3.5.15
LUX MARKER TECHNOLOGY FOR THE OPTIMISATION OF BIOCONTROL INVOLVEMENT IN SEED PATHOLOGY


A MCINTOSHl, OGG KNOXl, D WHITEl, LA GLOVER2 and C LEIFERTl

lDepartment of Plant and Soil Science, Aberdeen University, Aberdeen, AB24 2UU, UK; 2Department of Molecular and Cell Biology, Aberdeen University, Aberdeen, UK

Background and objectives
The use of the lux marker technology based on the tranformation of environmental rhizobacteria with genes from the lux operon of Vibrio fischeri and V. harveyi has greatly increased the understanding of the various interactions which occur in the plant-soil system. It has allowed for the study of microbial populations within undisturbed environments and also to track inocula when introduced into different environments. Monitoring populations and the activity of biocontrol agents is important in assessing their effectiveness in colonising and maintaining metabolic activity on plant roots. To complete the development of biocontrol products it is desirable to remove the variability which is observed at present in field trials. This can be achieved by looking at the relationship between the antagonists and the pathogens in terms of their activity and spatial arrangement. Lux is a well understood [1] and suitable marker system as it allows the spatial arrangement of microbes to be observed whilst also indicating which cells are metabolically active.

Results and Conclusions
The A and B genes of the lux operon of Vibrio harveji were introduced into antagonistic Pseudomonas fluorescens and Pseudomonas corrugate via a mini-Tn5 luxAB transposon through mating experiments with Escherischia coli carrying the transposon chromosomally. These lux marked antagonists have proved useful: in studying effects of environmental conditions, such as pH, soil water and nutrient availability, on the ability of these biocontrol agents to colonise the rhizosphere [2]. Experiments were also carried out to lux mark an antagonisic Bacillus subtilis strain. This could not be achieved by mating, transformation of competent cells or protoplast transformation. Successful transformation of this antagionist was achieved with electroporation using a plasmid vector for the lux A and B genes. A broad range of methods and vectors are now available in our Departments for marking bacterial biocontrol products with lux genes and this technology has greatly aided our understanding of the biocontrol of soil diseases.

References
1. Meighen EA, 1991. Microbiological Reviews 55, 123-142.
2. White D, Leifert C, Ryder MH and Killham K, 1996. New Phytologist 133, 173-181.