Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

International audienceA Chemiluminescence Enzyme-Linked Immuno-Sorbent Assay (CL-ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti-thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC50 and the limit of detection (LOD) values were 60 ng mL-1 and 9 ng mL -1, respectively, similar to those of colorimetric ELISA with a calibration range of 9 – 15,000 ng mL-1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid-liquid extraction or the graphitized carbon-based solid phase extraction. The calibration curves in honeybee extracts from liquid-liquid procedure gave an IC50 of 141 ng mL-1 and a LOD of 17 ng mL-1. In case of extracts obtained by SPE these values were 139 ng mL-1 and 15 ng mL-1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL-1 of thiram was 72% for SPE, higher than for liquid-liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid-liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia

Fonctionnement et caracterisation immunologique des glucane synthases de Saprolegnia monoica

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Pythium F induce a minor but ubiquitous disease in tomato soilless cultures [Lycopersicon esculentum]

International audienceAn immunoenzymatic staining procedure was used to assess the level of Pythium spp. colonization on the root surface of tomato plants growing in commercial hydroponic cultures. Sampling was performed with roots free of distinct necrosis or other symptoms. It was shown that 40% of the root segments on average were colonized by Pythium spp. Hyphae formed loose or dense mycelium networks on the root epidermis. The frequent occurence of the minor pathogen, Pythium F in soiless cultures was pointed out because it represented 82% of all the Pythium spp. isolates. It was observed by ultrastructural and cytochemical studies that, under such cultural conditions, Pythium F penetrated the cortical root areas and induced stricking alterations within the walls and the cytoplasmic contents of the invaded host cells. These host cell damages led to tomato yield losses although the roots looked macroscopically healthy. The ubiquitous occurrence of Pythium F and its pathogenic abilities can be of relevance, since such a minor pathogen can have a key impact in soilless culture

Cellulose and ß-glucan synthesis in Saprolegnia

International audienc

Congo red inhibits in vitro β-glucan synthases of Saprolegnia

International audience1,3-β- and 1,4-β-glucan synthases from Saprolegnia were inhibited in vitro by Congo red. This dye was found to be a non competitive inhibitor which prevented initiation and elongation of polymer chains

Morphogenetic effects of Congo red on hyphal growth and cell wall development of the fungus Saprolegnia monoica

International audienceCultures of Saprolegnia monoica were treated with Congo red, a dye which prevents glycan microfibril assembly. In the presence of the dye, the fungus developed cell wall thickenings and exhibited aberrant hyphal tips which expanded into spherical swellings. In the thickened cell walls, cellulose was produced as short microfibrils or amorphous material. These morphological anomalies were accompanied by changes in the localization of glucose incorporation into the cell wall which was not limited to the hyphal tips. Congo red uncouples the polymerization and crystallization of cellulose into normal microfibrils and it is suggested that the regular assembly of glucan chains into microfibrils is needed for normal hyphal morphogenesis

Malondialdehyde cannot be related to lipoperoxidation in habituated sugarbeet plant cells

International audienceMalondialdehyde (TBARS: thiobarbituric acid reactive substances) has been investigated in non-organogenic and organogenic habituated and normal sugarbeet cell lines and compared with lipid hydroperoxides, conjugated diene formation and lipoxygenase activity in order to estimate the reality of lipid peroxidation processes which have been postulated in the habituated non-organogenic cell line. The results presented here exhibit a strong discrepancy between TBARS and the other indices tested. Neither hydroperoxide index, nor conjugated dienes, nor lipoxygenase activity could confirm that the non-organogenic habituated cells studied were submitted to permanent stress due to free radical attacks. Moreover, these results underlined that the use of TBARS to estimate lipid peroxidation must be considered with extreme caution in plants and can lead to misinterpretations

Dot-blot assays and their use as a direct antigen-binding method to screen monoclonal antibodies to 1,4-β-and 1,3-β-glucan synthases

International audienceA rapid method has been developed to assay β-glucan synthases spotted on a nitrocellulose sheet. The sensitivity of this method allows screening of hybridoma-making monoclonal antibodies in a direct antigen-binding assay by measurement of the activity of the enzymes retained by the antibodies previously fixed on nitrocellulose

Immuno-enzymatic staining procedure for Pythium species with filamentous non-inflated sporangia in soilless cultures

International audiencePolyclonal antibodies were prepared against crude cell wall fractions of a Pythium isolate with filamentous non-inflated sporangia, associated with root rot of tomato in soilless cultures. In ELISA tests, the antiserum showed sensitivity and specificity against original antigens and those from closely related isolates with filamentous sporangia (such as Pythium aquatile and Pythium coloratum). The antiserum reacted weakly with mycelial preparations of several fungi commonly found on tomato roots, such as Fusarium spp. or Penicillium spp. An immunocytochemical staining test was developed in order to study the colonization by filamentous non-inflated sporangial isolates of Pythium in the presence of other fungi in soilless cultures

First Report of Apple Bitter Rot Caused by Colletotrichum fioriniae in Brittany, France

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