A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.

ABSTRACT

A simple method to enhance the staining of cell wall components for fluorescence microscopy is described. In stems of Nicotiana tabacum and needles of Pinus eldarica lignin, the cuticle and unsaturated lipids are indicated by a purple-red fluorescence while pectocellulosic components fluoresce pale blue.

The manner in which the bacterium Pseudomonas savastanoi pv. savastanoi ( Pss ), the causal agent of knot disease, infects olive plants is erratic and has not been fully documented. To investigate the process of Pss invasion, olive explants were inoculated in vitro and examined visually and by light microscopy at 2-weekly intervals for 10 weeks. In all host genotypes tested, interaction with the pathogen resulted in: (i) a progressive collapse of the stem, originating at the inoculation site at the apex of the explant, and proceeding downwards; and (ii), localized outgrowths on the stem located at various distances from the inoculation site. Histological analysis revealed that the anatomy of the outgrowths closely resembled that of knots formed in vivo ; they showed that Ps. savastanoi also diffused within the olive explants through the xylem vessels, and that the olive host reacted to pathogen invasion, possibly by producing substances of polysaccharidic and/or phenolic nature.