Taxonomy and symbiotic effectiveness of Mesorhizobium associated with chickpea

Abstract

Chickpea (Cicer arietinum L.) is one of the most produced pulses worldwide, with India being the biggest producer. The production of chickpea is spread throughout the world from its native range in Southeast Turkey to Australia and North and Central America. It comprises two major types, Desi and Kabuli, of which the former is adapted to winter conditions, while the latter is adapted to summer conditions. The genetic differences among each type have been used as part of breeding and improvement efforts. Chickpea production in Africa is mostly limited to Northern and Eastern Africa. Sub-Saharan Africa, which has enough arable land for the potential production of chickpea, remains unexplored. In addition, to overcome the overreliance on synthetic fertilizers, the application of nitrogen fixers collectively called legume nodulating bacteria (LNB) is encouraged. For chickpea, LNB have been found to belong to the genus Mesorhizobium. Two identified species (Mesorhizobium ciceri, and Mesorhizobium mediterraneum) sharing similar symbiosis loci are mainly associated with this interaction. The first research chapter of this study focused on screening a geographically diverse collection of chickpea symbionts in terms of nodulation efficacy and extra plant growth promotion traits. This was achieved by performing a glasshouse pot experiment where 21 Mesorhizobium strains (i.e., LMG14989, CC1192, USDA3392, SEMIA396, DSM30133, DSM1978, XAP4, XAP10, XAP11, LMG15046, LMG17147, LMG17149, IC59, ICCV3110.1, P16.1, P13.3, P17.1, P17.2, P20.1, P1.2, and IC3110.2) were tested on six genotypes (i.e., Dark variety, Light variety, ICCV3110, ICCV3111, ICCV3203, and ICCV4105) of the Desi variety. Several vegetative growth parameters (i.e., nodule fresh weights, shoot dry weights, average plant heights and symbiotic effectiveness) were measured and subjected to statistical analyses of variance using R software. This was followed by determining the plant growth promotion traits of the Mesorhizobium strains. These included siderophores production, ACC deaminase production, indole acetic acid (IAA) production and phosphate solubilization. Statistically, the factors, ‘chickpea genotype’ and ‘Mesorhizobium strain,’ had significant effects on the measured parameters but lacked significant interactions based on the analysis of variance. The Light variety examined outperformed the other chickpea genotypes on all tested parameters. In general, inoculation with strains LMG15046, CC1192, XAP4, XAP10, and LMG14989 resulted in the best performance for all the tested parameters. Furthermore, all the strains were able to produce IAA and solubilize phosphate except the South African field isolates, which could not solubilize phosphate. However, none of the 21 strains produced siderophores or showed ACC deaminase activity. These findings showed that nodulation and growth promotion of the strains varied broadly based on the six genotypes used, while their ability to produce IAA and solubilize phosphate supports previous findings for Mesorhizobium species. Interestingly, however, this study revealed the differential ability to solubilize phosphate by chickpea Mesorhizobium since the South African isolates failed to solubilize phosphate, a trait that has always been associated with the Mesorhizobium genus. The work presented in this chapter was published in the journal Microorganisms [10(12):2467] under the title “Nodulation and growth promotion of chickpea by Mesorhizobium isolates from diverse sources”. The second research chapter focussed on the taxonomic affiliations of the LNB examined. For this purpose, five housekeeping genes were sequenced to identify known species as well as to delineate new species. These analyses revealed five clusters, of which three represented novel species. They were B (XAP11, LMG17147 and IC59), C (SEMIA396, and LMG17149) and D (ICCV3110.1, P16.1, P13.3, P17.1, P17.2, P20.1, P1.2, and IC3110.2). The existence of these taxa were supported by genome-based Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (dDDH) values. Although the evaluated phenotypic data contained little information to conclusively differentiate between them, the strains in cluster D had a unique and consistent pattern for some traits (e.g., growth temperatures, pH, salinity, heavy metals sensitivity and antibiotic resistance). Investigation of the origin of the strains' symbiotic loci (i.e., nodA, nodC and nifH) revealed that they shared high sequence similarity with those from known chickpea Mesorhizobium nodulators, thus corroborating previous work on this legume. In conclusion, this study revealed varied performance in nodulation and growth promotion among different Mesorhizobium isolates on different genotypes of chickpea used. Since this study was conducted under controlled conditions, field experiments are recommended to test the promising Mesorhizobium strains under South African environmental conditions.