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Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves -). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves -). Also available on the Internet.

The usefulness of RAPDs (Random Amplified Polymorphic DNA) to distinguish among different taxa of Lotus was evaluated. The following species were included: L. corniculatus, L. tenuis, L. alpinus, L. japonicus, and L. uliginosus. Several accessions for each species were studied. Following DNA extraction, amplification reactions were performed in a Hybaid DNA Thermal Cycler, and the product visualized according to a standard procedure. Twenty primers were used for each species/accession. Clear bands and several polymorphisms were obtained for all primers. A phenogram was drawn based on the genetic distance among the species. L. alpinus appears as the most distant species from L. corniculatus, followed by L. uliginosus, L. tenuis, and L. japonicus. With the exception of L. alpinus, these findings are in agreement with previous experimental studies in the L. corniculatus group. The use of a greater number of primers and increased number of species may provide a greater resolution of the systematics of these taxa.

A existência de rizóbios nativos eficientes na fixação simbiótica do nitrogênio em leguminosas introduzidas nos campos do Rio Grande do Sul, como as forrageiras do gênero Lotus, é um fator determinante para adaptação ou o fracasso destas novas espécies. Este trabalho visou estudar a compatibilidade simbiótica de rizóbios simbiontes em Desmodium incanum, uma planta nativa dos campos do Rio Grande do Sul, e em L. corniculatus, L. uliginosus, L. glaber e L. subbiflorus. Para isto 35 isolados e 17 estirpes de rizóbios foram estudadas. Os isolados foram caracterizados fenotípicamente quanto a morfologia colonial, produção de ácido indol-acético e sideróforos. Os rizóbios foram estudados quanto a compatibilidade simbiótica pela inoculação cruzada com as espécies de plantas estudadas. Observou-se existem rizóbios nativos dos solos do Rio Grande do Sul capazes de produzir auxinas e sideróforos. Os rizóbios estudados neste trabalho foram sensíveis a salinidade. Também observou-se que todos os rizóbios isolados de Lotus foram capazes de induzir nodulação em pelo menos duas das cinco espécies estudadas. Os rizóbios de L. uliginosus que apresentaram compatibilidade com as cinco espécies de plantas estudadas, já os de D. incanum mostraram alta especificidade. Em experimento em casa de vegetação as plantas de L. corniculatus inoculadas com os o isolado UFRGS Lu 2 e as estirpes EEL 698 e SEMIA 816 produziram maior massa seca da parte aérea. Este isolado também foi eficiente em plantas de D. incanum. Os resultados deste trabalho mostram que existem rizóbios autóctones dos solos do Rio Grande do Sul que apresentam baixa especificidade hospedeira sendo capazes de formar simbiose com plantas de D. incanum, L. corniculatus, L. subbiflorus, L. glaber e L. uliginosus. A compatibilidade simbiótica com diferentes hospedeiros dificultou o estabelecimento de grupos de compatibilidade. A técnica de REP-PCR com o oligonucleotídeo iniciador BOX A1 mostrou que existe variabilidade entre os rizóbios estudados, sendo estes, diferentes das estirpes fornecidas pelas coleções de cultura. / The presence of native Rhizobia which are efficient in symbiotic nitrogen fixation in legumes introduced in the fields of Rio Grande do Sul, such as the forages plants of the genus Lotus, is one of the factors determining the success or the failure of the adaptation of these new plant species. This work aimed to evaluate the symbiotic compatibility of Rhizobia in association with Desmodium incanum, a native plant from the fields of Rio Grande do Sul, as well as with L. corniculatus, L. uliginosus, L. glaber and L. subbiflorus species. For this, 35 isolates and 17 lineages of Rhizobia were analyzed. The isolates were characterized phenotypically based on the colony morphology and on the indole acetic acid and siderophore production. The Rhizobia were studied for symbiotic compatibility by cross-inoculation with the plant species studied. It was observed that there are indigenous Rhizobia in the soils of Rio Grande do Sul capable of producing siderophores and auxins. All of Lotus Rhizobia were able to induce nodulation in at least two of the five studied species. Rhizobia strains isolated from L. uliginosus have shown a broad specificity, being able to induce nodulation in all studied species. On the other hand, Rhizobia isolated from D. incanum have shown to be highly specific. In a green-house experiment the plants of L. corniculatus inoculated with the isolate UFRGS Lu 2 and strains EEL 698 and SEMIA 816 produced higher shoot dry mass. This isolate was also efficient in D. incanum plants. The results show that there are indigenous Rhizobia in the soils of Rio Grande do Sul, which have low host specificity and are capable of forming symbiosis with D. incanum, L. corniculatus, L. subbiflorus, L. glaber and L. uliginosus plants. The symbiotic compatibility with different hosts made difficult the establishment of compatibility groups. The technique of REP-PCR using primer BOX A1 showed differences among the Rhizobia studied, which are different from strains provided by culture collections.

Integrative and conjugative elements (ICEs) are a newly appreciated group of mobile elements that contribute immensely to bacterial evolution. The symbiosis island ICEMlSym[R7A] of Mesorhizobium loti strain R7A is the largest known mobile ICE (502 kb) and confers the ability to form a nitrogen-fixing symbiosis with plants of the genus Lotus. Transfer of ICEMlSym[R7A] to non-symbiotic bacteria has been demonstrated both in the laboratory and the environment and involves chromosomal integration of ICEMlSym[R7A] adjacent to the phe-tRNA gene in the recipient bacterium. Integration of ICEMlSym[R7A] requires the ICEMlSym[R7A]-encoded protein IntS which likely catalyses recombination between the attachment sites attP and attB, present on excised ICEMlSym[R7A] and phe-tRNA gene respectively. In this study it was shown that intS was required for both excision and integration and was required in both the donor and recipient bacterium for efficient transfer. A minimal attP region required for integration was defined and a repetitive sequence motif that likely represents the IntS-binding sequence identified. intS is expressed from two alternate promoters, a weak promoter on attL (left ICEMlSym[R7A]-chromosome junction) and a strong promoter formed on attP. Analysis of nested deletions of the attP region demonstrated that an unusually large region of attP, corresponding to that required for integration, was required for full gene expression. A novel recombination directionality factor RdfS was identified using bioinformatics. A quantitative PCR assay developed to detect the presence of excised ICEMlSym[R7A] in bacterial populations showed that excision of ICEMlSym[R7A] was almost undetectable in an rdfS mutant. Constitutive expression of rdfS resulted in curing of ICEMlSym[R7A] from R7A, creating a non-symbiotic derivative R7ANS. Bioinformatic analyses of rdfS and associated genes on ICEMlSym[R7A] led to discovery of 26 putative ICEs (ICESym family) in 12 α-proteobacterial species. The ICESym clusters contained homologues of up to 25 ICEMlSym[R7A] proteins, encoding functions involved in mating pore formation, DNA processing and regulation, of which 17 proteins were universally conserved. Introduction of pJR174 containing a copy of the ICEMlSym[R7A]-encoded quorum sensing (QS) gene traR induced excision in 100% of cells and stimulated a 100-fold increase in ICEMlSym[R7A] transfer. Stable maintenance of ICEMlSym[R7A] in these cells required the DNA relaxase-encoding gene rlxS. Introduction of pJR174 also induced a 1000-fold increase in the production of 3-oxo-C6-homoserine lactone and several other acyl homoserine lactones; this induction required traI1. The induction of QS was accompanied by various growth-inhibitory effects, similar to those observed in other rhizobial QS systems. The QS system activated ICEMlSym[R7A] excision through expression of msi172 and msi171, genes that are downstream of QS gene traI2. Expression of the adjacent gene msi170 separately repressed both QS and excision. The expression of msi170 was negatively regulated by Msi169, while msi169 was positively autoregulated. msi169 encodes an Xre family DNA-binding protein and homologues of it were identified on the ICESym clusters and on QS-regulated plasmids, suggesting that they have a conserved role as regulators of transfer- and QS-related genes. Overall, the results suggest that ICEMlSym[R7A] excision and transfer are subject to both population-density and cell-cycle-dependent regulation.

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A method was developed for rapid plant regeneration from protoplasts of birdsfoot trefoil (Lotus corniculatus cv. Leo) using in vitro cotyledons and a new enzyme formula. Protoplasts of a transgenic Nicotiana tabacum cv. Xanthi line KCR were isolated from leaves, in vitro shoots, and calli by an enzyme formula consisting of 0.5 % Cellulase R-10 and 0.05 % Pectolyase Y23 with either 0.4 M sucrose or 0.5 M mannitol as the osmoticum. Nine asymmetric fusion experiments were conducted between iodoacetate inactivated birdsfoot trefoil and irradiated transgenic tobacco protoplasts. The fusion products underwent several divisions but no visible colonies were obtained. Resistance analyses revealed the inactivation of the herbicide resistant gene in the transgenic tobacco calli. A putative chlorsulfuron resistant line has been obtained by in vitro selection from birdsfoot trefoil cotyledonous protoplasts. Their regenerants had normal ploidy, and expressed improved tolerance when sprayed with chlorsulfuron at the rate of 30 g/ha.

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