FEMS Microbiol. Ecology

RESEARCH ARTICLE Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes Rosella Muresu1, Elisa Polone2, Leonardo Sulas1, Barbara Baldan3, Alessandra Tondello3, Giuseppe Delogu4, Piero Cappuccinelli4, Sara Alberghini2, Yacine Benhizia5, Hayet Benhizia6, Ammar Benguedouar5, Bruno Mori7, Roberto Calamassi7, Frank B. Dazzo8 & Andrea Squartini2 1C.N.R., Sezione di Sassari, c/o Dipartimento di Scienze Agronomiche e Genetica Vegetale Agraria, Universita` degli Studi di Sassari, Sassari, Italy; 2Dipartimento di Biotecnologie Agrarie, Universita` di Padova, Padova, Italy; 3Dipartimento di Biologia, Universita` di Padova, Padova, Italy; 4Dipartimento di Scienze Biomediche, Sassari, Italy; 5Laboratoire des Biotechnologies, Departement´ des Sciences de la Nature et de la Vie, Universite´ Mentouri, Constantine, Algeria; 6Laboratoire de Gen´ etique,´ Biochimie et Biotechnologies Veg´ etales,´ Departement´ des Sciences de la Nature et de la Vie, Universite´ Mentouri, Constantine, Algeria; 7Dipartimento di Biologia Vegetale, Universita` di Firenze, Firenze, Italy; and 8Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA Downloaded from Correspondence: Andrea Squartini, Abstract Dipartimento di Biotecnologie Agrarie, Universita` di Padova, Viale dell’Universita` 16, A previous analysis showed that Gammaproteobacteria could be the sole recover- 35020 Legnaro, Padova, Italy. Tel.: able bacteria from surface-sterilized nodules of three wild species of Hedysarum.In http://femsec.oxfordjournals.org/ 1390498272923; fax: 1390498272929; this study we extended the analysis to eight Mediterranean native, uninoculated e-mail: [email protected] legumes never previously investigated regarding their root-nodule microsym- bionts. The structural organization of the nodules was studied by light and Received 12 December 2006; revised 1 October electron microscopy, and their bacterial occupants were assessed by combined 2007; accepted 3 November 2007. cultural and molecular approaches. On examination of 100 field-collected nodules, First published online 11 January 2008. culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates DOI:10.1111/j.1574-6941.2007.00424.x could nodulate the original host when reinoculated in gnotobiotic culture. Despite by guest on November 20, 2015 Editor: Kornelia Smalla 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 Keywords these nodules contained rhizobia as the predominant population. The presence of rhizobia; endophytes; wild legumes; nodular endophytes colocalized with rhizobia was verified by immunofluorescence nonculturable. 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. light of these findings, and various studies have coupled the Introduction visual approach with the molecular characterization of The nitrogen-fixing symbiosis between plants of the family symbionts (Chen et al., 2005; Elliott et al., 2007). The Leguminosae and prokaryotic partners is typically character- traditional strategy used to investigate nodule-associated ized by the formation of root or stem nodules that are microbial symbionts involves their isolation and cultivation induced and subsequently invaded by the specific micro- from internal tissues of surface-sterilized nodules (Vincent, symbionts. These include the well-known alphaproteobac- 1970). The description of symbiotic partnerships for the terial group of Rhizobiaceae containing the genera various legumes has therefore traditionally relied, as a Rhizobium, Bradyrhizobium, Sinorhizobium (Ensifer), Meso- starting point, on the culturability of the bacterial occupant rhizobium, Azorhizobium, and Allorhizobium, along with within the nodule when streaked on yeast–mannitol-based other taxa such as Methylobacterium (Sy et al., 2001) and agar plates. Despite the physiological transformation of Devosia (Rivas et al., 2002), and members of the Betaproteo- vegetative bacteria into nondividing bacteroids, it is nor- bacteria such as Burkholderia (Moulin et al., 2001) and mally observed that rhizobia are regularly cultured from Ralstonia (Chen et al., 2001). The support of microscopy to surface-disinfected crushed nodules. This implies either that examine nodule symbioses has gained new importance in some vegetative rods (still confined or recently released from FEMS Microbiol Ecol 63 (2008) 383–400 c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 384 R. Muresu et al. infection threads) have not undergone the bacteroid con- fixation. Philipson & Blair (1957) found diverse species, version (Paau et al., 1980; Timmers et al., 2000), or that including Gram-positive bacteria, in roots and nodules of some bacteroids can be resuscitated back to culturable state, healthy red and subterranean clover plants. Sturz et al. (1997) or both of these possibilities. showed that rhizobia recovery from red clover nodule tissue The vast majority of studies that have detected the taxa so could yield up to 4.3 Â 109 CFUgÀ1 fresh weight, but that, at far assigned have, however, dealt with cultivated species of the same time, 3.0 Â 105 CFUgÀ1 of nonrhizobial endo- legumes or with man-exploited natural plants. In contrast, phytes, belonging to 12 different species, could be cultured little attention has been paid to the root-nodule symbionts from the same nodules. Agrobacterium sp. has been reported of truly wild legumes, that is, those whose ecology is not in nodules of tropical legumes (De Lajudie et al., 1999). In directly affected by human action. As the Leguminosae bean nodules, Mhamdi et al. (2005) found, along with family includes over 18 000 species (http://www.ildis.org/ Rhizobium, Agrobacterium-like bacteria, and proved that Leguminosae/), only a minor portion has been examined these could invade new nodules upon coinoculation with (mostly representing crops of agricultural interest), and rhizobia and affect their nodulation performance (Mrabet knowledge of the biological diversity of interactions between et al., 2006). Actinobacteria such as Streptomyces lydicus have legumes and microorganisms is still very limited. We been reported to colonize pea nodules (Tokala et al., 2002). previously addressed this issue by describing the unusual Furthermore, Bai et al. (2003) showed that Bacillus subtilis Downloaded from diversity of Gammaproteobacteria as the sole culturable and Bacillus thuringiensis can naturally coinhabit soybean nodule occupants within three wild Hedysarum species nodules along with Bradyrhizobium japonicum, and that collected in various locations in Algeria (Benhizia et al., these Gram-positive bacteria can enhance plant productivity 2004a, b). In those studies, rhizobia could not be cultured in coinoculation experiments. A more recent report (Zakhia from any of the 52 nodules examined, leading to the et al., 2006) described the association of 14 bacterial genera http://femsec.oxfordjournals.org/ hypothesis that some Gammaproteobacteria may represent with wild legume nodules in Tunisia. an alternative endosymbiotic partner to rhizobia for these In order to gain a better understanding of the incidence nodulated plants. and diversity of natural legume–endophyte associations, we In addition to the theories and research on Rhizobium– examined the microbial occupants inside nodules and other legume interactions, a parallel and rarely converging field of plant tissues of 11 wild legume species collected in Sardinia knowledge is that of microbial endophytes. Evidence that (Italy) and Algeria, using both the standard colony isolation the healthy plant interior can normally contain bacteria or method and a direct PCR amplification of prokaryotic DNA fungi not necessarily related to a pathogenic context was first from nodules and other tissues. In parallel, microscopy- put forward by Perotti (1926), subsequently revisited by Old based approaches were undertaken to document the micro- by guest on November 20, 2015 & Nicolson (1978), and is now well documented by many bial colonization within these legume tissues. studies that have been reviewed over the years (Hallmann et al., 1997; Sattelmacher, 2001). The majority of reports Materials and methods deal with culturable endophytes. Stems and roots of most plant species tested harbour a range of 103–106 live internal Plant collection and nodule examination bacteria per gram of fresh weight, whose roles are related to various interactive phenotypes. Legumes are in this respect An extensive search was conducted to locate plants of interest no exception: Gagne´ et al. (1987) found bacteria in the in suitable biotopes within Sardinia during the springs of xylem of alfalfa roots; and Sturz et al. (1998) found 22 2004 and 2005. The legumes sampled, the nearest urban species within red clover. Sturz et al. (1997) showed that red settlement, the geographical coordinates of the sampling site, clover harbours rhizobia of different species (not limited to and the number of nodules collected and analysed were as the endosymbiont, Rhizobium leguminosarum bv. trifolii) follows: Hedysarum spinosissimum (Gio`scari, 401420N, not just in nodules but systemic

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