Ronald J Yates

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.

Symbiotic specificity and nodule morphology are characteristics that can be used as taxonomic markers in the legume genus Lotononis and that support its division into two separate genera. Lotononis (from the Crotalarieae tribe in the Genistoid clade of the sub-family Fabaceae) is of mainly southern African origin, comprising some 150 species of herbs and small shrubs. Our work has shown that Lotononis is nodulated by phylogenetically diverse root nodule bacteria and that different specificity groups exist within the genus.

Lotononis is a genus of approximately 150 shrubs, herbaceous perennials and annuals belonging to the subfamily Fabaceae (Van Wyk, 1991). They are distributed mainly in southern Africa, with some species extending throughout Africa, southern Spain, Turkey, south-eastern Bulgaria and part of the Arabian Peninsula to the north-west of the Indian sub-continent (Van Wyk, 1991). Lotononis species have shown potential as perennial pasture legumes that can be used to help reduce the risk of dryland salinity in southern Australian agricultural systems. Species in the section Listia in particular may be useful as pasture legumes as they are perennial, stoloniferous, and lack the poisonous cyanogenic or alkaloid compounds found in some species of Lotononis. L. bainesii, from the Listia section, has been shown to grow well in southern Australia (Roberts & Carbon, 1969) and will grow on acid, sandy soils (R. Yates, pers. comm.). L. bainesii is nodulated by pink-pigmented root-nodule bacteria. Jaftha et al. (2002) characterised nine L. bainesii isolates and found them to be related to Methylobacterium. The genus Methylobacterium, often referred to as pink-pigmented facultative methylotrophs (PPFMs), are capable of growth on C1 compounds such as formate and methanol as sole carbon sources. PPFMs are ubiquitous in the plant phyllosphere and rhizosphere, where they utilize methanol and other C1 compounds that are the products of plant metabolism (Trotsenko et al, 2001). They can promote the germination or the growth of plants, probably because of their ability to synthesise auxins, cytokinins and other plant growth promoting substances (Holland & Polacco, 1994; Ivanova et al., 2000; Trotsenko et al., 2001). However, until the paper by Sy et al. (2001), describing Methylobacterium nodulans, which was isolated from nodules of Crotalaria species found in Senegal, no Methylobacterium species had been known to nodulate legumes, or indeed to fix nitrogen. The objectives in this study were to characterise root-nodule bacteria isolated from four species from the Listia section of Lotononis (L. angolensis, L. bainesii, L. listii and L. solitudinis) using a range of phenotypic and genetic techniques.