In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

Abstract

The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar-or soil-grown 10-and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4,6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.

Investigations of Rhizobium biofilm formation

“…This may have resulted from a depletion of carbon in the soil substrates or alternatively, from a decrease in cultivability of the bacterial cells over time. Rhizospheres, although rich in carbon compounds from the activity of roots and associated microbes (Foster et al, 1983), often contain slow-growing microbes that accumulate few ribosomes (Assmus et al, 1995). Because no plant tissues or sources of carbon were included in the buried slide assays, bacteria inoculated into these conditions are not likely to be actively dividing over the course of the experiment, and hence could have become recalcitrant to being cultured.…”

CELL‐CELL INTERACTION IN THE EUKARYOTE‐PROKARYOTE MODEL OF THE MICROALGAE CHLORELLA VULGARIS AND THE BACTERIUM AZOSPIRILLUM BRASILENSE IMMOBILIZED IN POLYMER BEADS1

“…In situ hybridization. This assay was based on the technique described by Assmus et al (1995), with numerous small modifications. Hybridization was performed at 35% formamide stringency at 46°C for 2 h. The final concentration of the probe was 3 ng AE lL )1 .…”

Numbers and locations of native bacteria on field‐grown wheat roots quantified by fluorescence in situ hybridization (FISH)

“…To date, FISH has been applied to roots grown in media and soil in controlled environments (e.g. Assmus et al ., 1995;Eller and Frenzel, 2001;Briones et al ., 2002), and both native and applied organisms have been quantified either directly on the roots or on rhizosphere biofilm and soil isolated from roots. In this study we quantify and localize bacteria in general, and Pseudomonas in particular, on wheat roots growing in the field.…”