Use of lux-marked genes to monitor antagonistic Pseudomonas syringae on citrus fruits

Pseudomonas syringae strain 48SR2, effective as biological control agent, was genetically tagged with the promoterless lux operon Tn4431 to monitor the population dynamic in in vivo assay. Four hundred thirteen mutants were obtained and diverse bioluminescent activities were observed according to the insertion of Tn4431 into a wide variety of regions of the chromosome. A selected strongly bioluminescent mutant (lux 176) and the wild-type strain were tested for their antagonistic properties to control the post-harvest pathogen Penicillium digitatum. Both the wild type and the luxmarked strain equally reduced the growth of P. digitatum in vitro and the severity and incidence of citrus decay in vivo when the biocontrol agents were applied in wounds 24 h before challenging P. digitatum. The persistence of the genetically engineered bacteria on citrus wounds was monitored over the time with bioluminescence detection systems as well as by dilution plating techniques. Population sizes of both wild-type and lux-mutant strain were comparable. These results indicate that P. syringae strain 48SR2 could be considered a biological control agent for citrus green mould and that bioluminescence can be a sensitive detection method to study population dynamics and antagonistic behaviour during fruit storage.