Real-time polymerase chain reaction based assays for quantitative detection of barley, rice, sunflower, and wheat

Quality assurance is a major issue in the food industry. The authenticity of food ingredients and their traceability are required by consumers and authorities. Plant species such as barley (Hordeum vulgare), rice (Oryza sativa), sunflower (Helianthus annuus), and wheat (Triticum aestivum) are very common among the ingredients of many processed food products; therefore the development of specific assays for their specific detection and quantification are needed. Furthermore, the production and trade of genetically modified lines from an increasing number of plant species brings about the need for control within research, environmental risk assessment, labeling/legal, and consumers' information purposes. We report here the development of four independent real-time polymerase chain reaction (PCR) assays suitable for identification and quantification of four plant species (barley, rice, sunflower, and wheat). These assays target gamma-hordein, gos9, helianthinin, and acetyl-CoA carboxylase sequences, respectively, and were able to specifically detect and quantify DNA from the target plant species. In addition, the simultaneous amplification of RALyase allowed bread from durum wheat to be distinguished. Limits of detection were 1 genome copy for barley, sunflower, and wheat and 3.3 copies for rice real-time PCR systems, whereas limits of quantification were 10 genome copies for barley, sunflower, or wheat and approximately 100 haploid genomes for rice real-time PCR systems. Real-time PCR cycling conditions of the four assays were stated as standard to facilitate their use in routine laboratory analyses. The assays were finally adapted to conventional PCR for detection purposes, with the exception of the wheat assay, which detects rye simultaneously with similar sensitivity in an agarose gel.     

Application of para-nitrophenol (pNP) enzyme assays in degraded tropical soils

Enzyme activities have been used as indicators of soil quality and changes in biogeochemical function due to management or perturbations. The objective of this study was to answer a number of methodological questions regarding sampling schemes, sample handling recommendations, and assay procedures to facilitate the use of enzyme assays in the tropical highlands of East Africa. We used para-nitrophenol (pNP) based substrates for five enzymes: β-glucosidase, cellobiohydrolase, chitinase, acid phosphatase, and alkaline phosphatase. In the first experiment, we examined sampling procedures and compared the results of determining enzyme activities on a plot using composite or discrete samples. Composite samples usually had higher activities than the means of individual cores (P<0.05), but relative ranking of sites was the same if analyses were based on composite or discrete samples. In the second experiment, we examined the effects of storage time and conditions on enzyme activity. Enzyme activity degraded rapidly in frozen samples, but was better maintained in samples stored at 4 °C. Phosphatase and cellobiohydrolase activity declined after 14 days of storage, while the activity of the other enzymes remained close to the values of fresh samples for 28 or more days. In the third experiment, we examined the effect of the addition of an antiseptic, toluene, to prevent bacterial growth during the assay. We found no consistent toluene effect (P>0.4), probably because the assays were of short duration and microbial growth was minimized. Finally, we looked at the incubation time necessary to produce reliable results. Phosphatases, with relatively high activities could reliably be determined in 2 h incubations, but the other enzymes had much lower activities and required longer incubation times for reliable determination. For the enzymes we looked at, 4 h was a good standard time for determining the activity of even the lowest activity enzymes. The results of this study provide practical guidelines for applying these enzyme assays in the degraded tropical soils.     

Validation of a mass spectrometry method to quantify oak ellagitannins in wine samples

Detection and individual quantification of oak wood ellagitannins in oak barrel aged red wine samples are difficult mainly due to their low levels and the similarity between their structures. In this work, a quantification method using mass spectrometry has been developed and validated to quantify wine ellagitannins after sample fractionation with a previously reported method. The use of an internal standard is a requirement to correct mass signal variability. (-)-Gallocatechin, among the different tested compounds, was the only one that proved to be a suitable internal standard making possible the accurate and individual quantification of the main oak wood ellagitannins. The developed methodology has been used to detect and quantify these ellagitannins in different Spanish commercial wines, proving its usefulness.     

Late nitrogen fertilization affects nitrogen remobilization in wheat

A pot experiment with wheat plants was carried out to study how late application of nitrogen (N) fertilizer affects the use of pre‐anthesis N reserves during the grain‐filling period. Increasing doses of N fertilizer were applied (0, 40, and 52 mg N plant^–1), either in two amendments (growth stages GS20 and GS30, according to Zadoks scale) or in three amendments (GS20, GS30, and GS37). The experiment was arranged in a complete randomized three‐block design with 129 plants per treatment. The plants were watered daily, harvested every 2 d between anthesis and maturity, and were separated into roots, leaf sheaths, leaf blades, and ears for further N determination. Grain N concentration improved due to a late N application in GS37 by 14% (higher N dose) and by 7% (further splitting the same N‐fertilizer dose, respectively). The higher the N‐fertilizer dose applied, the greater was the amount of pre‐anthesis reserves in vegetative organs, these reserves became later available for remobilization. Although splitting the same N dose in three amendments did not increase the N reserves, these reserves were more efficiently remobilized allowing an improvement in grain N concentration. The fertilizer management did not change the temporary pattern of N accumulation in the ear, but did induce a change in the amount of N remobilized and in the contribution of each organ (root, leaf sheath, leaf blade) to this remobilization. Late N amendment allowed a greater N availability of leaf blades and ear N reserves (from 20% up to 26% and from 19% up to 22%, respectively) for remobilization towards the grain, decreasing the root contribution from 28% down to 15%, while the contribution of leaf sheaths was maintained around 35% irrespective of the N applied.     

Microbial dynamics after adding bovine manure effluent together with a nitrification inhibitor (3,4 DMPP) in a microcosm experiment

The application of animal manure effluents in agriculture in combination with nitrification inhibitors should be beneficial for nutrient recycling, soil quality, plant productivity, and greenhouse gas emission and offer economic advantages to make them an alternative to conventional fertilizers. The present study aims to estimate the effects of the addition of bovine manure effluent alone or together with a nitrification inhibitor (3,4-dymethylpyrazol-phosphate (3,4 DMPP)) on the microbial community dynamics in a Mediterranean soil in an incubation experiment over 28 days. The application of the bovine manure effluent increased respiration, microbial biomass carbon, fungal and bacterial growth, and enzyme activities and changed the microbial community structure evaluated by the phospholipid fatty acid pattern. Adding the bovine manure effluent together with the nitrification inhibitor, although partly negating the positive effect of the effluent on soil microbial activity, still resulted in higher or similar growth and activity as in the control. Our results indicate that the addition of the nitrification inhibitor 3,4 DMPP together with a bovine manure effluent could be a promising solution to control the animal manure effluent application effects on soil microbiological properties and microbial dynamics, as well as counteracting direct inhibiting effects of 3,4 DMPP on the soil heterotrophic community.     

IMPACT OF SALT STRESS ON MICRONUTRIENTS IN CORDYLINE FRUTICOSA VAR. ‘RED EDGE'

This trial was carried out to study the influence of the nutrient solution on the microelements concentration and distribution in C. fruticosa var. ‘Red Edge' plants. Four treatments were tested: T₁ [control, 1.5 dS m^?1, 14.3 mmol L^?1 sodium chloride (NaCl)], T₂ (2.5 dS m^?1, 22.2 mmol L^?1 NaCl), T₃ (3.5 dS m^?1, 32.7 mmol L^?1 NaCl) and T₄ (4.5 dS m^?1, 38.2 mmol L^?1 NaCl). In roots and stems, iron (Fe) concentrations were lower in the no saline treatment. Stems accumulated more Fe with treatments T₃ and T₄. Copper concentration and extraction were not affected by salinity. The highest manganese (Mn) concentration in roots was observed in T₂, while in petioles was higher in T₃ and T₄. Manganese extraction reached higher levels in the saline treatments in roots and stems, while in petioles it was lower in T₁, T₂ and T₃. In roots, zinc (Zn) concentration was lower with the extreme treatments. Micronutrients concentration in leaves was unaffected by salinity, because an exclusion mechanism that consists on accumulation in roots and stems was developed.     

Fungistatic activity of bicyclo[4.3.0]-γ-lactones

Five optically active and sixteen racemic lactones (nine of them new) of bicyclo[4.3.0]nonane structure were synthesized. IC(50) values for the following phytopathogens were determined: Aspergillus ochraceus AM 456, Fusarium culmorum AM 282, Fusarium oxysporum AM 13, Fusarium tricinctum AM 16. Effect of compound structures, especially stereogenic centers, on fungistatic activity has been discussed. The highest fungistatic activity was observed for trans-7,8-dibromo-cis-3-oxabicyclo[4.3.0]nonan-2-one (3c), IC(50) = 30.1 μg/mL (0.10 μM/mL), and cis-7,8-epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (3b), IC(50) = 72.2 μg/mL (0.47 μM/mL), toward F. oxysporum AM 13.     

Beef authentication and retrospective dietary verification using stable isotope ratio analysis of bovine muscle and tail hair

Stable isotope ratio analysis (SIRA) was used as an analytical tool to verify the preslaughter diet of beef cattle. Muscle and tail hair samples were collected from animals fed either pasture (P), a barley-based concentrate (C), silage followed by pasture (SiP), or silage followed by pasture with concentrate (SiPC) for 1 year (n = 25 animals per treatment). The (13)C/(12)C, (15)N/(14)N, (2)H/(1)H, and (34)S/(32)S isotope ratios in muscle clearly reflected those of the diets consumed by the animals. By applying a stepwise canonical discriminant analysis, a good discrimination of bovine meat according to dietary regimen was obtained. On the basis of the classification success rate, the (13)C/(12)C and (34)S/(32)S ratios in muscle were the best indicators for authentication of beef from animals consuming the different diets. Analysis of (13)C/(12)C and (15)N/(14)N in tail hair sections provided an archival record of changes to the diet of the cattle for periods of over 1 year preslaughter.