Ultrastructural aspects of tomato leaves infected by Tomato torrado virus (ToTV) and co‐infected by other viruses

Optical and electron microscopy studies were carried out to investigate the cytopathology induced in tomato leaves infected by Tomato torrado virus (ToTV), a new picorna‐like virus associated with the ‘Torrado’ disease. Infected leaves, showing typical Torrado disease symptoms were surveyed in commercial greenhouses in the main tomato production areas of Spain. The effect of the co‐infection of ToTV with other viruses which commonly infect tomato crops was also studied. Ultra‐thin sections of ToTV‐infected tomato leaves did not show a strong cellular alteration. However, crystalline arrays of isometric virus‐like particles (VLPs) of 20–30 nm in the inclusion bodies were observed in phloem parenchyma cells of the infected tissues. Tissues co‐infected by ToTV and either Tomato chlorosis virus (ToCV) or Pepino mosaic virus (PepMV) presented more severe cellular alterations. The most deleterious consequences for tomato cells were found in triple infections of ToTV, PepMV and Tomato spotted wilt virus (TSWV), where characteristic cell wall overgrowth was distinguishable, together with a large amount of necrotic cells.     

A combined procedure to evaluate the global antioxidant response of bread

Baking is the most representative manufacturing process applied to bread, involving thermal and moisture conditions that facilitate the Maillard reaction (MR) and, at the same time, the destruction-formation of natural-labile and thermally-induced antioxidant compounds respectively. In the present paper, the use of a new approach to measure the Global Antioxidant Response (GAR) of cereal derivatives is proposed: a combination of in vitro digestion – which enables measurement of the bioaccessible fraction – and the QUENCHER, which makes it possible to determine the antioxidant activity of the insoluble fraction, since it is a simple and direct procedure for determining the total antioxidant capacity of solid products. After digestion, the results obtained by the antioxidant assays are up to 20-fold higher than those reported using the standard extraction methods. The non-extractable residue displayed significant antioxidant activity that accounted for up to 17% of the total antioxidant activity. Moreover, the GAR obtained in some of the assays developed was 10–40% higher than the antioxidant activity registered by the QUENCHER procedure in wheat bread, and the difference was even higher in wheat bran bread. Therefore, the use of the GAR approach could avoid underestimation of the antioxidant activity of cereal derivatives.     

Evaluation of an in-clinic Serum Amyloid A (SAA) assay and assessment of the effects of storage on SAA samples

Background

An in-clinic assay for equine serum amyloid A (SAA) analysis, Equinostic EVA1, was evaluated for use in a clinical setting. Stability of SAA in serum samples was determined.

Methods

Intra- and inter- assay variation of the in-clinic method was determined. The in-clinic method (EVA1) results were compared to a reference method (Eiken LZ SAA) with 62 patient samples. For samples with SAA concentrations within the assay range of EVA1 (10-270 mg/L), differences between the methods were evaluated in a difference plot. Linearity under dilution was evaluated in two samples. Stability of SAA in three serum pools stored at 4°C and approximately 22°C was evaluated with the reference method day 0, 1, 2, 4, 7, 17 and analysed with a two-way ANOVA.

Results

The imprecision (coefficient of variation, CV) for the in-clinic method was acceptable at higher SAA concentrations with CV values of 7,3-12%, but poor at low SAA concentrations with CV values of 27% and 37% for intra- and inter-assay variation respectively. Recovery after dilution was 50-138%. The in-clinic assay and the reference method identified equally well horses with low (<10 mg/L) and high (>270 mg/L) SAA concentrations. Within the assay range of the in-clinic method, 10-270 mg/L, the difference between the two methods was slightly higher than could be explained by the inherent imprecision of the assays. There were no significant changes of serum SAA concentrations during storage.

Conclusions

The in-clinic assay identified horses with SAA concentrations of <10 mg/L and >270 mg/L in a similar way as the reference method, and provided an estimate of the SAA concentration in the range of 10-270 mg/L. The imprecision of the in-clinic method was acceptable at high SAA concentrations but not at low concentrations. Dilution of samples gave inconsistent results. SAA was stable both at room temperature and refrigerated, and thus samples may be stored before analysis with the reference method.     

Validation of a Commercial Enzyme Immunoassay for Detection of Clostridium difficile Toxins in Feces of Horses with Acute Diarrhea

Background: Clostridium difficile infection (CDI) is a recognized cause of colitis in the horse. Identification of its toxins is important for management of individual cases and for prevention of transmission and zoonosis. In humans, CDI diagnosis is performed with enzyme immunoassays, none of which have been validated for horses. Hypothesis/Objectives: (1) Establish which test for CDI diagnosis was more frequently used by diagnostic laboratories, (2) determine the identified test's performance, sensitivity, and specificity, and (3) validate its use in diarrheic horses. Animals: Samples were obtained from 72 horses presented with acute diarrhea and hospitalized at the Ontario Veterinary College, University of Guelph. Methods: A survey was conducted to establish which of the tests for CDI diagnosis in horses is most commonly used throughout North America. A questionnaire was sent to all laboratories registered in the Veterinary Infection Control Society and the American Association of Veterinary Laboratory Diagnosticians. The performance of the test was evaluated by comparison to a cell cytotoxicity assay (CTA), the accepted Gold Standard for C. difficile toxin detection. Results: The Techlab C. difficile Tox A/B II ELISA was the most frequently used test. Compared with the CTA, no significant difference was observed, and a good level of agreement (93%) was obtained. The diagnostic performance of the ELISA test was adequate (84% sensitivity and 96% specificity). Conclusions and Clinical Importance: Results demonstrate that the Techlab C. difficile Tox A/B II ELISA is a reliable, adequate, and practical tool for identification of C. difficile toxins in horse feces.     

Participatory organic certification in Mexico: an alternative approach to maintaining the integrity of the organic label

Over the past two decades the growth of the organic sector has been accompanied by a shift away from first party, or peer review, systems of certification and towards third party certification, in which a disinterested party is responsible for the development of organic standards and the verification of producer compliance. This paper explores some of the limitations of the third party certification model and presents the case of Mexico as an example of how an alternative form of participatory certification has emerged. The paper suggests that participatory guarantee systems (PGS) are reflective of the growing “beyond organic” movement, which focuses on reconstructing the local and re-embedding food systems into their socio-ecological contexts. It argues that PGS offers a number of benefits for producers and consumers, particularly in the South, but that it faces a number of challenges as well, such as a lack of formal recognition, social conflicts and dependence on donated resources.     

Pathotypes of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> causing damage to oilseed rape in the Czech Republic and Poland

Winter oilseed rape (Brassica napus) is an important crop in the Czech Republic and Poland. Clubroot disease caused by the pathogen Plasmodiophora brassicae is a serious and still-growing problem for oilseed rape growers in both countries. The aim of this study was to evaluate the pathotype composition of P. brassicae populations from the Czech Republic and Poland, according to the three evaluation systems, and to determine soil inoculum loads for representative fields via traditional end-point PCR as well as quantitative PCR analysis. There were considerable differences between the populations of P. brassicae from both countries, and the number of pathotypes varied depending on the evaluation system and the threshold used to distinguish susceptible vs. resistant plant reactions. This is the first study comparing the effect of different thresholds. Using an index of disease (ID) of 25 % to distinguish susceptible vs. resistant reactions, there was a total of seven pathotypes identified based on the differentials of Williams, five with the system of Somé et al., and 18 with the European Clubroot Differential (ECD) set. However, based on a threshold of 50 %, there were nine pathotypes according to the evaluation system by Williams, four based on the differentials of Somé et al., and 15 with the ECD set. Changing of the thresholds led to the reclassification of some pathotypes. Several pathotypes were common in both countries. High amounts of pathogen DNA were found in many of the field soils analysed by quantitative PCR. There was a weak correlation between soil pH and infestation of P. brassicae for the Polish soils.     

Proteome profiling of the compatible interaction between wheat and stripe rust

Over the last decade, comparative molecular profiling studies between compatible and incompatible plant-pathogen interactions have shown that susceptible response of the host to a pathogen requires factors that promote disease development. In this study, we examined proteome profiles during a compatible interaction between wheat and stripe rust. A 2D-LC system (ProteomeLab PF2D) was used for protein separation and to compare the proteome from infected and control samples. More than 700 protein peaks at each time point were compared between pathogen- and mock-inoculated samples. Selected proteins, with significant differences in abundance were identified by nanoLC-ESI- MS/MS and generated spectra were searched against the wheat protein databases from UniProt, and NCBI and the Puccinia database from The Broad Institute. In total, the identified proteins comprised of 62 % wheat and 38 % Pst proteins. All identified proteins were searched by bioinformatics-based algorithms to detect their subcellular localization and signal peptide motifs which have the potential to catch the candidate effector proteins. The wheat proteins were classified based on their function. Although a compatible interaction, many wheat proteins, such as antioxidants, PRs and cold-responsive proteins, are implicated in defense and stress tolerance. On the pathogen side, 64 proteins were identified, and included some important pathogenicity proteins that can play role in pathogen virulence and suppress the host defense. In addition, we discovered that nine proteins have a signal sequence and three of the hypothetical fungal proteins, PGTG_11681T0, PGTG_07231T0 and CBH50687.1, have been tentatively identified as candidate effectors.     

When structure means conservation: Effect of aggregate structure in controlling microbial responses to rewetting events

Rewetting events after a drought produce a pulse of soil respiration (the “Birch Effect”) that leads to a loss of carbon from soil, especially in Mediterranean ecosystems. Two main hypotheses have developed to explain the Birch effect: the “metabolic explanation”, based on the rapid consumption of intracellular osmolytes previously accumulated to survive to dry conditions, and the “physical explanation”, based on the consumption of carbon made accessible by physical destruction of internal structures of the soil.Here, we compared the respiration response of intact and crushed 9–4 mm aggregates from a California grassland soil under two different rewetting schemes: (1) successive short dry/wet events and (2) increased drought periods followed by a single rewetting. In intact aggregates, both microbial biomass and respiration rates were relatively stable through both experimental treatments. In crushed aggregates, through multiple short dry/wet cycles, both respiration rate and microbial biomass increased, while as drought length increased, biomass was unaffected but the magnitude of the following rewetting pulse increased. A mechanism that explains both these results is that crushing aggregates exposes occluded particular material that must be degraded into an immediately bioavailable form for microbes to take it up and metabolize it. Nitrification was generally higher in intact than crushed aggregates, suggesting the importance of physical association between nitrifiers and resources in regulating overall soil nitrification.This work suggests that physical processes are most important in driving respiration pulses through multiple rewetting cycles and that the physical association of organisms, substrates, and mineral particles are critical in controlling the functioning of the “microbial landscape”.