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L-form bacteria can be induced in vitro by the treatment of cell walled bacteria with compounds which suppress cell wall synthesis. It has previously been demonstrated that a variety of bacterial L-forms could invade plant-cells and establish a novel symbiotic association within the plant-host, with no apparent host limitations. Such associations were mainly recognised by direct microscopy and immunological detection techniques. The main aim of this project was to develop and optimise a reporter gene system for the detection of genetically modified Bacillus subtilis and Pseudomonas syringae pv. phaseolicola L-form associations in planta . Associations were attempted between B.subtilis L-forms chromosomally marked with -glucuronidase ( gus ) genes and Chinese cabbage. PCR detection indicated the lack of stable plant-L-form associations. Plasmid vectors encoding the genes for luciferase ( lux ) from Vibrio fischeri were introduced into the French bean pathogen Pseudomonas syringae pv. phaseolicola and L-forms were then induced. Symbiotic associations were established between these lux -modified L-forms and Chinese cabbage. Associations were detected at 7 days after germination by PCR amplification of a lux -gene fragment, using 100 ng of total plant DNA as a template. In addition the distribution of lux -modified P.syringae L-forms into various tissues of the plant was monitored using PCR amplification of the lux -gene and Staphylococcus agglutination, these techniques provided information on the presence of DNA and protein-antigens respectively. In contrast, bioluminescence, determined by luminometry, X-ray film imaging and charge-coupled device enhanced microscopy, permitted the detection of intact, active and viable populations of P.syringae L-forms. The bioluminescence based detection assays were non destructive and rapid (5 min) providing direct visualisation of microbial activity in planta . The data suggests that the distribution of lux -modified L-forms is non-uniform, predominantly in the roots and primary leaves of 14 day old Chinese cabbage.

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The aim of this work was twofold: to construct plasmids with a gene encoding a pesticidal protein expressed from an Agrobacterium tumefaciens virulence promoter and to determine, in planta, the sites of Agrobacterium vir-induction. A number of methods were employed to detect in situ vir-induction and, to this end, genes encoding β-glucuronidase (GUS) and bioluminescence (lux) were linked in plasmid constructs to Agrobacterium vir-promoters. In each case, expression of the gene was shown to be induced by the v/r-inducing phenolic compound acetosyringone. An existing plasmid, in which the lacZ gene was under control of the virB promoter was utilised to demonstrate v/r-induction occurring at sites of injury on the roots of mung bean seedlings. Pesticidal genes expressed from Agrobacterium vimlence promoters would form the basis of a biological control system. A microbial inoculant harbouring such a construct would produce the pesticidal protein only when in the presence of vi-inducing compounds in plant wound exudates. A chitinase gene, chiB, from Serratia marcescens was characterised and sequenced and, following removal of its promoter region, was linked to an Agrobacterium virB promoter. Plasmids were also constructed in which the chiA gene of S. marcescens was brought under the control of a virB or virE promoter. All the constructs specified acetosyringone- inducible production of chitinase. Chitinase is effective in the biological control of chitin containing organisms such as fungi.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1930. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 78-83).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1947. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 82-88).