Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

An immunocytochemical procedure for protein localization in various nematode life stages combined with plant tissues using methylacrylate-embedded specimens

Plant-parasitic nematodes possess a large number of proteins that are secreted in planta, allowing them to be successful parasites of plants. The majority of these proteins are synthesized mainly in the nematode subventral and dorsal glands as well as in other organs. To improve the immunovisualization of these proteins, we adapted a methacrylate embedding method for the localization of proteins inside nematode tissues, and extracellularly when secreted in planta or within plant cells. An important advantage is that the method is applicable for all nematode stages: preparasitic as well as parasitic stages, including large mature females. Herein, the method has been successfully applied for the localization of four nematode secreted proteins, such as Mi-MAP-1, Mi-CBM2-bearing proteins, Mi-PEL3, and Mi-6D4. In addition, we could also localize 14-3-3 proteins, as well as two cytoskeletal proteins, by double-immunolabeling on preparasitic juveniles. Superior preservation of nematode and plant morphology, allowed more accurate protein localization as compared with other methods. Besides excellent epitope preservation, dissolution of methacrylate from tissue sections unmasks target proteins and thereby drastically increases antibody access.     

Comparison of the anthocyanin composition during ripening of Syrah grapes grown using organic or conventional agricultural practices

The anthocyanin composition of Syrah grapes harvested at different stages of ripening and produced using organic or conventional agriculture was studied. Samples of grapes were collected from veraison to full maturity in each plot, and the content in nine anthocyanins was determined by high-performance liquid chromatography with diode array detection. The total content in anthocyanins during ripening of the conventionally grown grapes was significantly higher compared to that found in the organic production. The accumulation of anthocyanins reached a maximum 28 days after veraison (in agreement with high temperature) and then decreased until harvest. In all samples, grapes from the conventional agriculture presented higher proportions of delphinidin, petunidin, malvidin, and acylated malvidin glucosides compared to grapes from organic agriculture. In contrast with other comparative studies of organically and conventionally grown plants, the results demonstrated a higher content in anthocyanins in conventionally grown grapes.     

Kinetics of formation and functional properties of conjugates prepared by dry-state incubation of beta-lactoglobulin/acacia gum electrostatic complexes

The formation of conjugates between beta-lactoglobulin and acacia gum based on electrostatic complexes formed at pH 4.2 was investigated upon dry-state incubation for up to 14 days at 60 degrees C and 79% relative humidity (RH). By means of SEC-HPLC and RP-HPLC, it was shown that the beta-lactoglobulin incubated alone was able to form polymers with molecular masses higher than 200 kDa until 50% of the initial monomeric protein disappeared after 14 days. In the presence of acacia gum at initial protein to polysaccharide weight mixing ratios of 2:1 and 1:2, only 35% of the initial beta-lactoglobulin monomers disappeared after 14 days. Using RP-HPLC, an apparent reaction order of 2 was found for the disappearance of monomeric beta-lactoglobulin both in the presence or absence of acacia gum. However, the reaction rate was faster in the absence of acacia gum. SDS-PAGE electrophoresis with silver staining confirmed the formation of beta-lactoglobulin/acacia gum conjugates. The solubility curves of the incubated beta-lactoglobulin showed a minimum around pH 4-5. By contrast, the minimum of solubility of the beta-lactoglobulin/acacia gum incubated mixtures shifted to lower pH values compared to initial mixtures. The conjugates exhibited higher foam capacity than the incubated protein as well as lower equilibrium air/water surface tension. Conjugation at ratio 1:2 led to increased interfacial viscosity (300 mN s m(-1) at 0.01 Hz) compared to beta-lactoglobulin alone (100 mN s m(-1) at 0.01 Hz), but similar interfacial elasticity (30-40 mN m(-1)). The foam capacity of the conjugates was significantly higher than that of the incubated beta-lactoglobulin as well as foam expansion and drainage time, especially at pH 5.3, i.e., higher than the pH of formation of the conjugates.     

Toward metrological traceability for DNA fragment ratios in GM quantification. 2. Systematic study of parameters influencing the quantitative determination of MON 810 corn by real-time PCR

This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.     

Are Phenological Observations Sufficient to Estimate the Quality of Seed Crops from a <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> Open-pollinated Seed Orchard? Consequences for Seed Collections

Reproductive phenology was observed over three years in a Eucalyptus grandis seed orchard in Madagascar to determine the impact of geographic differences of parental selections on the expected genetic composition of seed crops. Pollen flow (observed pollen cloud) was deduced from a paternity assignment based on sampling from one year’s seed production. The two approaches were used to verify whether knowledge of phenology is sufficient to predict the genetic quality of the seeds collected. Despite the high flowering level, with a constant cycle over three years, the results demonstrated wide reproductive phenological differences associated with the parents’ origin, suggesting putative pollination disequilibrium. From both observed and expected pollen clouds, the results showed preferential mating among different provenances, which has consequences for seed crop composition. Phenological observations, which can be made inexpensively, can be used to promote methods of effective seed orchard management in order to improve the genetic quality of seeds.     

The effect of water movement on the transport of dicyandiamide,ammonium and urea in unsaturated soils

A laboratory experiment was conducted to investigate the relative mobility of dicyandiamide (DCD) and jointly applied ammoniacal salts or urea in three different soils of lower Egypt, and to determine the extent to which DCD separates from N-fertilizer in unsaturated soil undergoing leaching. The experimental results suggest that, under conditions of water flow, DCD is readily separated from NH₄⁺ but parts from urea to a far lesser extent. The large difference in mobility between DCD and NH₄⁺ should severely limit the effectiveness of DCD as a nitrification inhibitor in the three soils considered when applied in conjunction with ammoniacal salts. In two out of three cases, the situation is similarly unfavorable in the case of joint DCD and urea application. However, the observation that DCD, in a low CEC sandy loam, moves within the soil solution at a slightly lower rate than urea suggests that joint application with urea would keep at least part of the DCD in contact with the NH₄⁺ ions and, therefore, would preserve some of the effectiveness of DCD under leaching conditions in this soil.     

Characterization using Raman microspectroscopy of arabinoxylans in the walls of different cell types during the development of wheat endosperm

The time course and pattern of arabinoxylan deposition in the wheat (Triticum aestivum) endosperm during grain development were studied using Raman spectroscopy. The presence of arabinoxylans (AX) is detected at the beginning of grain filling. At this stage, AX appear more substituted than at the later stages. Feruloylation of AX increases during the grain-filling stage, especially in the case of the aleurone layer. Whatever the stage of grain development, four populations of cells could be defined according to Raman arabinoxylan signatures. In the walls of the aleurone cells, AX appeared to be little substituted and highly esterified with phenolic acids. In the walls of prismatic cells, AX were found to be highly substituted and poorly esterified. Apart from aleurone and prismatic cells, the substitution degree of AX in endosperm was in the same range. Cells in the crease region were distinguished from cells in the starchy endosperm by their lower amount of esterified phenolic compounds.     

Pesticide residues in heterogeneous plant populations, a model-based approach applied to nematicides in banana (Musa spp.)

Nematicides are widely used to control plant-parasitic nematodes in intensive export banana (Musa spp.) cropping systems. Data show that the concentration of fosthiazate in banana fruits varies from zero to 0.035 g kg-1, under the maximal residue limit (MRL=0.05 mg kg-1). The fosthiazate concentration in fruit is described by a Gaussian envelope curve function of the interval between pesticide application and fruit harvest (preharvest interval). The heterogeneity of phenological stages in a banana population increases over time, and thus the preharvest interval of fruits harvested after a pesticide application varies over time. A phenological model was used to simulate the long-term harvest dynamics of banana at field scale. Simulations show that the mean fosthiazate concentration in fruits varies according to nematicide application program, climate (temperature), and planting date of the banana field. This method is used to assess the percentage of harvested bunches that exceed a residue threshold and to help farmers minimize fosthiazate residues in bananas.