Maria P. Fernandez

Sinorhizobium medicae sp. nov., Isolated from Annual Medicago spp.

The taxonomic position of isolates of a new genomic species (designated genomic species 2) obtained from several annual Medicago species and originating from different geographical locations was established through the results of phenotypic tests (including the results of auxanographic and biochemical tests and symbiotic properties) and 16S rRNA phylogenetic inferences. A comparison of the complete 16S rRNA sequence of a representative of genomic species 2 (strain A 321T [T = type strain]) with the 16S rRNA sequences of other members of the Rhizobiaceae and closely related taxa showed that genomic species 2 was phylogenetically related to Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, and Sinorhizobium teranga. The levels of sequence similarity and observed numbers of nucleotide substitutions in Sinorhizobium strains indicated that A 321T and S. meliloti exhibited the highest level of sequence similarity (99.7%), with four nucleotide substitutions and one deletion. The results of a numerical analysis based on data from 63 auxanographic and biochemical tests clearly separated genomic species 2 isolates from S. meliloti. Genomic species 2 isolates nodulated and fixed nitrogen with Medicago polymorpha, whereas S. meliloti isolates were ineffective and formed rudimentary nodules on this host plant. On the basis of phenotypic and 16S sequence analysis data, genomic species 2 isolates cannot be assigned to a previously described species. We propose that these isolates belong to a new species, Sinorhizobium medicae.

Rhizobium ciceri sp. nov., consisting of strains that nodulate chickpeas (Cicer arietinum L.).

The taxonomic status of 16 collection strains of chickpea (Cicer arietinum L.) rhizobia which were previously determined to belong to two groups (groups A and B) were compared with reference strains belonging to different genera and species of the family Rhizobiaceae. We used the following taxonomic, phylogenetic, and phenotypic characteristics and approaches to study these organisms: DNA homology, guanine-plus-cytosine content, restriction fragment length polymorphism of the amplified 16S-intergenic spacer rRNA gene, partial 16S rRNA sequencing, and auxanographic tests performed with 147 carbon sources. Similar groups of chickpea strains were identified by the different approaches. The chickpea strains were found to belong to the genus Rhizobium regardless of the phylogenetic group to which they belonged (group A or B). All strains fell into a tight cluster which included Rhizobium loti and Rhizobium galegae, and the group B strains were closely related to R. loti. An analysis of partial 16S ribosomal DNA sequences revealed identical nucleotide sequences for the slowly growing strains and fast-growing strains that were used as representatives of groups A and B, respectively, and these organisms fell into the Rhizobium-Agrobacterium lineage. When the sequences of these organisms were compared with the partial sequences of Rhizobium huakuii and R. loti, one- and two-nucleotide mismatches were observed, respectively, indicating that the chickpea rhizobia are closely related to these two species. The DNA-DNA hybridization data revealed that the chickpea rhizobia exhibited low levels of homology (less than 17%) to previously described Rhizobium and Bradyrhizobium species. Moreover, when we compared chickpea strains to R. loti and R. huakuii, the most closely related species as determined by the partial 16S rRNA sequence analysis, the homology values ranged from 21 to 52% and the delta Tm values were greater than 5 degrees C (delta Tm is the difference between the denaturation temperatures of the heterologous and homologous duplexes). These results confirmed that the rhizobia that nodulate chickpeas cannot be assigned to a previously described species. Within the chickpea rhizobia, the DNA homology values obtained when members of groups A and B were compared were less than 38%, indicating that the group A and group B organisms belong to different species. Furthermore, these organisms can be distinguished from each other by the results of phenotypic tests. We propose that the group B chickpea rhizobia should be assigned to a new species, Rhizobium ciceri; UPM-Ca7 is the type strain of R. ciceri.

Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov.

The genetic diversity of chickpea strains was studied by using 30 isolates obtained from nodules on chickpeas growing in uninoculated fields over a wide geographic range. The following taxonomic approaches were used: DNA-DNA relatedness analysis, restriction fragment length polymorphism analysis of the amplified 16S ribosomal DNA (rDNA) intergenic spacer (IGS), and total 16S rRNA sequence analysis. The division of chickpea-infective strains into two major phylogenetic groups (groups A and B) that has been described previously was confirmed by the polymorphism of the 16S IGS rDNA. We identified a total of five genomic species, including the previously described species Rhizobium ciceri. All of the group B strains except one were homogeneous and belonged to a single genomic species corresponding to R. ciceri. Group A was heterogeneous, containing three genomic species and five strains that remained unclassified, and its members had very different PCR restriction fragment length polymorphism profiles. The complete 16S rRNA sequences of strains representing the two major groups, R. ciceri UPM-Ca7T (T = type strain) and genomic species 2 strain UPM-Ca36T, exhibited 19 mismatches. Both of these strains belonged to the Rhizobium loti-Rhizobium huakuii branch; R. ciceri UPM-Ca7T was closely related to R. loti, and strain UPM-Ca36T was clearly separated from R. ciceri and closely related to R. huakuii. Thus, genomic species 2 could be distinguished from R. ciceri by its 16S rRNA sequence, by DNA relatedness data, by the polymorphism of the 16S IGS rDNAs, and by previously described multilocus enzyme electrophoresis results and phenotypic characteristics. Therefore, we propose that strains belonging to genomic species 2 should be classified in a new species, Rhizobium mediterraneum, and that strain UPM-Ca36 should be the type strain.

Mesorhizobium chacoense sp. nov., a novel species that nodulates Prosopis alba in the Chaco Arido region (Argentina)

Low-molecular-weight RNA analysis was performed for the identification and classification of 20 Argentinian strains isolated from the root nodules of Prosopis alba. SDS-PAGE of total cellular proteins, determination of the DNA base composition, DNA-DNA reassociation experiments and physiological and biochemical tests were also carried out for these strains and the whole 16S rRNA gene was sequenced from one of the strains, strain LMG 19008T. Results of the genotypic and phenotypic characterization showed that the strains isolated in this study belong to a group that clustered in the genus Mesorhizobium. The results of DNA-DNA hybridizations showed that this group is a novel species of this genus. The name Mesorhizobium chacoense sp. nov. is proposed for this species. The type strain is LMG 19008T (= CECT 5336T).

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism.

ABSTRACT The identity of Frankia strains from nodules ofMyrica gale, Alnus incana subsp. rugosa, andShepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype.Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct fromFrankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from twoM. gale nodules at a second site in Wisconsin.Frankia strains from nodules of S. canadensisbelonged to a divergent subset of a cluster ofElaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankiapopulations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankiapopulations found in Myrica nodules.

In-planta sporulation capacity enhances infectivity and rhizospheric competitiveness of Frankia strains

Frankia Sp+ strains maintain their ability to sporulate in symbiosis with actinorhizal plants, producing abundant sporangia inside host plant cells, in contrast to Sp− strains, which are unable to perform in-planta sporulation. We herein examined the role of in-planta sporulation in Frankia infectivity and competitiveness for root infection. Fifteen strains belonging to different Sp+ and Sp− phylogenetic lineages were inoculated on seedlings of Alnus glutinosa (Ag) and A. incana (Ai). Strain competitiveness was investigated by performing Sp−/Sp+ co-inoculations. Plant inoculations were standardized using crushed nodules obtained under laboratory-controlled conditions (same plant species, age, and environmental factors). Specific oligonucleotide primers were developed to identify Frankia Sp+ and/or Sp− strains in the resulting nodules. Single inoculation experiments showed that (i) infectivity by Sp+ strains was significantly greater than that by Sp− strains, (ii) genetically divergent Sp+ strains exhibited different infective abilities, and (iii) Sp+ and Sp− strains showed different host preferences according to the origin (host species) of the inocula. Co-inoculations of Sp+ and Sp− strains revealed the greater competitiveness of Sp+ strains (98.3 to 100% of Sp+ nodules, with up to 15.6% nodules containing both Sp+ and Sp− strains). The results of the present study highlight differences in Sp+/Sp− strain ecological behaviors and provide new insights to strengthen the obligate symbiont hypothesis for Sp+ strains.

Alder and the Golden Fleece: high diversity of Frankia and ectomycorrhizal fungi revealed from Alnus glutinosa subsp. barbata roots close to a Tertiary and glacial refugium

Background Recent climatic history has strongly impacted plant populations, but little is known about its effect on microbes. Alders, which host few and specific symbionts, have high genetic diversity in glacial refugia. Here, we tested the prediction that communities of root symbionts survived in refugia with their host populations. We expected to detect endemic symbionts and a higher species richness in refugia as compared to recolonized areas. Methods We sampled ectomycorrhizal (EM) root tips and the nitrogen-fixing actinomycete Frankia communities in eight sites colonized by Alnus glutinosa subsp. barbata close to the Caucasus in Georgia. Three sites were located in the Colchis, one major Eurasian climatic refugia for Arcto-Tertiary flora and alders, and five sites were located in the recolonized zone. Endemic symbionts and plant ITS variants were detected by comparing sequences to published data from Europe and another Tertiary refugium, the Hyrcanian forest. Species richness and community structure were compared between sites from refugia and recolonized areas for each symbionts. Results For both symbionts, most MOTUs present in Georgia had been found previously elsewhere in Europe. Three endemic Frankia strains were detected in the Colchis vs two in the recolonized zone, and the five endemic EM fungi were detected only in the recolonized zone. Frankia species richness was higher in the Colchis while the contrary was observed for EM fungi. Moreover, the genetic diversity of one alder specialist Alnicola xanthophylla was particularly high in the recolonized zone. The EM communities occurring in the Colchis and the Hyrcanian forests shared closely related endemic species. Discussion The Colchis did not have the highest alpha diversity and more endemic species, suggesting that our hypothesis based on alder biogeography may not apply to alder’s symbionts. Our study in the Caucasus brings new clues to understand symbioses biogeography and their survival in Tertiary and ice-age refugia, and reveals that isolated host populations could be of interest for symbiont diversity conservation.

Absence of Cospeciation between the Uncultured Frankia Microsymbionts and the Disjunct Actinorhizal Coriaria Species

Coriaria is an actinorhizal plant that forms root nodules in symbiosis with nitrogen-fixing actinobacteria of the genus Frankia. This symbiotic association has drawn interest because of the disjunct geographical distribution of Coriaria in four separate areas of the world and in the context of evolutionary relationships between host plants and their uncultured microsymbionts. The evolution of Frankia-Coriaria symbioses was examined from a phylogenetic viewpoint using multiple genetic markers in both bacteria and host-plant partners. Total DNA extracted from root nodules collected from five species: C. myrtifolia, C. arborea, C. nepalensis, C. japonica, and C. microphylla, growing in the Mediterranean area (Morocco and France), New Zealand, Pakistan, Japan, and Mexico, respectively, was used to amplify glnA gene (glutamine synthetase), dnaA gene (chromosome replication initiator), and the nif DK IGS (intergenic spacer between nifD and nifK genes) in Frankia and the matK gene (chloroplast-encoded maturase K) and the intergenic transcribed spacers (18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA) in Coriaria species. Phylogenetic reconstruction indicated that the radiations of Frankia strains and Coriaria species are not congruent. The lack of cospeciation between the two symbiotic partners may be explained by host shift at high taxonomic rank together with wind dispersal and/or survival in nonhost rhizosphere.

Frankia canadensis sp. nov., isolated from root nodules of Alnus incana subspecies rugosa

Strain ARgP5T, an actinobacterium isolated from a root nodule present on an Alnus incana subspecies rugosa shrub growing in Quebec City, Canada, was the subject of polyphasic taxonomic studies to clarify its status within the genus Frankia. 16S rRNA gene sequence similarities and ANI values between ARgP5T and type strains of species of the genus Frankiawith validly published names were 98.8 and 82 % or less, respectively. The in silico DNA G+C content was 72.4 mol%. ARgP5T is characterised by the presence of meso-A2pm, galactose, glucose, mannose, rhamnose (trace), ribose and xylose as whole-organism hydrolysates; MK-9(H8) as predominant menaquinone; diphosphatidylglycerol, phosphatidylinositol and phosphatidylglycerol as polar lipids and iso-C16 : 0 and C17 : 1ω8c as major fatty acids. The proteomic results confirmed the distinct position of ARgP5T from its closest neighbours in Frankiacluster 1. ARgP5T was found to be infective on two alder (Alnus glutinosa and Alnusalnobetula subsp. crispa) and on one bayberry (Morella pensylvanica) species and to fix nitrogen in symbiosis and in pure culture. On the basis of phylogenetic (16S rRNA gene sequence), genomic, proteomic and phenotypic results, strain ARgP5T (=DSM 45898=CECT 9033) is considered to represent a novel species within the genus Frankia for which the name Frankia canadensis sp. nov., is proposed.

Draft Genome Sequence of Frankia Strain G2, a Nitrogen-Fixing Actinobacterium Isolated from Casuarina equisetifolia and Able To Nodulate Actinorhizal Plants of the Order Rhamnales

ABSTRACT Frankia sp. strain G2 was originally isolated from Casuarina equisetifolia and is characterized by its ability to nodulate actinorhizal plants of the Rhamnales order, but not its original host. It represents one of the largest Frankia genomes so far sequenced (9.5 Mbp).