Biserrula pelecinus L. has been introduced to Australia from the Mediterranean region, in the last decade due to many attractive agronomic features. This deep rooted, hard seeded, acid tolerant and insect resistant legume species provides high quality food for cattle and sheep, and grows well under the harsh edaphic and environmental conditions of Australia. In 1994, B. pelecinus was introduced to a site in Northam, Western Australia where there were no native rhizobia capable of nodulating this legume. The introduced plants were inoculated with a single inoculant strain of Mesorhizobium sp., WSM1271. This study investigated whether a diversity of rhizobia emerged over time. A second objective was to investigate the possible mechanisms involved in the diversification of rhizobia able to nodulate B. pelecinus. Eighty eight isolates of rhizobia were obtained from nodules on B. pelecinus growing at the Northam site in August 2000, six years after introduction. These plants were self-regenerating offspring from the original seeds sown. Molecular fingerprinting PCR with RPO1 and ERIC primers revealed that seven strains (novel isolates) had banding patterns distinct from WSM1271 while 81 strains had similar banding patterns to WSM1271. A 1400 bp internal fragment of the 16S rRNA gene was amplified and sequenced for four of the novel isolates (N17, N18, N45 and N87) and WSM1271. The phylogenetic tree developed using these sequences clustered the novel isolates in Mesorhizobium. There were >6 nucleotide mismatches between three of the novel isolates (N17, N18, N87) and WSM1271 while there were 23 nucleotide mismatches between N45 and WSM1271. When B. pelecinus cv. Casbah was inoculated with the novel isolates, five (N17, N18, N39, N46 and N87) yielded <40% of the shoot dry weight of the plants inoculated with the original inoculant (WSM1271). Novel isolates N15 and N45 were completely ineffective on B. pelecinus cv. Casbah. Physiological experiments to test the ability of the novel isolates and WSM1271 to grow on 14 different carbon sources (N acetyl glucosamine, arabinose, arbutine, dulcitol, β-gentiobiose, lactose, maltose, melibiose, D-raffinose, saccharose, L-sorbose, D-tagatose, trehalose and D-turanose) as the sole source of carbon, intrinsic resistance to eight different antibiotics (ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, spectinomycin, streptomycin and tetracycline) and pH tolerance (pH 4.5, 5.0, 7.0, 9.0) revealed that the novel isolates had significantly different carbon source utilization patterns to WSM1271. However, pH tolerance and intrinsic resistance to antibiotics were similar between the novel isolates and WSM1271 except for streptomycin (100 μg/ml). Novel isolates N17, N18, N46 and N87 were susceptible for this antibiotic while the other novel isolates and WSM1271 were resistant. Host range experiments were performed for the novel isolates N17, N18, N45, N87, WSM1271 and two other root nodule bacteria (RNB) previously isolated from B. pelecinus growing in the Mediterranean region (WSM1284 and WSM1497) for twenty one legumes (Amorpha fruticosa, Astragalus adsurgens, Astragalus membranaceus, Astragalus sinicus, Biserrula pelecinus cv Casbah, Dorycnium hirsutum, Dorycnium rectum, Glycyrrhiza uralensis, Hedysarum spinosissimum, Leucaena leucocephala, Lotus corniculatus, Lotus edulis, Lotus glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus parviflorus, Lotus pedunculatus, Lotus peregrinus, Lotus subbiflorus, Macroptilium atropurpureum, and Ornithopus sativus). Only isolate N17 have the same host range as WSM1271 in that they both nodulated B. pelecinus and A. membranaceus, while the other three novel isolates, WSM1284 and WSM1497 had a broader host range than WSM1271. Three isolates N18, N45 and N87 formed small white nodules on M. atropurpureum, in addition to nodulating the above hosts. Isolates N18 and N45 also nodulated A. adsurgens while N45 was the only isolate to nodulate L. edulis. Isolate N87 was the only isolate to nodulate A. fruticosa. WSM1497 nodulated A. adsurgens, A. membranaceus, B. pelecinus and L. corniculatus while WSM1284 was a promiscuous strain that nodulated 16 host species out of the 21 tested. A 710 bp internal region of nifH, a 567 bp internal region of nodA and a 1044 bp internal region of intS were sequenced for N17, N18, N45, N87 and WSM1271. The sequence comparison showed that the sequences of the above three genes of the four novel isolates were identical to that of WSM1271. Eckhardt gel electrophoresis revealed that WSM1271, three other RNB isolates from B. pelecinus from the Mediterranean region and isolate N18 each have a plasmid of approximately 500 kb while N17, N45 and N87 are plasmid free. Probing of the plasmid DNA from the Eckhardt gel with nifH and nodA probes indicated that these two genes were not located on the plasmid. Furthermore, the results of this study demonstrated that 92% of the nodules on B. pelecinus growing in the Northam site six years after the introduction of this plant were occupied by the inoculant strain and the N2 fixation efficiency of the progeny strains of WSM1271 remain similar to the mother culture. This study also showed that the carbon source utilization pattern, intrinsic antibiotic resistance and pH range of the progeny strains of WSM1271 remain relatively similar, except for few variations in carbon source utilization patterns. This thesis clearly demonstrated that phenotypicaly, genetically and phylogenetically diverse strains capable nodulating B. pelecinus evolved through symbiotic gene transfer from the inoculant strain to other soil bacteria within six years. The presence of intS, and the evidence of gene transfer between these Mesorhizobium strains indicates that transfer of symbiotic genes may have occurred via a symbiosis island present in WSM1271.

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. / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

The conversion of atmospheric dinitrogen (N2) into plant available nitrogen (N), by legumes and their prokaryotic microsymbionts, is an integral component of sustainable farming. A key constraint to increasing the amount of N2 fixed in agricultural systems is the prevalence of symbioses which fix little or no N. The biotic factors leading to this suboptimal N2 fixation have not been extensively analysed. Using the widely studied and cultivated perennial legume Medicago sativa and the model indeterminate annual legume Medicago truncatula with the sequenced bacterial microsymbiont Sinorhizobium meliloti 1021 (Sm1021) as a basis, the work presented in this thesis examined the effectiveness of N2 fixation in these associations and in other comparable systems and investigated factors which lead to the establishment of suboptimally effective symbioses. The ability of Sm1021, S. medicae WSM419 and the uncharacterised Sinorhizobium sp. WSM1022 to fix N with M. truncatula A17, M. sativa cv. Sceptre and a range of other Medicago spp. was evaluated in N-limited conditions. As measured by plant shoot dry weights and N-content, Sm1021 was partially effective with M. truncatula A17 whereas WSM1022 and WSM419 were both effective with this host in comparison to nitrogen-fed (N-fed) control plants. In contrast, Sm1021 and WSM1022 were effective with M. sativa while WSM419 was only partially effective. Nodules induced by Sm1021 on M. truncatula A17 were more numerous, paler, smaller in size and more widely distributed over the entire root system than in the two effective symbioses with this host. On the contrary, nodule number, size and distribution did not differ between these three strains on M. sativa. WSM1022 was effective on M. littoralis, M. tornata and two other cultivars of M. truncatula (Jemalong and Caliph) but Sm1021 was only partially effective on these hosts. These data indicate that the model indeterminate legume symbiosis between M. truncatula and Sm1021 is not optimally matched for N2 fixation and that Sm1021 possesses broader symbiotic deficiencies. In addition, the interaction of WSM1022 with M. polymorpha (small white nodules but does not fix N), M. murex (does not nodulate), M. arabica (partially effective N2 fixation) and M. sphaeorcarpus (partially effective N2 fixation), and the sequence of the 16S rDNA, are all consistent with this isolate belonging to the species S. meliloti. The colony morphology of TY, half-LA and YMA agar plate cultures of Sm1021, WSM419 and WSM1022 suggested differences in EPS profiles between these strains. Sm1021 is very dry, compared to the mucoid WSM419 and extremely mucoid WSM1022. Sm1021 is known to carry an insertion in expR rendering the gene non-functional and resulting in the dry colony phenotype. WSM419 has an intact copy of expR, while the expR status of WSM1022 is not known. Rm8530, a spontaneous mucoid derivative of Sm1021 with an intact expR, was significantly less effective with M. truncatula than Sm1021, but there was no difference in effectiveness between these strains on M. sativa. The effectiveness of Sm1021, when complemented with a plasmid-borne copy of expR from Rm8530, was significantly reduced on M. truncatula but not M. sativa, implicating a functional expR as being the cause of reduced N2 fixation observed with Rm8530 on M. truncatula. ExpR could reduce the effectiveness of Rm8530 by acting as a negative regulator of genes essential for symbiosis with M. truncatula, or by altering the quantity or structure of succinoglycan and/or galactoglucan produced. These data support the emerging view of ExpR being a central regulator of numerous cellular processes. The timing of nodulation between Sm1021 and WSM419 on M. truncatula and M. sativa was investigated. Compared to the other symbioses analysed, the appearance of nodule initials and nodules was delayed when M. truncatula was inoculated with Sm1021 by 3 and 4 days, respectively. To explore whether events during early symbiotic signalling exchange could account for these observed delays, leading to the establishment of a suboptimal N2-fixing symbiosis, a novel system was developed to compare the response of the Sm1021 transcriptome to roots and root exudates of M. truncatula A17 and M. sativa cv. Sceptre. This system consisted of a sealed 1 L polycarbonate chamber containing a stainless steel tripod with a wire mesh platform on which surface-sterilised seeds could be placed and allowed to germinate through the mesh, into a hydroponic medium below. After germination, Sm1021 cells were inoculated into the hydroponic solution, exposed to the roots and root exudates for 16 h, harvested and their RNA extracted. Comparison of Sm1021 mRNA from systems exposed to M. truncatula or M. sativa revealed marked differences in gene expression between the two. Compared to the no plant control, when M. sativa was the host plant, 23 up-regulated and 40 down-regulated Sm1021 genes were detected, while 28 up-regulated and 45 down-regulated genes were detected with M. truncatula as the host. Of these, 12 were up-regulated and 28 were down-regulated independent of whether M. truncatula or M. sativa was the host. Genes expressed differently when exposed to either M. truncatula or M. sativa included nex18, exoK, rpoE1 and a number of other genes coding for either hypothetical proteins or proteins with putative functions including electron transporters and ABC transporters. Characterisation of these differentially expressed genes along with a better understanding of the composition of M. truncatula root exudates would yield a clearer insight into the contribution of early signal exchange to N2 fixation. Comparison of the regulation of nodule number between Sm1021 and WSM419 on M. truncatula and M. sativa revealed nodule initials at 42 days post-inoculation (dpi) on M. truncatula inoculated with Sm1021. In contrast, no new nodule initials were present 21 dpi on any of the other interactions examined. Moreover, analysis of nodule sections revealed that the number of infected cells in M. truncatula-Sm1021 nodules was less than for comparable symbioses. These data suggest that nodule number is not tightly controlled in the M. truncatula-Sm1021 association, probably due to N2 fixation being insufficient to trigger the down regulation of nodulation. Quantification of N2 fixation activity in this and other more effective symbioses is required. The poor effectiveness of the M. truncatula-Sm1021 symbiosis makes these organisms unsuitable as the model indeterminate interaction and the implications for legume research are discussed. The recently sequenced WSM419 strain, revealed here to fix N2 more effectively with M. truncatula than Sm1021, may be a better model microsymbiont, although WSM419 is only partially effective for N2 fixation with M. sativa. The sequencing of S. meliloti WSM1022, a highly effective strain with both M. truncatula and M. sativa, would provide a valuable resource in indentifying factors which preclude the establishment of effective symbioses.