Rapid detection system for important plantpathogenic bacteria using luminescent phage

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For the detection of plant pathogenic bacteria, Ralstonia solanacearum which is a causal agent bacterial blight of Solanaceous plants, we first isolated the phage specifically infect this bacteria. Its genome was sequenced and the subclone containing the putative lysozyme-like encoding gene was obtained. The cassette containing luciferase genes (luxA and luxB) of Vibrio fisherii was inserted into this lysozyme gene. This construct was found to produce light in the presence of its substrate, tetra-decanal. Then, this plasmid was used as the feeder of lux-inserted lysozyme gene by marker exchanged into the phage lysozyme gene. When the constructed reporter phage was infected to various concentration of R. solanacearum, we found that the light production was detected even 100 cells in the sample. At the same time, we tested the applicability of the phage which was labeled with Biotin to use for the detection directly the pathogen after attachment with streptoavidin coupled with peroxidase. We could up to 100 bacterial cells detect by reading chemiluminescent product by the action of peroxidase. This latter method was shown to be equally effective for the detection of this bacteria.For the detection of Xanthomonas axonopodis pv. citri, a causative agent of citrus canker, we integrated the above luxA,B cassette into the gene encoding putative coat protein using similar method. When this reporter phage was used for the detection of this pathogen, we could detect even 100 of this bacteria. We also tried the phage labeled with Ethidium bromide. By infecting with high moi of the labeled phage, we could detect up to 1000 bacteria by measuring the fluorescence of the attached phages on the bacteria.