Phylogeography and Symbiotic Effectiveness of Rhizobia Nodulating Chickpea (Cicer arietinum L.) in Ethiopia

Abstract

Chickpea (Cicer arietinum L.) used to be considered a restrictive host that nodulated and fixed nitrogen only with Mesorhizobium ciceri and M. mediterraneum. Recent analysis revealed that chickpea can also establish effective symbioses with strains of several other Mesorhizobium species such as M. loti, M. haukuii, M. amorphae, M. muleiense, etc. These strains vary in their nitrogen fixation potential inviting further exploration. We characterized newly collected mesorhizobial strains isolated from various locations in Ethiopia to evaluate genetic diversity, biogeographic structure and symbiotic effectiveness. Symbiotic effectiveness was evaluated in Leonard Jars using a locally released chickpea cultivar "Nattoli". Most of the new isolates belonged to a clade related to M. plurifarium, with very few sequence differences, while the total collection of strains contained three additional mesorhizobial genospecies associated with M. ciceri, M. abyssinicae and an unidentified Mesorhizobium species isolated from a wild host in Eritrea. The four genospecies identified represented a subset of the eight major Mesorhizobium clades recently reported for Ethiopia based on metagenomic data. All Ethiopian strains had nearly identical symbiotic genes that grouped them in a single cluster with M. ciceri, M. mediterraneum and M. muleiense, but not with M. plurifarium. Some phylogeographic structure was observed, with elevation and geography explaining some of the genetic differences among strains, but the relation between genetic identity and symbiotic effectiveness was observed to be weak.

Keywords: Genetic diversity; Genospecies; Haplotypes; Mesorhizobial strains; Nucleotides; Spatial patterns.

Fig. 1

Sampling sites of Mesorhizobium strains collected from Ethiopia by Greenlon et al., 2019 and HwU 3 (Hawassa University, Soil Microbiology Laboratory) and included in the current analysis

Fig. 2

Phylogeny of multilocus sequence analysis (MLSA) of concatenated housekeeping genes of 16S rRNA, atpD and recA reconstructed using TN93 + I + G model in R. Strains with “*” were obtained from previous culture collection [30], and “T” at the end of some reference strains indicate type strains for that species

Fig. 3

Symbiosis gene phylogenies reconstructed using nifH and nodC gene sequences

Fig. 4

Spatial distribution of Mesorhizobia over space. Colour difference in the scatter plot indicates relative distribution of mesorhizobial genospecies to each other in space. “Plurifarium” represents R. plurifarium genospecies (see cluster I in Fig. 1), and “WSM3876” represents Mesorhizobium WSM3876 (see cluster II in Fig. 1).

Fig. 5

Effects of geographic and altitude components on the distribution of strains at nucleotide and haplotype levels of genetic level. On the x-axis, geographic or altitudinal distance components by which strain distribution is significantly affected. On the y-axis, principal coordinate distances at nucleotides or haplotypes that were influenced by the geographic distances is presented. Strains were scattered within the coordinate plane, showing their dispersal in space

Fig. 6

Hypothetical resampling of rhizobia in a sliding window of 400 km. Genetic distances for a pair of strains was estimated at every 10-km distance of sampling and plotted against geographic distance. (a) Nucleotide distances and (b) locus/haplotype distance. Genetic distance is plotted on the y-axis, and geographic distance is plotted on the x-axis