Characterization of rhizobia and their symbiotic performance on selected important legumes under abiotic stress conditions

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Introduction: Legumes are a source of proteins in human diet and a staple food for many cultures globally as an inexpensive meat alternative. They form a symbiotic relationship with rhizobia to form root nodules. Inside the nodules, the rhizobia fix atmospheric nitrogen (N2) by the biological nitrogen fixation (BNF) process. This provides a supply of nitrogen to plants. Lately, farmers are more open to the use of rhizobia inoculants due to the availability of effective and quality products in the market. These inoculants improve yields at a low cost when compared to chemical or artificial fertilizers. Symbiotic genes (nodA and nodC), the ACC deaminase enzyme (encoded by acdS gene) and exopolysaccharides (exoR gene) are essential for the rhizobium-legume association, particularly under abiotic stress conditions for effective nodulation. Aim: The current study aims at isolating stress tolerant SARCC (South African Rhizobium Culture Collection) isolates to be applied as inoculants under abiotic stress conditions. Materials and methods: Rhizobia spp. tolerance to various levels of temperature, acidity/alkalinity (pH5, pH7 and pH9), heavy metals (50mM, 100mM and 150mM concentrations of AlCl3.6H2O) and salinity (50mM, 100mM and 150mM concentrations of NaCl) stresses were investigated. Phylogenetic characterization of the isolates was determined using nucleotide sequence analysis of the 16S rRNA, exoR gene, acdS gene, the house keeping recA gene, as well as nodA and nodC symbiotic genes. A glasshouse nodulation efficacy test under normal and abiotic stress conditions was also conducted. Results: This study reports tolerance to abiotic stresses and the phylogenetic characterization of 40 rhizobia strains previously isolated from the root nodules of Medicago sativa, Trifolium repens, Lupinus albus, Vigna unguiculata and Phaseolus vulgaris. The isolates exhibited significant variations in their tolerance to abiotic stresses. Using molecular approaches, some of the rhizobia isolates have been detected to possess certain beneficial traits involved in the reduction of abiotic stresses such as the acdS (9 isolates) and exoR (5 isolates) genes. The symbiotic genes nodC (7 isolates) and nodA (16 isolates) were detected as well. Phylogenetic analyses based on 16S rRNA gene, housekeeping genes (recA) revealed that the isolates belongs to five genera: Sinorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium and Aminobacter. Under glasshouse nodulation efficacy test, effective isolates provided the highest plant biomass and number of nodules under normal conditions, acidity/alkalinity and salinity abiotic stresses when compared to the un-inoculated controls. Conclusion: Amid the increasing threats of the global climate change and the rising abiotic stresses, these current results provide baseline information in the selection of rhizobia for use as inoculants under abiotic stressed conditions in South Africa.
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023
Rhizobia, Abiotic stress, Biological nitrogen fixation