Michael Dilworth

Summary This manual brings together state-of-the-art methods for the study of root-nodule bacteria, both in the free-living state and in symbiosis with legumes. In each chapter, the manual introduces a topic and provides guidance on how study of the symbiosis might best be tackled. Detailed descriptions of the protocols that need to be followed, potential problems and pitfalls are provided. Topics covered include acquiring, recognising, growing and storing rhizobia, experimenting with strains in the laboratory, glasshouse and field, and applying contemporary molecular and genetic methodologies to assist in the study of rhizobia.

Background and Aims: The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. Methods: Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. Key Results: Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. Conclusions: Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.

Strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from nitrogen-fixing nodules of the native legumes Listia angolensis (from Zambia) and Lupinus texensis (from Texas, USA). Phylogenetic analysis of the 16S rRNA gene showed that the novel strains belong to the genus Microvirga, with 96.1 % or greater sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6T and WSM3557T was M. flocculans TFBT, with 97.6-98.0 % similarity, while WSM3693T was most closely related to M. aerilata 5420S-16T, with 98.8 % similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA, rpoB) and cellular fatty acid profiles confirmed the placement of Lut6T, WSM3557T and WSM3693T within Microvirga. DNA:DNA relatedness values and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6T, WSM3557T and WSM3693T from each other and from other validly published Microvirga species. The nodA sequence of Lut6T was placed in a clade that contained strains of Rhizobium, Mesorhizobium and Sinorhizobium, while the 100 % identical nodA sequences of WSM3557T and WSM3693T clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that Lut6T, WSM3557T and WSM3693T were most closely related to Rhizobium etli CFN42T nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Microvirga are proposed: Microvirga lupini (Lut6T = LMG26460T, = HAMBI 3236) Microvirga lotononidis (WSM3557T = LMG26455T, = HAMBI 3237) and Microvirga zambiensis (WSM3693T = LMG26454T, = HAMBI 3238).

Steady-state assays of nitrogenases share at least five requirements: an anaerobic environment, a consistent source of magnesium adenosine triphosphate (MgATP), a suitable source of reductant, a buffer system compatible with the product-quantification protocol to be used, and the desired substrate. The assay is initiated by injection of the component protein(s) of the enzyme or MgATP and terminated by injection of either acid or a solution of Na2EDTA. The various nitrogenases catalyze the reduction of a wide variety of substrates. This chapter outlines the methods used to analyze the products of nitrogenase-catalyzed reactions involving nitrogen-nitrogen bonds, nitrogen-oxygen bonds, carbon-nitrogen bonds, carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, and hydrogen only. The usefulness of measurements of residual amounts of other components of nitrogenase assays is also discussed.

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.

Ensifer (Sinorhizobium) medicae is an effective nitrogen fixing microsymbiont of a diverse range of annual Medicago (medic) species. Strain WSM419 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from a M. murex root nodule collected in Sardinia, Italy in 1981. WSM419 was manufactured commercially in Australia as an inoculant for annual medics during 1985 to 1993 due to its nitrogen fixation, saprophytic competence and acid tolerance properties. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first report of a complete genome se-quence for a microsymbiont of the group of annual medic species adapted to acid soils. We reveal that its genome size is 6,817,576 bp encoding 6,518 protein-coding genes and 81 RNA only encoding genes. The genome contains a chromosome of size 3,781,904 bp and 3 plasmids of size 1,570,951 bp, 1,245,408 bp and 219,313 bp. The smallest plasmid is a fea-ture unique to this medic microsymbiont.

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.