Michael Dilworth

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.

Exopolysaccharides (EPS's) play an important role in forming and extending infection threads in the symbiosis between Medicago and Sinorhizobium. S. meliloti 1021 (Sm1021) is able to produce one type of EPS (succinoglycan or EPS I) but not another (galactoglucan or EPS II), due to the presence of an insertion sequence (ISRm2011-1) in expR. ExpR forms part of the SinlR quorum sensing system and is a regulator of galactoglucan synthesis. Previous work in our laboratory determined that under N-limited conditions, Sm1021 was poorly effective at fixing N with the model indeterminate legume Medicago truncatula, while two other mucilaginous strains, S. medicae WSM419 and S. melitoti WSM1022, were significantly more effective on this host. While the expR status of WSM1022 is unknown, WSM419 possesses an intact expR gene. These data indicate that the interrupted expR gene in Sm1021 might account for the reduced effectiveness of this strain on the model legume.