Gene tagging systems for polymerase chain reaction based monitoring of bacteria released for biological control of weeds

A prerequisite to release of any modified pathogen into the environment, re gardless of origin, is a reliable method for it's absolute identification f rom other strains. Several techniques are available, including lacZY gene o f Escherichia coli, moc gene of Pseudomona fluorescens, green fluorescent p rotein, and lux gene. The latter has worked well for tracking plant pathoge nic Xanthomonas campestris pv. campestris in cabbage fields. We describe in detail a newly developed polymerase chain reaction (PCR)-based genomic sch eme utilizing a 0.52-kilobase (kb) fatty acid desaturase (DES) fragment of the unique tox-argK gene cluster of Pseudomonas syringae pv. phaseolicola f or tagging pathogenic xanthomonads released for biological control of weeds . The fragment is inserted into pGX15, a cosmid clone containing a 10.3-kb EcoR1-HindIII fragment with pigG of the xanthomonadin gene of pIG102 to mak e pGXP6. A 10.8-kb fragment of pGXP6 is then inserted into pLAFR3 to make p LXP22. Finally, the marked strain is constructed by inserting the unique 0. 52-kb DES fragment of P. syringae pv. phaseolicola into a XmaI site within the pigG transcriptional unit on chromosomal DNA using pLXP22 and marker ex change. Results show that DES-tagged strains of X. campestris pv. convolvul i are stable and easily identified by PCR.