin-O-deethylase (EROD), as indicator of cytochrome P4501A activity, didn't reveal induction/inhibition although its combination with a P4501A agonist (i.e. β-naphthoflavone) resulted in a synergic EROD response. Results suggest a low toxicity of PrP for fish and support the use of fish embryos and eleutheroembryos as alternatives of in vivo biomarkers indicative of exposure/toxicity. © 2013 Elsevier Ltd. All rights reserved.

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