title: Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes.
abstract: 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.
authors.affiliation: Dipartimento di Scienze Agronomiche e Genetica Vegetale Agraria, Università degli Studi di Sassari, Sassari, Italy.
authors.forename: Rosella
authors.initials: R
authors.lastname: Muresu
authors.forename: Elisa
authors.initials: E
authors.lastname: Polone
authors.forename: Leonardo
authors.initials: L
authors.lastname: Sulas
authors.forename: Barbara
authors.initials: B
authors.lastname: Baldan
authors.forename: Alessandra
authors.initials: A
authors.lastname: Tondello
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Delogu
authors.forename: Piero
authors.initials: P
authors.lastname: Cappuccinelli
authors.forename: Sara
authors.initials: S
authors.lastname: Alberghini
authors.forename: Yacine
authors.initials: Y
authors.lastname: Benhizia
authors.forename: Hayet
authors.initials: H
authors.lastname: Benhizia
authors.forename: Ammar
authors.initials: A
authors.lastname: Benguedouar
authors.forename: Bruno
authors.initials: B
authors.lastname: Mori
authors.forename: Roberto
authors.initials: R
authors.lastname: Calamassi
authors.forename: Frank B
authors.initials: FB
authors.lastname: Dazzo
authors.forename: Andrea
authors.initials: A
authors.lastname: Squartini
mesh_terms: D000462:Algeria; D001419:Bacteria / Q000145:classification* / Q000254:growth & development*; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D004754:Enterobacter / Q000254:growth & development; D007887:Fabaceae / Q000145:classification / Q000382:microbiology*; D007558:Italy; D008856:Microscopy, Fluorescence; D009586:Nitrogen Fixation; D018517:Plant Roots / Q000382:microbiology* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012230:Rhizobiaceae / Q000145:classification / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D013559:Symbiosis
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S