Détection par piégeage du Ceratocystis fimbriata f. platani

Application of the technique of trapping Ceratocystis fimbriata f. platani for the study of the persistance in affected trees . The study of the persistance of Ceratocystis fimbriata f. platani in affected trees is achieved by trapping of the fungus which is carried out on small branches of London plane, stripped of their bark. If C. fimbriata is present in samples of wood to be tested, it becomes visible after a few days when numerous perithecia form on the trap. Trapping the fungus by this method has been systematically achieved with young, artificially inoculated trees: in many cases, C. fimbriata persists in the wood after a period of about 19 to 22 months.     

Assessment of PCR-based tools for the specific identification of some temperate Meloidogyne species including M. chitwoodi,M. fallax and M. minor

Several conventional PCR tests have been developed for the identification of the European quarantine root-knot nematodes Meloidogyne chitwoodi and M. fallax but data are lacking for the evaluation of their performance in terms of sensitivity, repeatability, reproducibility and specificity against a large range of populations. This study evaluated the performance criteria of three conventional PCR tests recommended by the consensus diagnostic protocol for Meloidogyne chitwoodi and Meloidogyne fallax published by the European and Mediterranean Plant Protection Organization (EPPO): a species-specific PCR (IGS target), a SCAR PCR, and a rDNA ITS PCR-RFLP. Evaluation was carried out with DNA extracts from juveniles, males and females according to EPPO recommendations for test validation. A minimum of 34 populations of target and non target nematode species were tested to check the specificity of these three PCR assays. The three PCR tests were ranked according to their specificity (with regard to cross reaction with other nematodes species or genus) and their sensitivity (detection of a single juvenile or mixed with other species). The species-specific PCR proved to be more sensitive but less specific than the SCAR PCR. The PCR-RFLP enables the identification of several Meloidogyne species but profile analysis can be difficult when several species are present in the mixture. Specific PCR products and RFLP profiles were also observed for M. arenaria and M. enterolobii, and described for M. minor and M. artiellia.     

Degradation of [14C]Terbuthylazine and [14C]Atrazine in Laboratory Soil Microcosms

The degradation and formation of major chlorinated metabolites of terbuthylazine and atrazine in three soils (loamy clay, calcareous clay and high clay) were studied in laboratory experiments using molecules labelled with ¹⁴C on the s-triazine ring. Soil microcosms were treated with the equivalent of 1 kg ha^-1 of herbicide and incubated in the dark for 45 days at 20(±1)°C. The quantity of [¹⁴C]carbon dioxide evolved in the soils treated with atrazine was negligible and could not be attributed to mineralization of the parent molecule. The mineralization of terbuthylazine accounted for 0·9–1·2% of the initial radioactivity. In the soils studied, the extrapolated half-lives varied from 88 to 116 days for terbuthylazine and 66 to 105 days for atrazine, with no significant differences for the three soils and the two molecules. The deethyl metabolites of the two s-triazines and the deisopropyl-atrazine metabolite appeared during the incubation in the three soils. The completely dealkylated metabolite was not detected in any of the soils. After 45 days of incubation, the non-extractable soil residues for the high clay, loamy clay and calcareous clay soils represented for terbuthylazine, 33·5, 38·3 and 43·1% and for atrazine, 19·8, 20·8 and 22·3% of the initial radioactivity. © 1997 SCI.     

Simultaneous and selective detection of two major soft rot pathogens of potato: Pectobacterium atrosepticum (Erwinia carotovora subsp. atrosepticum) and Dickeya spp. (Erwinia chrysanthemi)

Dickeya spp. and Pectobacterium atrosepticum are major pathogens of potato. Current methods to detect these soft-rotting bacteria require separate identification steps. Here we describe a simple method allowing simultaneous detection of both pathogens based on multiplex PCR. The sensitivity of the primer sets was first examined on purified genomic DNA of the type strains Dickeya chrysanthemi 2048^T and P. atrosepticum 1526^T. The specificity and detection limits of the primer sets were successfully tested on 61 strains belonging to various Dickeya and Pectobacterium species, on artificially inoculated and on naturally contaminated potato plants. This new method provides a gain in time and materials, the main advantages for large-scale processes such as pathogen-free seed certification.     

Effect of Mixing Conditions on the Behavior of Lipoxygenase,Peroxidase, and Catalase in Wheat Flour Doughs

The effect of mixing has been tested on the extractable activities of lipoxygenase, peroxidase, and catalase from dough after 2, 5, and 20 min of mixing, and 30 min of rest period after 20 min of mixing. Different mixing conditions have been studied including temperature, atmosphere, speed, amount of water added to the dough, buffer solutions between pH 3.6 and 7.5 added to the dough, and different additives (linoleic acid, guaiacol, hydrogen peroxide, ascorbic acid, cysteine, yeast, and sodium chloride). In all the mixing conditions tested, the dough peroxidase activity remains equivalent to the initial flour activity, whereas losses in lipoxygenase and catalase activities largely varied according to mixing conditions. The results show that a self-destruction mechanism as well as physicochemical denaturation are responsible for these losses. Lipoxygenase losses seem mainly associated with the former mechanism, whereas catalase losses are highly increased in acidic conditions (physicochemical denaturation). Therefore, the relative impact of the three oxidoreducing enzymes may be largely modulated by mixing conditions.     

Topography of the casein micelle surface by surface plasmon resonance (SPR) using a selection of specific monoclonal antibodies

Several theoretical models of the casein micelle structure have been proposed in the past, but the exact organization of the four individual caseins (α(s1), α(s2), β, and κ) within this supramolecular structure remains unknown. The present study aims at determining the topography of the casein micelle surface by following the interaction between 44 monoclonal antibodies specific for different epitopes of α(s1)-, α(s2)-, β-, and κ-casein and the casein micelle in real time and no labeling using a surface plasmon resonance (SPR)-based biosensor. Although the four individual caseins were found to be accessible for antibody binding, data confirmed that the C-terminal extremity of κ-casein was highly accessible and located at the periphery of the structure. When casein micelles were submitted to proteolysis, the C-terminal extremity of κ-casein was rapidly hydrolyzed. Disintegration of the micellar structure resulted in an increased access for antibodies to hydrophobic areas of α(s1)- and α(s2)-casein.     

Characterization and quantification of proteins in lecithins

Several methods for extraction and quantification of proteins from lecithins were compared. Extraction with hexane-2-propanol-water followed by amino acid analysis is the most suitable method for isolation and quantification of proteins from lecithins. The detection limit of the method is 15 mg protein/kg lecithin, and the quantification limit is 50 mg protein/kg. The relative repeatability limits for samples containing 0-500 and 500-5000 mg protein/kg sample were 12.6 and 7.5%, respectively. The protein recovery ranged between 101 and 123%. The protein content has been determined in different kinds of lecithins. The results were as follows: standard soy lecithins (between 232 and 1338 mg/kg), deoiled soy lecithin (342 mg/kg), phosphatydylcholine-enriched soy lecithins (not detectable and 163 mg/kg), sunflower lecithins (892 and 414 mg/kg), and egg lecithin (50 mg/kg). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns of the standard soy and sunflower lecithins are very similar to those of soy flour. The protein profile of the egg lecithin shows several bands with a broad range of molecular masses. The molecular masses of the main proteins of soy lecithins and soy flour have been determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and ranged from 10.5 to 52.2 kDa. Most of the major proteins from soy and sunflower lecithins identified by MALDI-MS and electrospray tandem MS belong to the 11S globulin fraction, which is one of the main fractions of soy and sunflower seeds. In addition, the seed maturation protein P34 from the 7S globulin fraction of soy proteins has also been identified in soy lecithins. This protein has been reported as the most allergenic protein in soybean.     

Genetically encoded sensors for ions and metabolites

Many of the genes encoding important trans porters and metabolic enzymes have been identified over the last ten years. Moreover it has been possible to study the biochemical properties of the corresponding proteins in great detail. It is expected that by 2,010 biochemical functions will have been assigned to many of the products of the approximately 30,000 Arabidopsis genes. We will get closer to understanding the biological function of the gene products by systematic analysis of mutants using knock-out and TILLING approaches. Metabolomics initiatives complement these approaches by providing insight into the changes in cellular ion and metabolite profiles in the mutants, thus giving information essential for the construction of cellular and whole plant models. However, one important dataset especially relevant to multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of ions and metabolite levels at cellular and subcell ular levels. To address this issue, we have developed protein-based nanosensors for several metabolites, providing a set of tools for the determination of cytosolic and subcellular ion (e.g. iron and zinc) and metabolite levels in real time using fluorescence-based microscopy. The prototypes of these sensors were shown to function in vitro and also in vivo, i.e. in yeast and in mammalian cell cultures. One future goal is to expand the set of sensors to a wider spectrum of targets by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets. Application of nanosensor technology to plant cells and tissues will help to elucidate the special and temporal distribution of ions and metabolites.     

A new equation to simulate the contact between soil and maize residues of different sizes during their decomposition

The availability of soil nutrients, which are recycled through the decomposition of crop residues, is important for the management of cropped soils. However, knowledge regarding the influence of contact between crop residues and soil on the dynamics of carbon (C) and nitrogen (N) in soil is limited. In particular, the effect of particle size on decomposition is not well-known, and conceptual approaches for modelling the soil-residue contact in a decomposition model remain scarce. Therefore, we analysed and modelled the effect of maize stem particle length on decomposition. We incubated maize stem residues with particle sizes of 0.02, 0.5, 2 and 5 cm in length in a loamy soil at 25 °C over 301 days. We continuously measured the mineralisation of C and N and determined the chemical evolution of the remaining particles. We used the decomposition model CANTIS which takes into account the soil-residue contact, using a contact factor, K_MZ. The decomposition rates of smaller maize particles were higher than those of larger particles during the early phases of decomposition. However, these differences were not maintained after 301 days. These results suggest that a larger size of the maize particles only slowed the rate of mineralisation in the short term but did not modify decomposition in the medium term. We proposed a new formalism for expressing the changes in soil-residue contact with different particle sizes. The contact factor K_MZ was calculated using the standardised specific surface area and can be applied more widely to residues that differ in morphology and density.     

Evaluation of sample pre-treatment and potential contamination of soluble carbon in soil extracts during lyophilisation

Lyophilisation of K₂SO₄ soil extracts has been proposed as a sample preparation technique before elemental analysis of carbon or nitrogen. However, previous measurements, based on wet oxidation or catalytic combustion, indicated that C measurements in lyophilised samples not always proved to be accurate. To determine whether the C analysis was affected by the lyophilisation process, an exploratory study was conducted to investigate the potential effects of the sample pre-treatment and of the lyophilisation process itself. This paper puts forward that the use of soil extracts, previously stored at −20 °C, may affect the recovery of salt in the samples and that contamination of the soluble carbon with exogenous C during lyophilisation is feasible. Therefore we recommend to use freshly prepared soil extracts for lyophilisation and always to include an internal standard among the unknown samples to account for a possible contamination.