Genetic characterization of root-nodule bacteria associated with Acacia salicina and A. stenophylla (Mimosaceae) across south-eastern Australia Mohammad S. Hoque, Linda M. Broadhurst and Peter H. Thrall Correspondence Mohammad S. Hoque Mohammad.Hoque@csiro.au CSIRO Plant Industry, PO Box 1600, Canberra ACT 2601 Australia Symbiotic relationships between legumes and nitrogen-fixing soil micro-organisms are of ecological importance in plant communities worldwide. For example, nutrient-poor Australian soils are often dominated by shrubby legumes (e.g. species of Acacia). However, relatively few studies have quantified patterns of diversity, host-specificity and effectiveness of these ecologically important plant–microbe interactions. In this study, 16S rRNA gene sequence and PCR-RFLP analyses were used to examine bacterial strains isolated from the root nodules of two widespread south-eastern Australian legumes, Acacia salicina and Acacia stenophylla, across nearly 60 sites. The results showed that there was extensive genetic diversity in microbial populations, including a broad range of novel genomic species. While previous studies have suggested that most native Australian legumes nodulate primarily with species of the genus Bradyrhizobium, our results indicate significant associations with members of other root-nodule-forming bacterial genera, including Rhizobium, Ensifer, Mesorhizobium, Burkholderia, Phyllobacterium and Devosia. Genetic analyses also revealed a diverse suite of non-nodulating bacterial endophytes, only a subset of which have been previously recorded. Although the ecological roles of these endosymbionts are not well understood, they may play both direct and indirect roles in promoting plant growth, nodulation and disease suppression. INTRODUCTION Legumes are one of the largest angiosperm groups and are broadly distributed across the globe (Polhill et al., 1981). In many regions, native legumes are important contributors to ecological community structure and function, often supporting a broad range of insects (Bernhardt, 1989), providing food and habitat resources (Jackson, 2000; Sharpe & Goldingay, 1998; Thums et al., 2005), and forming important symbiotic relationships with nitrogen- fixing soil micro-organisms (Sprent, 2001). Despite the importance of these associations in generating available ni- trogen for legumes, as well as co-occurring non-leguminous species (Khanna, 1997), relatively few comparative studies of interactions involving native legumes and their symbionts exist, particularly on broad geographical scales (Weir et al., 2004). In Australia, shrubby legumes are a major component of almost all ecosystems (Maslin, 2001) and associations with nitrogen-fixing symbionts are particularly advantageous to plants growing in nutritionally poor Australian soils. As with other regions of the world, Australia is now experiencing chronic soil and water degradation and a decline in biodiversity associated with broadscale vegeta- tion clearing, necessitating prompt and cost-effective restoration. Legumes can play an important role in restoring degraded landscapes, particularly in regions where they are prolific or important components of the ecological community. Despite good evidence showing that introducing native rhizobia can improve restoration outcomes for legumes as well as associated species (Thrall et al., 2005), little effort has been made to reintroduce appropriate and effective symbionts with their legume hosts simultaneously. This may be due partly to the paucity of information with respect to the diversity, distribution, abundance, host-specificity and effectiveness of these ecologically important plant–microbe interactions. Known legume–symbiont associations involve both alpha- and betaproteobacteria (Moulin et al., 2001). Alpha- proteobacteria involved in these interactions include species of the genera Azorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium, Ensifer (previously Sino- rhizobium but now reinstated; http://www.bacterio.cict.fr/ e/ensifer.html; Martens et al., 2007), Devosia, Phyl- lobacterium and Methylobacterium (Chen et al., 2001; de Lajudie et al., 1998; Dreyfus et al., 1988; Rivas et al., 2002; Valverde et al., 2005). Recent reports of betaproteobacterial Abbreviation: CNI, close-neighbour interchange. The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are FJ687955–FJ688005 and FJ719310– FJ719366 as indicated in Table 1. International Journal of Systematic and Evolutionary Microbiology (2011), 61, 299–309 DOI 10.1099/ijs.0.021014-0 021014 G 2011 IUMS Printed in Great Britain 299