Julie Ardley

Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. and Microvirga zambiensis sp. nov. are alphaproteobacterial root-nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts.

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% sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6(T) and WSM3557(T) was Microvirga flocculans TFB(T), with 97.6-98.0% similarity, while WSM3693(T) was most closely related to Microvirga aerilata 5420S-16(T), with 98.8% similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA and rpoB) and cellular fatty acid profiles confirmed the placement of Lut6(T), WSM3557(T) and WSM3693(T) within the genus Microvirga. DNA-DNA relatedness values, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6(T), WSM3557(T) and WSM3693(T) from each other and from other Microvirga species with validly published names. The nodA sequence of Lut6(T) was placed in a clade that contained strains of Rhizobium, Mesorhizobium and Sinorhizobium, while the 100% identical nodA sequences of WSM3557(T) and WSM3693(T) clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that the sequences of Lut6(T), WSM3557(T) and WSM3693(T) were most closely related to that of Rhizobium etli CFN42(T) nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Microvirga are proposed: Microvirga lupini sp. nov. (type strain Lut6(T) =LMG 26460(T) =HAMBI 3236(T)), Microvirga lotononidis sp. nov. (type strain WSM3557(T) =LMG 26455(T) =HAMBI 3237(T)) and Microvirga zambiensis sp. nov. (type strain WSM3693(T) =LMG 26454(T) =HAMBI 3238(T)).

Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419

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 sequence 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 feature unique to this medic microsymbiont.

Burkholderia sprentiae sp. nov., isolated from Lebeckia ambigua root nodules

Seven Gram-stain-negative, rod-shaped bacteria were isolated from Lebeckia ambigua root nodules and authenticated on this host. Based on the 16S rRNA gene phylogeny, they were shown to belong to the genus Burkholderia, with the representative strain WSM5005(T) being most closely related to Burkholderia tuberum (98.08 % sequence similarity). Additionally, these strains formed a distinct group in phylogenetic trees based on the housekeeping genes gyrB and recA. Chemotaxonomic data including fatty acid profiles and analysis of respiratory quinones supported the assignment of the strains to the genus Burkholderia. Results of DNA-DNA hybridizations, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from the closest species of the genus Burkholderia with a validly published name. Therefore, these strains represent a novel species for which the name Burkholderia sprentiae sp. nov. (type strain WSM5005(T) = LMG 27175(T) = HAMBI 3357(T)) is proposed.

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers.

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp.

Complete genome sequence of Rhizobium leguminosarum bv trifolii strain WSM2304, an effective microsymbiont of the South American clover Trifolium polymorphum.

Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.

Biogeography of nodulated legumes and their nitrogen-fixing symbionts

Contents 40 I. 40 II. 41 III. 44 IV. 48 V. 49 VI. 49 VII. 52 VIII. 53 53 References 53 SUMMARY: In the last decade, analyses of both molecular and morphological characters, including nodulation, have led to major changes in our understanding of legume taxonomy. In parallel there has been an explosion in the number of genera and species of rhizobia known to nodulate legumes. No attempt has been made to link these two sets of data or to consider them in a biogeographical context. This review aims to do this by relating the data to the evolution of the two partners: it highlights both longitudinal and latitudinal trends and considers these in relation to the location of major land masses over geological time. Australia is identified as being a special case and latitudes north of the equator as being pivotal in the evolution of highly specialized systems in which the differentiated rhizobia effectively become ammonia factories. However, there are still many gaps to be filled before legume nodulation is sufficiently understood to be managed for the benefit of a world in which climate change is rife.

A Genomic Encyclopedia of the Root Nodule Bacteria: assessing genetic diversity through a systematic biogeographic survey

Root nodule bacteria are free-living soil bacteria, belonging to diverse genera within the Alphaproteobacteria and Betaproteobacteria, that have the capacity to form nitrogen-fixing symbioses with legumes. The symbiosis is specific and is governed by signaling molecules produced from both host and bacteria. Sequencing of several model RNB genomes has provided valuable insights into the genetic basis of symbiosis. However, the small number of sequenced RNB genomes available does not currently reflect the phylogenetic diversity of RNB, or the variety of mechanisms that lead to symbiosis in different legume hosts. This prevents a broad understanding of symbiotic interactions and the factors that govern the biogeography of host-microbe symbioses.Here, we outline a proposal to expand the number of sequenced RNB strains, which aims to capture this phylogenetic and biogeographic diversity. Through the Vavilov centers of diversity (Proposal ID: 231) and GEBA-RNB (Proposal ID: 882) projects we will sequence 107 RNB strains, isolated from diverse legume hosts in various geographic locations around the world. The nominated strains belong to nine of the 16 currently validly described RNB genera. They include 13 type strains, as well as elite inoculant strains of high commercial importance. These projects will strongly support systematic sequence-based studies of RNB and contribute to our understanding of the effects of biogeography on the evolution of different species of RNB, as well as the mechanisms that determine the specificity and effectiveness of nodulation and symbiotic nitrogen fixation by RNB with diverse legume hosts.

Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the Southern African crotalarioid clade Lotononis s.l.

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.

Nodulation of legumes from the Thar desert of India and molecular characterization of their rhizobia

AimsTo survey the occurrence of nodulated legumes in the arid and semi-arid areas of Western Rajasthan and to characterize their associated symbiotic bacteria.MethodsHerbaceous annual species were excavated whole, while tree species were studied as seedlings in the field or as trap plants in pot experiments. Nodules were examined by microscopy to confirm their effectiveness and to determine their internal structure. Bacteria isolated from the nodules were authenticated on their original hosts and were identified on the basis of 16S rRNA sequencing. Phylogenetic trees were inferred using the neighbour-joining method.ResultsWe studied 35 of more than 50 species of native legume reported from these areas. Legumes are drought escaping (annual species), drought tolerant perennials or trees possessing deep root systems and other adaptations to arid conditions. Nodulation was recorded in all members of the Papilionoideae and Mimosoideae, but only one species of Caesalpinioideae. Internal structure of nodules varied within these groups, especially with respect to the presence or absence of uninfected cells in the infected region. Full 16S rRNA gene sequencing revealed that the nodules harboured a range of nodulating bacteria belonging to the genera Sinorhizobium, Rhizobium and Bradyrhizobium, within which they formed separate sub clades.ConclusionsThis study extends the range of legumes known to grow and nodulate in semi-arid regions, and provides information about their endosymbionts.

Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazonian rainforest soils

Root nodule bacteria were trapped within cowpea (Vigna unguiculata) in soils with different cultivation histories collected from the Amazonian rainforest in northern Brazil. Analysis of the 16S rRNA gene sequences of six strains (BR 3351(T), BR 3307, BR 3310, BR 3315, BR 3323 BR and BR 3361) isolated from cowpea nodules showed that they formed a distinct group within the genus Bradyrhizobium, which was separate from previously identified type strains. Phylogenetic analyses of three housekeeping genes (glnII, recA and rpoB) revealed that Bradyrhizobium huanghuaihaiense CCBAU 23303(T) was the most closely related type strain (96% sequence similarity or lower). Chemotaxonomic data, including fatty acid profiles (predominant fatty acids being C16 : 0 and summed feature 8), the slow growth rate and carbon compound utilization patterns supported the assignment of the strains to the genus Bradyrhizobium. The results of DNA-DNA hybridizations, antibiotic resistance and physiological tests differentiated these novel strains from the most closely related species of the genus Bradyrhizobium with validly published names. Symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) grouped the novel strains of the genus Bradyrhizobium together with Bradyrhizobium iriomotense strain EK05(T), with 94% and 96% sequence similarity, respectively. Based on these data, these six strains represent a novel species for which the name Brabyrhizobium manausense sp. nov. (BR 3351(T) = HAMBI 3596(T)), is proposed.