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Item Enhancement of abiotic stress tolerance of lessertia frutescents using plant growth promoting rhizobacteria.(Tshwane University of Technology, 2024-03-01) Hlongwane, Mokgadi Miranda; Prof NS Mokgalaka; Prof FD DakoraLessertia (L.) frutescens is among the medicinal plants which caught the interest of conservationists as it is over exploited and risks becoming extinct. Moreover, the worsening climate conditions in South Africa may negatively impact the efficacy and yield of L. frutescens. Despite the multipurpose medicinal benefits associated with L. frutescens, little has been done to conserve the plant. To provide an environmentally friendly solution to the challenges surrounding L. frutescens, this study aimed to explore the use of plant growth promoting rhizobacteria to induce drought and salt stress tolerance in L. frutescens. The rhizobacteria was trapped using a homologous host L. frutescens from rhizosphere soils sampled in the Western Cape and Northern Cape, South Africa, under glasshouse conditions. A total of 111 bacterial isolates with varying morphological characteristics were obtained and 67 authenticated with the homologous host. The genetic diversity, phylogenetic relationships, tolerance to extreme conditions (drought, pH and salinity) and plant growth promoting properties (phosphorus solubilising and indole acetic acid capability) of the authenticated microsymbionts were evaluated for accurate selection of three isolates with superior biomass yield, drought and salt tolerance for further studies. Genetic diversity dendogram clustered the 67 test isolates into 7 major clades, with most isolates grouping according to their province of origin. Meanwhile, the phylogenetic study that targeted multiple genes (16s rRNA, glnII, gyrB, atpD, nifH and nodC) identified genus Mesorhizobium (M.) as the species that formed symbiotic relationship with Lessertia frutescens in the Western Cape and Northern Cape provinces. The specific species involved in the nodulation of L. frutescens had high sequence similarity with M. atlanticum, M. australicum and M. shonense. Furthermore, the tested microsymbionts exhibited markedly different indole acetic acid (IAA) content and phosphorus solubilising (PSB) ability. Majority (81%) of the isolates recorded IAA value less than 10%, while only 48% exhibited ability to solubilise phosphorus. Interestingly, the isolates from the Western Cape province recorded outstanding performance in the pH and salt stress tolerance assay, while the Northern Cape isolates performed better in the drought tolerance assay. These observations were attributed to the ability of rhizobacteria isolates to evolve and device mechanism for survival under extreme conditions, demonstrating the significance of exploring the microsymbionts in the Western Cape and Northern Cape provinces to combat drought and salt induced stress. Ultimately, three rhizobial microsymbionts TUTLFNC33 (biomass yield), TUTLFNC37 (drought tolerance) and TUTLFWC74 (salt tolerance) were selected to ascertain their remarkable traits in a glasshouse pot trial experiment. A glasshouse pot trial experiment was set up to evaluate the three selected rhizobacteria isolates for their performance when used to inoculate L. frutescens under drought and salt stress. Isolate TUTLFNC37 was found to be the most effective to enhance drought and salt stress tolerance in Lessertia frutescens. The rhizobacteria isolate applied various mechanisms including root proliferation, stomatal closure and osmolyte proline accumulation, to enhance tolerance of L. frutescens to drought and salt stress. Most importantly, the concentrations of the metabolites that are unique to Lessertia frutescens (sutherlandiosides A – D and sutherlandins A – D) remained stable under various levels of stress when the plant was inoculated with TUTLFNC37. This study is the first to explore microsymbionts in the Western Cape and Northern Cape provinces for enhancement of drought and stress tolerance in L. frutescens. Although further studies are required to verify the performance of TUTLFNC37 under a field setting and abiotic stress conditions, the findings of this study form significant part of the preliminary work towards developing biological inoculants for Lessertia frutescens and possibly other leguminous medicinal plants.