The diversity of root nodule bacteria associated with Lebeckia species in South Africa

Abstract

Nitrogen-fixing diazotrophic root-nodule bacteria are of immense economic importance because of their symbiosis with leguminous plants. Diazotrophic bacteria infect the host legume root inducing the formation of nodules in which the bacteria (also termed rhizobia) replicate and synthesize the enzyme nitrogenase. This enzyme catalyzes the reduction of atmospheric dinitrogen to ammonia for subsequent use by the plant as a major source of nitrogen. Nitrogen is an essential element required by plants for growth and synthesis of protein and is usually the most limiting element in agricultural production as well as being the most expensive component of fertilizer. The aim of my study was to determine the diversity and taxonomy of a specific group of root nodule bacteria associated with different species of Lebeckia. The genus Lebeckia Thunb. (Family Leguminosae, subfamily Papilionoideae, tribe Crotalarieae) comprises some 33 plant species. These plants are mainly indigenous to the southern and Western Cape regions of South Africa. They are divided into shrubby trifoliate-leaf species in the sections Calobota, Stiza and Viborgioides and suffrutescent unifoliate needle-leaf species in the section Lebeckia. Plants of this genus are adapted to harsh environmental conditions such as are found in the Karoo and Namaqualand. Several Lebeckia species are beneficial, such as L. spinescens and L. multiflora, which are valuable as pasture legumes and well grazed by animals especially in winter. All the species have ecological value because of their nitrogen-fixing symbiosis with rhizobia. To my knowledge, no attempts have been made in the past to investigate these microsymbionts of Lebeckia. Root nodules were collected from Lebeckia species at a wide variety of localities in the western and southern Cape regions of South Africa. Indigenous rhizobia isolated from these nodules were investigated for their nodulation abilities on their respective host plants as well as on non-host promiscuous legumes, cowpea and siratro. The isolates were then characterized using random amplified DNA fingerprinting followed by DNA sequencing of selected isolates. Results presented in this study showed that the indigenous South African genus Lebeckia is nodulated by diverse rhizobia from both α- and β-Proteobacteria. The first chapter contains a literature review of symbiotic nitrogen fixation that includes a general description of the biology, inoculant technology and the taxonomy of legumes and their rhizobia. The genera within the tribe Crotalarieae (such as Crotalaria, Lotononis, and Aspalathus) were discussed with special reference to the genus Lebeckia. Technical methods used for the classification of rhizobia were also reviewed. Non-DNA-based methods such as host specificity, substrate utilization, antibiotic resistance, morphological characters and biochemical properties as well as DNA based fingerprinting methods (ARDRA, RFLP RAPD, and AFLP), DNA sequence information, analysis of whole genomes, DNA-DNA hybridization and polyphasic approaches were outlined. The second chapter describes the isolation of 79 rhizobial isolates from the root nodules of 10 Lebeckia species. The isolates were purified and tested for nodulation and nitrogen fixation on cowpea and siratro as well as their host plants. All the isolates fixed nitrogen on their respective Lebeckia hosts, whereas 56% of the strains were effective on cowpea and 77% on siratro. The third chapter describes initial comparison and screening of the isolates by SP-PCR fingerprinting analysis. DNA profiles showed that most of the isolates grouped according to host plant species rather than geographical location. Isolates selected from different clusters were subjected to partial 16S rDNA gene sequencing to confirm their taxonomic identity. This revealed that Lebeckia is nodulated by diverse genera of root nodule bacteria from both the α-Proteobacteria (Bradyrhizobium, Mesorhizobium, and Sinorhizobium) as well as the β-Proteobacteria (Burkholderia). The final chapter (Chapter 4) provides concluding remarks on my results and possible future avenues of research on the Lebeckia rhizobia.