Possible involvement of salicylic acid in systemic acquired resistance ofCucumis sativus againstSphaerotheca fuliginea

The possible involvement of salicylic acid in systemic acquired resistance ofCucumis sativus againstSphaerotheca fuliginea was studied. Cucumber plants were inoculated with tobacco necrosis virus on the cotyledons and the level of endogenous salicylic acid in the first true leaf was determined by gas chromatography. Salicylic acid increased continously from the second day after virus inoculation to the fifth day, when the same leaf was inoculated withSphaerotheca fuliginea. In healthy plants, the efficiency of exogenous salicylic acid in inducing resistance was assayed by applying aqueous solutions at different times beforeSphaerotheca fuliginea inoculation. To evaluate the level of induced resistance, the following parameters were examined by light microscopy: percentage of conidial germination, length of the hyphae derived from single conidia, number of haustoria, percentage of epidermal cells with lignified walls and of necrotic cells underlying fungal hyphae. In treated plants conidial germination was reduced, the total length of the hyphae was shorter, the number of haustoria was lower and the haustorium-containing epidermal cells had more frequently lignified walls. Moreover, an evident increase in callose deposition was observed leading to the formation of oversized papillae around the penetration pegs. These results indicate that the application of salicylic acid before inoculation withSphaerotheca fuliginea reduces the intensity of the infectious process and that salicylic acid is involved in the expression of systemic resistance in cucumber challenged by the biotrophic pathogenSphaerotheca fuliginea.     

A dynamic risk assessment model (FUMAgrain) of fumonisin synthesis by Fusarium verticillioides in maize grain in Italy

Fumonisin contamination of maize grain starts in the field. Forecasting Fusarium infection and fumonisin synthesis could allow operators in the field to control contamination during the growing season and to make the best agronomic decisions for high quality yields while respecting the limits imposed by the European Union.A research project to develop a decision support system for the control of field-phase fumonisin contamination began in Italy in 2003. This paper presents a preliminary version of the engine of the decision support tool: FUMAgrain, a dynamic risk assessment model developed with data from the north of Italy. The structure of FUMAgrain is based on the pathosystem formed by maize, F. verticillioides and Ostrinia nubilalis (European Corn Borer). The elements of the pathosystem are simulated by three sub-models: (i) maize development, (ii) F. verticillioides infection and fumonisin synthesis, (iii) European Corn Borer wounding activity on maize grain. Inputs to the model are (i) planting date, (ii) hourly meteorological data including temperature, relative humidity, wind speed and rain intensity, (iii) information on the phenological development of the hybrid planted (flowering and dry-down), and (iv) information about the chemical treatment against European Corn Borer. FUMAgrain gives an initial risk alert at the end of flowering based on the meteorological conditions during this phase. A second alert follows maturation when an assessment is made from (i) maize grain moisture, (ii) European Corn Borer damage to the ear, and (iii) fumonisin synthesis risk. Following calibration and validation with data FUMAgrain demonstrated its good capability to simulate fumonisin synthesis in maize grain in Italy (calibration: R² = 0.70; validation: R² = 0.71) and its usefulness for determining the optimal harvest date while respecting grain safety levels required by the international market and limiting moisture content, hence drying costs.     

Acquisition capability of the grapevine Flavescence dorée by the leafhopper vector Scaphoideus titanus Ball correlates with phytoplasma titre in the source plant

Flavescence dorée (FD) is one of the most economically important grapevine diseases in Southern Europe, and it is associated with phytoplasmas, phloem-limited wall-less bacteria. Recovery from disease naturally occurs in infected grapevines during the following seasons after infection. The capability of the leafhopper vector Scaphoideus titanus to acquire FD phytoplasma (FDP) from recovered and infected grapevines of Barbera and Nebbiolo varieties was investigated in North-western Italy vineyards monitored from 2007 to 2011. Pathogen concentration was quantified by real-time PCR in FDP-infected grapevines and broad beans, also used as source plants under controlled conditions, to correlate acquisition capabilities and phytoplasma titre in source plants. S. titanus acquired FDP from infected, but not from recovered, grapevines. FDP titre was higher in Barbera than in Nebbiolo and higher in summer than in spring, and acquisition efficiency and pathogen titre in source plants were positively correlated, both in field and laboratory conditions. Recovered plants do not represent a source of inoculum for the vector and therefore do not contribute to FDP spread. The inability of recovered plants to serve as FDP acquisition sources for the vector as well as the effect of the season and of the two grapevine varieties on the FDP acquisition efficiency are relevant results to re-design disease management practices, especially since insecticide treatments against the vector are not fully effective, and newly designed successful control strategies are required.     

Direct measurement of the reaction front in chemically amplified photoresists

The continuing drive by the semiconductor industry to fabricate smaller structures using photolithography will soon require dimensional control at length scales comparable to the size of the polymeric molecules in the materials used to pattern them. The current technology, chemically amplified photoresists, uses a complex reaction-diffusion process to delineate patterned areas with high spatial resolution. However, nanometer-level control of this critical process is limited by the lack of direct measurements of the reaction front. We demonstrate the use of x-ray and neutron reflectometry as a general method to measure the spatial evolution of the reaction-diffusion process with nanometer resolution. Measuring compositional profiles, provided by deuterium-labeled reactant groups for neutron scattering contrast, we show that the reaction front within the material is broad rather than sharply defined and the compositional profile is altered during development. Measuring the density profile, we directly correlate the developed film structure with that of the reaction front.     

Multinucleated giant cells with an osteoclast phenotype derived from caprine peripheral blood mononuclear cells

Formation of multinucleated giant cells (MGCs) by macrophage fusion is a typical cytopathic effect of lentiviral replication in caprine monocytes and MGC formation from cultured caprine peripheral blood mononuclear cells (PBMCs) has been considered to be diagnostic for small ruminant lentivirus (SRLV) infection. In this study, formation of MGCs was observed after 7–14 days when PBMCs were cultured from healthy goats free from SRLV infection. These MGCs expressed tartrate-resistant acid phosphatase, calcitonin receptor, integrin αVβ3, cathepsin K and matrix metalloproteinase 9 and were able to resorb bone in vitro in the absence of RANKL and macrophage colony stimulating factor, consistent with an osteoclast phenotype.     

Use of electrochemical biosensor and gas chromatography for determination of dichlorvos in wheat

Two methods for the determination of dichlorvos in durum wheat by electrochemical assay and gas chromatography, respectively, have been developed. Dichlorvos, an organophosphorus anticholinesterase pesticide, was extracted from wheat with hexane, and the filtered extract was directly analyzed by gas chromatography with nitrogen-phosphorus flame detection (NPD). Recoveries of dichlorvos from milled wheat spiked at 0.25-1.5 microg/g ranged from 96.5 to 100.9%, and the limit of detection was 0.02 microg/g. The electrochemical assay was based on the detection of choline, the acetylcholinesterase product, via a choline oxidase biosensor. An aliquot of the filtered hexane extract was partitioned with phosphate buffer solution, and the organic layer was evaporated prior to electrochemical analysis. A limit of detection of 0.05 microg/g of dichlorvos was obtained with mean recoveries of 97-103% at spiking levels of 0.25-1.5 microg/g. A good correlation (r = 0.9919) was found between the results obtained with the electrochemical and those obtained with the gas chromatographic methods. The electrochemical method was peer-validated in two laboratories that analyzed 10 blind samples (5 duplicates), including a blank and 4 spiked samples with dichlorvos at levels of 0.25, 0.60, 1.00, and 1.50 microg/g. Within-laboratory repeatability (RSDr) and between-laboratory reproducibility (RSDR) ranged from 5.5 to 7.8% and from 9.9 to 17.6%, respectively.     

Existence of different physiological forms within genetically diverse strains of <Emphasis Type="Italic">Ampelomyces quisqualis</Emphasis>

Powdery mildew fungi are parasitized by strains of the genetically distinct Ampelomyces quisqualis. To investigate whether differences in the phylogeny and other cultural, morphological and physiological characteristics of these different strains are related to differences in their geographic origins or the host species from which they were isolated, several A. quisqualis strains isolated from different species of Erysiphaceae collected in different countries and possessing different ITS rDNA sequences were selected and characterized. The results revealed some significant variation among the selected strains, which provides evidence for the existence of different physiological forms within the A. quisqualis species. Two groups that display differential growth on artificial media were identified. These groups also differ in the morphology of their mycelium, but not in the morphology of their pycnidia and conidia. Temperature greatly affected the in vitro growth of the A. quisqualis strains and growth rate was closely correlated to colony color. Differences in the conidial germination of distinct strains were observed during the recognition phase of the parasitic relationship. The germination of each of the investigated strains was greatly stimulated by all of the examined powdery mildew species and not only by the conidia of their original hosts. An Italian strain isolated from grapevine in the Trentino Alto-Adige region was identified as the strain that germinates the most quickly in the presence of powdery mildew conidia. Phylogenetic analysis revealed that these A. quisqualis strains can be classified into five different genetic groups, which generally correlate with the fungal host of origin and morphological and growth characteristics.     

Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum

Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities.     

Novel Prunus rootstock somaclonal variants with divergent ability to tolerate waterlogging

Plants require access to free water for nutrient uptake, but excess water surrounding the roots can be injurious or even lethal because it blocks the transfer of free oxygen between the soil and the atmosphere. Genetic improvement efforts in this study were focused on the increased tolerance in roots to waterlogging. Among a pool of clones generated in vitro from leaf explants of rootstock Mr.S.2/5 of Prunus cerasifera L., the S.4 clone was flood tolerant whereas the S.1 clone was sensitive. The S.4 clone formed adventitious roots on exposure to flooding. Moreover, the chlorophyll content and mitochondrial activity in the leaf and root, soluble sugar content, alcohol dehydrogenase activity and ethylene content were different between the clones. The sorbitol transporter gene (SOT1) was up-regulated during hypoxia, the alcohol dehydrogenase genes (ADH1 and ADH3) were up-regulated in the leaves and down-regulated in the roots of the S.4 clone during hypoxia, and the 1-aminocyclopropane-1-oxidase gene (ACO1) was up-regulated in the leaves and roots of the S.4 clone during hypoxia and down-regulated in the wild-type roots. In addition, in the S.4 root, hypoxia induced significant down-regulation of a glycosyltransferase-like gene (GTL), which has a yet-undefined role. Although the relevant variation in the S.4 genome has yet to be determined, genetic alteration clearly conferred a flooding-tolerant phenotype. The isolation of novel somaclonals with the same genomic background but with divergent tolerance to flooding may offer new insights in the elucidation of the genetic machinery of resistance to flooding and aid in the selection of new Prunus rootstocks to be used in various adverse environments.     

Effects of fly attack (Bactrocera oleae) on the phenolic profile and selected chemical parameters of olive oil

The phenolic fraction of virgin olive oil influences both its quality and oxidative stability. One of the principal threats of the quality of olive fruit is the olive fly ( Bactrocera oleae) as it alters the chemical composition. The attack of this olive pest has been studied in order to evaluate its influence on the quality of virgin olive oil (free acidity, peroxide value, fatty acid composition, water content, oxidative stability, phenols, and antioxidant power of phenolic fraction). The study was performed using several virgin olive oils obtained from olives with different degrees of fly infestation. They were acquired in different Italian industrial mills from the Abruzzo region. Qualitative and quantitative analyses of phenolic profiles were performed by capillary electrophoresis-diode array detection, and electrochemical evaluation of the antioxidant power of the phenolic fraction was also carried out. These analyses demonstrated that the degree of fly attack was positively correlated with free acidity ( r = 0.77, p < 0.05) and oxidized products ( r = 0.58, p < 0.05), and negatively related to the oxidative stability index ( r = -0.54, p < 0.05) and phenolic content ( r = -0.50, p < 0.05), mainly with secoiridoid compounds. However, it has been confirmed that the phenolic fraction of olive oil depends on several parameters and that a clear correlation does not exist between the percentages of fly attack and phenolic content.