Sofie De Meyer

The rhizobia-legume symbiosis is a highly important source of nitrogen (N) in both natural and agricultural systems. Nodulated legumes are found in nearly all terrestrial and even some aquatic ecosystems. Rhizobial microsymbionts are phylogenetically and genetically diverse. Their symbiotic ability is conferred by a group of approximately 400 genes, which enable nodulation and N2-fixation with the legume host and can be acquired by horizontal gene transfer. While certain rhizobial species are known to be specifically associated with particular legume hosts, recent studies also provide evidence of emerging trends in rhizobial biogeography, in which edaphic and climatic factors strongly influence rhizobial distribution patterns. Research at the Centre for Rhizobium Studies (CRS) has shown that an understanding of these distribution patterns is critical in assessing the suitability of novel legumes and their associated rhizobia for introduction into agricultural systems. Rhizobial biogeography also appears to play a role in determining which species of microsymbionts are nodule occupants of several invasive legume weeds. We present here an overview of the adaptation of different rhizobial species and genera to specific environments, with case studies of species of Burkholderia and Sinorhizobium (Ensifer). The sequenced genomes of more than 142 strains of rhizobia from 9 different genera are now available, largely through joint ventures between the CRS, the DoE Joint Genome Institute (JGI) and an international consortium of scientists (http://genome.jgi.doe.gov/programs/bacteria-archaea/GEBA-RNB.jsf). These can be used to determine connections between rhizobial genetic backgrounds and patterns of biogeographic distribution.

The closely related papilionoid tribes Mirbelieae and Bossiaeeae are a large (ca. 750 species) group of endemic Australasian legumes. Typically they are ericoid shrubs that are conspicuous understorey members of sclerophyll communities growing on poor soils of the southwest and southeast temperate biomes (Crisp et al. 2004). The centre of diversity is in Southwest Australia, a biodiversity hotspot, unglaciated since the Permian (ca. 250 Mya) and dominated by old landscapes with nutrient-deficient soils (Hopper and Gioia 2004). The crown clade age for the mirbelioids and their sister tribe Hypocalypteae is estimated to be ca. 55 Mya, suggesting that mirbelioids evolved in isolation, shortly after Australia separated from the Antarctic continent, having lost contact with other Gondwanan land masses (Sprent et al. 2013). Molecular-dated phylogenies indicate that the group radiated rapidly during the Mid-Cenozoic (ca. 25–10 Mya) period of climatic cooling and drying (Crisp et al. 2004). In common with most papilionoid legumes, Mirbelieae and Bossiaeeae species form nitrogen-fixing associations with rhizobia, usually with strains of Bradyrhizobium (Lafay and Burdon 1998; Stępkowski et al. 2012). Nodule morphology and structure has not yet been studied. This character trait has previously been found to be a useful phylogenetic and taxonomic marker (Sprent et al. 2013). Phylogenetic analyses place the mirbelioid legumes between the Dalbergioid and Indigoferoid clades, two groups that have quite different nodule structures (Sprent et al. 2013). This study aimed to characterise rhizobial microsymbionts of diverse mirbelioid species collected from sites across southwest Australia and to determine the mirbelioid nodule morphology and structure.

Root nodule bacteria isolated from Zambian Lotononis angolensis (1) and southern USA Lupinus texensis (2) form a group that is distinct from other named and described legume root nodule bacteria. A phylogenetic tree based on the sequence of nearly full-length portions of the 16S rRNA gene indicates these isolates are affiliated to the α-proteobacterial genus Microvirga. Microvirga spp isolated from soil, air and thermal waters or hot springs have previously been formally described but none has been reported as capable of symbiotic nitrogen fixation. These isolates therefore represent a new lineage of nitrogen-fixing legume symbionts. We present here a polyphasic description of these novel species. A phylogenetic tree based on rpoB sequences supports the topography of the 16S rRNA tree in affiliating these isolates with Microvirga. Sequences of nifD and nifH are closely related in the L. angolensis and L. texensis strains, and there is no indication of horizontal gene transfer. In contrast, the nodA sequence of a L. angolensis strain grouped with Burkholderia tuberum and Methylobacterium nodulans nodA sequences, while that of a L. texensis strain was placed within a different group of rhizobia. The 16S rRNA phylogenetic tree indicates that L. texensis strains do not form a single lineage that is phylogenetically divergent from the African L. angolensis strains; rather, multiple lineages of these root nodule bacteria seem to be distributed across both geographic regions. DNA:DNA hybridization, fatty acid composition and % G+C data are consistent with these isolates forming several novel species. This study presents additional characterisation of these isolates, such as morphology, physiology, substrate utilisation, antibiotic resistance and legume host range that differentiates them from other Microvirga spp. These novel root nodule bacteria tolerate comparatively high temperatures and may have potential as inoculants in hot climates.

Root nodule bacteria isolated from native legumes in various biogeographical areas have demonstrated that rhizobia are more phylogenetically diverse than originally supposed. We present here an overview of our studies on novel species, isolated from nodules of legume hosts in Australia and Africa, which are affiliated to Burkholderia, Methylobacterium and Microvirga. The microsymbionts’ physiological adaptations to their environment, host specificity and phylogeny of nodulation and nitrogen fixation genes were examined, along with the modes of plant infection and nodule formation. Important findings include the apparent adaptation of Burkholderia spp. to infertile soils of wide pH range, the confirmation of specificity in the non-root hair-mediated Lotononis/Methylobacterium symbiosis and the potential of legume nodule morphology as a taxonomic aid. These species’ inclusion in the Genomic Encyclopedia for Bacteria and Archaea sequencing project will aid elucidation of the diverse rhizobial genomic architecture that underlies symbiotic ability and specificity.