Detection and quantification of native microbial populations on soil-grown rice roots by catalyzed reporter deposition-fluorescence in situ hybridization

Abstract

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) was applied to detect microbial cells on the rhizoplane of wetland rice (Oryza sativa L.). Fluorescent signals of high intensity and specificity allowed for a reliable quantification of selected microbial phyla. Absolute cell numbers of archaea and bacteria were observed to be highest at flowering stage of rice plant development (P < 0.05) showing values of 1.32 and 6.26 × 104 cells mm-2 rhizoplane, respectively. Highest colonization densities shifted from the root tip toward more mature regions with increasing plant age. Significant differences between cell numbers observed within a short distance (0-15 mm) indicated irregular distribution patterns of microbiota. Root tips, elongation zones, and openings at the base of lateral roots represented preferential areas for microbial colonization, which were often covered with iron coatings and densely colonized with potential iron-oxidizing Betaproteobacteria (59% of bacteria). Methanogenic archaea were abundant on the rhizoplane (up to 0.96 × 103 cells mm-2 rhizoplane), and the decline of their relative abundance with plant age was also found in the associated rhizosphere soil. Cell numbers of methanotrophic bacteria significantly increased at flowering (6.38 × 103 cells mm-2 rhizoplane; P < 0.05), indicating their stimulation by root-derived substrates which was less pronounced in the rhizosphere soil. A protocol is presented that allowed the routine localization, visualization, and quantification of spatial distribution patterns of microbial phyla associated with the rhizoplane of soil-grown rice roots in situ. © 2014 Federation of European Microbiological Societies.