TaqMan Chemistry for Phytophthora ramorum Detection and Quantification, with a Comparison of Diagnostic Methods

ABSTRACT The choice of detection method for phytopathogens can be critically important in determining the success or failure of pest regulation systems. We present an assay for Phytophthora ramorum that uses 5' fluorogenic exonuclease (TaqMan) chemistry to detect and quantify the pathogen from diseased tissue, and include a universal primer and probe set for an internal positive control. This method is sensitive, detecting as little as 15 fg of target DNA when used in a nested design or 50 fg when used in a single round of polymerase chain reaction. None of the 17 other Phytophthora spp. tested was amplified by this assay. A comparison of the nested and non-nested TaqMan assays, and of one other nested assay, showed nested methods to be significantly more sensitive than nonnested and showed that host substrate significantly affected sensitivity of all assays. The nested TaqMan protocol was successfully field-tested; P. ramorum was detected in 255 of 874 plants in California woodlands, whereas the single-round TaqMan protocol detected significantly fewer positive samples. Finally, we documented increases in the quantity of pathogen DNA in Umbellularia californica leaves in initial stages of infection.     

Use of near-infrared spectroscopy for fast fraud detection in seafood: application to the authentication of wild European sea bass (Dicentrarchus labrax)

The possibility of using near-infrared spectroscopy (NIRS) for the authentication of wild European sea bass ( Dicentrarchus labrax ) was investigated in this study. Three different chemometric techniques to process the NIR spectra were developed, and their ability to discriminate between wild and farmed sea bass samples was evaluated. One approach used spectral information to directly build the discrimination model in a latent variable space; the second approach first used wavelets to transform the spectral information and subsequently derived the discrimination model using the transformed spectra; in the third approach a cascaded arrangement was proposed whereby very limited chemical information was first estimated from spectra using a regression model, and this estimated information was then used to build the discrimination model in a latent variable space. All techniques showed that NIRS can be used to reliably discriminate between wild and farmed sea bass, achieving the same classification performance as classification methods that use chemical properties and morphometric traits. However, compared to methods based on chemical analysis, NIRS-based classification methods do not require reagents and are simpler, faster, more economical, and environmentally safer. All proposed techniques indicated that the most predictive spectral regions were those related to the absorbance of groups CH, CH(2), CH(3), and H(2)O, which are related to fat, fatty acids, and water content.     

Molecular and phenotypic characterization of a collection of white grain sorghum [Sorghum bicolor (L.) Moench] for temperate climates

Genetic Resources and Crop Evolution - Sorghum [Sorghum bicolor (L.) Moench] is a subsistence crop and the main food for populations in arid or semiarid regions and it is appreciated for the...     

Qualitative and semiquantitative analysis of phenolic compounds in extra virgin olive oils as a function of the ripening degree of olive fruits by different analytical techniques

Capillary electrophoresis (CE) can be effectively used as a fast screening tool to obtain qualitative and semiquantitative information about simple and complex phenolic compounds of extra virgin olive oil. Three simple phenols (tyrosol, hydroxytyrosol, and vanillic acid), a secoiridoid derivative (deacetoxy oleuropein aglycon), and two lignans (pinoresinol and acetoxypinoresinol) were detected as the main compounds in extra virgin olive oils by high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). Spectrophotometric indices, radical scavenging activity, and oxidative stability of extra virgin olive oil samples obtained from olives hand-picked at different ripening degrees were statistically correlated with the CZE and HPLC quantification. The concentration of phenols in extra virgin olive oil decreased with ripeness of olive fruits. The high correlations found between CZE and the other analytical results indicate that CE can be applied as a rapid and reliable tool to routinely determine phenolic compounds in extra virgin olive oils.     

Differentiation of natural and synthetic phenylalanine and tyrosine through natural abundance 2H nuclear magnetic resonance

The natural abundance deuterium NMR characterization of samples of the amino acids tyrosine (1) and phenylalanine (2), examined as the acetylated methyl esters 4 and 6, has been performed with the aim to identify by these means the contribution in animals of the hydroxylation of the diet l-phenylalanine (2) to the formation of l-tyrosine (1), a feature previously revealed on the same samples through the determination of the phenolic delta(18)O values. The study, which includes also the NMR examination of benzoic acid (5) from 2 and of tyrosol (7) from 1, substantially fails in providing the required information because the mode of deuterium labeling of tyrosine samples of different origins is quite similar but indicates a dramatic difference in the deuterium labeling pattern of the two amino acids 1 and 2. The most relevant variation is with regard to the deuterium enrichments at the CH(2) and CH positions, which are inverted in the two amino acids of natural derivation. Moreover, whereas the diastereotopic benzylic hydrogen atoms of l-tyrosine (1) appear to be equally deuterium enriched, in l-phenylalanine (2) the (D/H)(3)(R)() > (D/H)(3)(S)(). Similarly, benzoic acid (5) shows separate signals for the aromatic deuterium nuclei, which are quite indicative of the natural or synthetic derivation. The mode of deuterium labeling of the side chain of 1 and 2 is tentatively correlated to the different origins of the two amino acids, natural from animal sources for l-tyrosine and biotechnological probably from genetically modified microorganisms for l-phenylalanine.     

Effects of intra-vineyard variability and soil heterogeneity on vine performance,dry matter and nutrient partitioning

Three vigor zones, identified in a Barbera vineyard by remote sensing at full canopy, were carefully ground-truthed to determine, over 2 years, the relative weight of soil factors in affecting within-field variability, and to investigate vigor zone influence on dry matter (DM) and nutrient partitioning into different vine organs. Regardless of season, high vigor (HV) achieved stronger vine capacity as total vegetative growth and yield while resulting in markedly less ripened fruits than low vigor (LV) vines. PCA analysis carried out on ten different soil and vine variables clearly separated the three vigor levels and the correlation matrix highlighted that the factors mostly contributing to HV were soil depth, soil K and P concentration, total available water, clay fraction and N_leaf concentration. Conversely, sand fraction was the main marker for LV. When annual DM retrieved in clusters, canes, leaves, and shoot clippings was calculated for each vigor level and expressed as content (i.e. kg/ha) there was a general decreasing trend moving from HV to LV. However, when DM partitioned to each organ was given on a relative basis (i.e. percentage over total) results were similar across vigor levels. Similarly, when nutrients were given as content (e.g. kg or g/ha) out of 120 within-vigor combinations (12 nutrients, 2 seasons, 5 organs), 65 showed a significant difference between HV and LV. Conversely, with data expressed on a concentration basis (i.e. % DM) the number of significant differences between the vigor level means fell to 15. The study strengthens the causal link between soil properties and intra-vineyard spatial variability and clarifies that patterns of dry matter and nutrient partitioning to different vine organs are mildly affected by vine vigor when referred on a relative basis.

     

Heterologous expression of the glucose oxidase gene in Trichoderma atroviride leads enhanced ability to attack phytopathogenic fungi and induction of plant systemic disease resistance

A transgenic strain of Trichoderma atroviride that expresses the Aspergillus niger glucose oxidase gene goxA under a homologous pathogen-inducible promoter (nag1) has been constructed, with the aim of increasing the ability of this biocontrol agent (BCA) to attack phytopathogenic fungi and enhance plant systemic disease resistance. The sporulation and growth rate of the transgenic progenies were similar to the wild-type strain P1. goxA expression occurred immediately after contact with the p…     

Survival and feeding activity of Protaphorura armata in different composts

Effects of compost products, enriched or not-enriched with a strain of the mycoparasitic fungus Trichoderma atroviride, on the survival of the collembolan Protaphorura armata and the viability of fungal conidia after the transit through the springtail gut were investigated. The effect of compost enriched with Ca-Lignosulphonate (Ca-Ls), a low cost by-product of the acid sulphite pulping process, with lignin-like structure, on P. armata was also evaluated. All compost products enriched or not with the mycoparasitic fungus or Ca-Ls did not affect P. armata survival. No statistical differences were found in animal survival for different types of product or in enriched and not-enriched products. In addition to adults, live juveniles were also observed in all compost products. The gut content of animals, collected at the end of the survival test from compost enriched with T. atroviride, was examined under the light microscope, and in a few cases observations revealed the presence of some T. atroviride conidia. Subsequent tests carried out to study the viability of conidia after the transit through the springtail gut showed that colonies of the fungus developed from all faecal pellets produced by adult and juveniles specimens of P. armata previously fed on conidia of T. atroviride. These results suggest compatibility between Collembola and Trichoderma or Ca-Ls in the composts.     

Determination of free flavan-3-ol content in barley (Hordeum vulgare L.) air-classified flours: comparative study of HPLC-DAD/MS and spectrophotometric determinations

The determination of free flavan-3-ol compounds in barley flours (cv. Gotic) and two resulting milling fractions (fine fraction 57% and coarse fraction 43%, w/w) obtained by air classification of dehulled grain is herein described. The determinations were carried out using reversed phase high-performance liquid chromatography coupled with both diode array detection and ESI-MS compared with conventional spectrophotometric determinations (total phenolic compounds by the Folin-Ciocalteu method and free radical scavenging activity with the DPPH assay). Significant correlations among the HPLC quantification, the spectrophotometric data, and the antioxidant capacity of extracts were revealed by Pearson's analysis. Nine flavan-3-ols were identified by HPLC-MS. Catechins and their derivatives were found to make a substantial contribution to the antioxidant power of extracts. The coarse fraction showed larger concentrations of flavan-3-ols (221%) with respect to the fine fraction. This was confirmed by the antioxidant activity of the analyzed flours. The coarse fraction showed the greatest antioxidant activity (1200.1 +/- 66.2 micromol of Trolox equiv/100 g of flour) with respect to whole meal and fine fraction (1025.9 +/- 18.3 and 761.7 +/- 55.3 micromol of Trolox equiv/100 g of flour, respectively).