In Situ Localization and Strain-Specific Quantification of Azospirillum and Other Diazotrophic Plant Growth-Promoting Rhizobacteria Using Antibodies and Molecular Probes

Abstract

A central issue in the understanding of the interaction and symbiotic function of diazotrophic bacteria with non-leguminous crop plants is detailed knowledge about the localization of the associated diazotrophic bacteria within the plant, their in situ activities in the plant-associated niches, and strain-specific quantification of inoculated bacteria. In addition to the colonization of rhizosphere soil and the rhizoplane, it has become apparent that an endophytic location of a diazotroph would provide it with a higher potential to interact more closely with the plant, particularly with respect to increasing the availability of carbon and energy nutrients derived from the plant, as well as the possibility, in return, of improving the transfer of bacterial-derived metabolites to the plant. Detailed localization of bacteria was successfully performed using fluorescence labeled ribosome-directed oligonucleotide probes in the fluorescence in situ hybridization (FISH) approach coupled to the use of confocal laser scanning microscopy (CLSM), and via immunolocalization with specific antibodies using transmission electron microscopy (TEM). Furthermore, the fate of inoculated bacteria could be traced by using specifically marked strains by applying the genes for the green or red fluorescent protein (GFP, RFP) and β-glucuronidase (GUS). Strain-specific quantification approaches for inoculants based on quantitative PCR using sequence characterized amplified regions (SCARs) and other genomic marker sequences have been developed and successfully applied. In this chapter major achievements and existing obstacles using these high resolution approaches to analyze bacteria in situ are presented together with some basic protocols.