Chromosomal and symbiotic relationships of rhizobia nodulating Medicago truncatula and M. laciniata

Abstract

Multilocus sequence typing (MLST) is a sequence-based method used to characterize bacterial genomes. This method was used to examine the genetic structure of Medicago-nodulating rhizobia at the Amra site, which is located in an arid region of Tunisia. Here the annual medics Medicago laciniata and M. truncatula are part of the natural flora. The goal of this study was to identify whether distinct chromosomal groups of rhizobia nodulate M. laciniata because of its restricted requirement for specific rhizobia. The MLST analysis involved determination of sequence variation in 10 chromosomal loci of 74 isolates each of M. laciniata and M. truncatula. M. truncatula was used as a control trap host, because unlike M. laciniata, it has relatively unrestricted rhizobial requirements. Allelic diversity among the plasmid nodC alleles in the isolates was also determined. The 148 isolates were placed into 26 chromosomal sequence types (STs), only 3 of which had been identified previously. The rhizobia of M. laciniata were shown to be part of the general Medicago-nodulating population in the soil because 99.95% of the isolates had chromosomal genotypes similar to those recovered from M. truncatula. However, the isolates recovered from M. laciniata were less diverse than those recovered from M. truncatula, and they also harbored an unusual nodC allele. This could perhaps be best explained by horizontal transfer of the different nodC alleles among members of the Medicago-nodulating rhizobial population at the field site. Evidence indicating a history of lateral transfer of rhizobial symbiotic genes across distinct chromosomal backgrounds is provided.

FIG. 1.

Snapshot of a population of 378 Medicago-nodulating rhizobia derived from allelic variation at 10 chromosomal loci. A matrix of the STs followed by the allele labels for each ST was used in eBURST (14) to generate a diagram of the evolutionary patterns among the strains and isolates. The snapshot was produced by setting the group definition to 0/10 genes. The sizes of the circles are related to the numbers of strains and isolates in each ST. The founder and cofounder genotypes are blue and yellow, respectively. The lengths of connecting lines between STs are arbitrary.

FIG. 2.

NeighborNet for 378 Medicago-nodulating rhizobia derived from allelic variation at 10 chromosomal loci. The inset shows the NeighborNet for the 148 Tunisian isolates. Matrices of the strain or isolate identification and the ST followed by the allele designations for each taxon was used in the START program (version 1.05) (19) to generate a Nexus file for input in Splitstree (18) to generate the NeighborNet (8).

FIG. 3.

Diagram illustrating the host origins of rhizobia that belong to each of the STs. STs in the yellow, blue, and green areas originated from M. laciniata, from M. truncatula, and from both host species, respectively.

FIG. 4.

Phylogeny of nodC in rhizobia that form symbioses with the legume genus Medicago. The sequence alignment, created in Genedoc (http://www.nrbsc.org/gfx/genedoc/index.html) with the nodC genes of R. leguminosarum (accession number Y00548) and R. tropici (accession number X98514) used as outgroups, was 1,285 bp long. The msf file was converted to Mega format with Mega version 2.1 (21), which was also used to obtain nucleotide (A) and derived amino acid (B) sequence similarities for construction of trees using the UPGMA algorithm.