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Symbiotic specificity and nodulation in the southern African legume clade Lotononis s. l. and description of novel rhizobial species within the Alphaproteobacterial genus Microvirga

Ardley, Julie (2011) Symbiotic specificity and nodulation in the southern African legume clade Lotononis s. l. and description of novel rhizobial species within the Alphaproteobacterial genus Microvirga. PhD thesis, Murdoch University.

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Lotononis s. l. is a legume clade within the Crotalarieae tribe, with a centre of origin in South Africa. After taxonomic revision, the three genera Listia, Leobordea and Lotononis s. str. are now recognised. The N2-fixing symbiosis between Listia bainesii and pigmented Methylobacterium rhizobia is known to be highly specific, while a recent study has shown that Listia angolensis is effectively nodulated by a novel lineage of root nodule bacteria. The symbiotic relationships of Lotononis s. l. species outside the Listia genus have not yet been examined. The work presented in this thesis sought to determine the identity of rhizobia isolated from Listia, Leobordea and Lotononis s. str. hosts, to examine the phylogeny of their nodA genes and to quantify the nodulation and N2 fixation capabilities of Lotononis s. l.- associated rhizobia on eight taxonomically diverse Lotononis s. l. hosts. Additionally, this research sought to examine the processes of infection and nodule initiation in L. angolensis and L. bainesii and to validly name and characterise the novel L. angolensis rhizobia.

Amplification and sequencing of nearly full length fragments of the 16S rRNA gene showed that the rhizobia isolated from nodules of Lotononis s. l. species were phylogenetically diverse. Strains isolated from Leobordea and Lotononis s. str. hosts were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. The Listia angolensis microsymbionts, together with closely related Lupinus texensis rhizobia, were identified as novel species of the Alphaproteobacterial genus Microvirga. The xiii phylogeny of the nodA genes correlated more with the rhizobial 16S rRNA genes than with the taxonomy of the host plants.

The nodulation and effectiveness trials confirmed the symbiotic specificity of the genus Listia. L. bainesii nodulated only with the representative pigmented Methylobacterium strain WSM2598. As measured by plant shoot dry weight, this symbiosis was highly effective. L. angolensis was effectively nodulated only by Microvirga rhizobia, but formed ineffective nodules with the pigmented Methylobacterium and M. nodulans strains. WSM2598 was effective only on Listia species (other than L. angolensis). In contrast, the Microvirga strain WSM3557 was partially effective on some Leobordea and Lotononis s. str. species. No clear pattern of symbiotic association was seen in the Leobordea and Lotononis s. str. hosts. Nodulation in these species was usually promiscuous and often ineffective for N2 fixation.

The nodules formed on Listia species were lupinoid, whereas the nodules of Leobordea and Lotononis s. str. species were indeterminate. Micrographs of all sectioned nodules, whether lupinoid or indeterminate, showed a mass of central, uniformly infected tissue, with no uninfected interstitial cells. Rhizobial infection and nodulation in L. angolensis and L. bainesii did not appear to involve root hair curling or the development of infection threads. Nodule organogenesis followed a process similar to that observed in Lupinus species, with nodule primordia developing in the outer cortex.

The polyphasic characterisation of the Microvirga rhizobia associated with Listia angolensis and Lupinus texensis resulted in the description of three new rhizobial species: Microvirga lotononidis, M. zambiensis and M. lupini. Microvirga species possess several phenotypic properties that are unusual in rhizobia, including the ability to grow at relatively elevated temperatures and the presence of pigmentation in most strains. The rhizobial Microvirga strains WSM3557T and Lut6T have been included in the Genomic Encyclopedia for Bacteria and Archaea Root Nodule Bacteria (GEBA-RNB) sequencing project (Kyrpides & Reeve, collaborative CSI project ( and should provide new insights into the evolution of and genomic architecture required for rhizobial symbionts.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): Howieson, John, O'Hara, Graham, Reeve, Wayne and Yates, Ron
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