Pasteuria penetrans is a Gram-positive endospore-producing bacterium that is a parasite of root-knot nematodes. Attachment of endospores to the cuticle of the nematode is the first stage in the infection process. Western blot analysis with monoclonal and polyclonal antibodies that recognize the 30 kDa heparin-binding domain (HBD) and the 45 kDa gelatin-binding domain (GBD) fragments of human fibronectin (Fn) revealed a series of polypeptides of approximately 40, 45 and 55 kDa present in crude cuticle extracts of Meloidogyne javanica 2nd-stage juveniles. The results suggest that the structure of the nematode fibronectin is different to the fibronectins so far characterized. Pre-treatment of endospores of Pasteuria with either the HBD or the GBD was found to inhibit binding to the nematode cuticle. The larger GBD fragment was the most effective at blocking adhesion. Pre-treatment of the GBD fragment with gelatin prevented the GBD fragment from inhibiting endospore attachment to the nematode cuticle.
Pasteuria penetrans isolates sampled from different geographical areas were characterised both for the heterogeneity of the endospore surface using monoclonal antibodies and for the ability of spores to attach to different isolates of Meloidogyne spp. The efficacy of these different Pasteuria isolates as biological control agents was tested in a glasshouse experiment with M. incognita from Senegal on Acacia holosericea. The immunoprofiles divided the P. penetrans isolates broadly differently from the attachment tests. Isolate PP16 from Senegal was associated with better seedling development of M. incognita-inoculated A. holoceria than were other isolates. Substantial variation in root and shoot biomass was not related to the observed variation in spore attachment tests. The difficulties involved in obtaining consistent biological control with Pasteuria are discussed in relation to the high degree of variability of this bacterium
Currently, the abundance of Pasteuria penetrans in soils, an unculturable bacterial parasite of root-knot nematodes (Meloidogyne spp,). is estimated by the percentage of nematode juveniles infected with bacteria and the number of spores attached to their cuticle. Indirect immunofluorescence led to detection of free spores directly in soil suspensions using UV light and polyclonal antibodies raised against two P. penetrans populations (ORS-21414-Sen and PPI). Three extraction methods were compared in order to improve spore recovery. A gentle shaking/sieving method recovered more than 90% of the spores inoculated in soils and was more efficient and simple than aqueous two-phase partitioning and polyethylene glycol extractions. All the spores inoculated in sandy or sandy-clay soils were detected with immunofluorescence microscopy. The quantification of the spores was improved using an ELISA technique that showed a good correlation between optical density and spore concentration in inoculated soils. Specific antibodies provide a suitable method to quantify A penetrans and may be used to follow the evolution of the real pool of bacteria either in native suppressive soils or in inoculated ones. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved
Currently, the abundance of Pasteuria penetrans in soils, an unculturable bacterial parasite of root-knot nematodes (Meloidogyne spp.), is estimated by the percentage of nematode juveniles infected with bacteria and the number of spores attached to their cuticle. Indirect immunofluorescence led to detection of free spores directly in soil suspensions using UV light and polyclonal antibodies raised against two P. penetrans populations (ORS-21414-Sen and PP1). Three extraction methods were compared in order to improve spore recovery. A gentle shaking/sieving method recovered more than 90% of the spores inoculated in soils and was more efficient and simple than aqueous twophase partitioning and polyethylene glycol extractions. All the spores inoculated in sandy or sandy^clay soils were detected with immunofluorescence microscopy. The quantification of the spores was improved using an ELISA technique that showed a good correlation between optical density and spore concentration in inoculated soils. Specific antibodies provide a suitable method to quantify P. penetrans and may be used to follow the evolution of the real pool of bacteria either in native suppressive soils or in inoculated ones. ß
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