Bacterial L-form associations with plants
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.