Simultaneous detection of the establishment of seed-inoculated Pseudomonasfluorescens strain DR54 and native soil bacteria on sugar beet root surfaces using fluorescence antibody and in situ hybridization techniques

Colonization at sugar beet root surfaces by seedling-inoculated biocontrol strain Pseudomonas fluorescens DR54 and native soil bacteria was followed o ver a period of 3 weeks using a combination of immunofluorescence (DR54-tar geting specific antibody) and fluorescence in situ hybridization (rRNA-targ eting Eubacteria EUB338 probe) techniques with confocal laser scanning micr oscopy. The dual staining protocol allowed cellular activity (ribosomal num ber) to be recorded in both single cells and microcolonies of strain DR54 d uring establishment on the root. After 2 days, the population density of st rain DR54 reached a constant level at the root basis. From this time, howev er. high cellular activity was only found in few bacteria located as single cells, whereas all microcolony-forming cells occurring in aggregates were still active. In contrast, a low density of strain DR54 was observed at the root tip, but here many of the bacteria located as single cells were activ e. The native population of soil bacteria, comprising a diverse assembly of morphologically different forms and size classes, initiated colonization a t the root basis only after 2 days of incubation. Hence the dual staining p rotocol allowed direct microscopic studies of early root colonization by bo th inoculant and native soil bacteria, including their differentiation into active and non-active cells and into single or microcolony-forming cells. (C) 2000 Federation of European Microbiological Societies. Published by Els evier Science B.V. All rights reserved.