ARS | Publication request: Development of an engineered ‘bioluminescent’ reporter phage for the detection of bacterial blight of crucifers

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 7, 2012
Publication Date: May 1, 2012
Citation: Schofield, D.A., Bull, C.T., Rubio, I., Wechter, W.P., Westwater, C., Molineux, I.J. 2012. Development of an engineered ‘bioluminescent’ reporter phage for the detection of bacterial blight of crucifers. Applied and Environmental Microbiology. 78:3592-3598.

Interpretive Summary: The control of bacterial plant pathogens is fundamental to providing high quality food and fiber to consumers. Disease control and prevention is dependant upon understanding what pathogens cause disease and their epidemiology. However microorganisms are small and it is difficult to determine whether they are present in the environment or on plants. This manuscript describes a new method that can rapidly indicate if a plant pathogen is present in levels high enough to cause disease. Furthermore, data generated using these tools will allow researchers to design preventative disease control strategies.

Technical Abstract: Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassica family. The disease is often misdiagnosed as peppery leaf spot, a much less severe disease caused by the closely related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage based diagnostic that can diagnose the causative agent of bacterial blight, and can differentiate the two pathogens. A recombinant “light”-tagged reporter phage was generated by integrating bacterial luxAB genes encoding the luciferase enzyme into the genome of P. cannabina pv. alisalensis phage PBS1. The reporter phage is viable, stable and retains similar properties to the wild-type and is able to detect P. cannabina pv. alisalensis rapidly and sensitively by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Non-alisalensis bacterial pathogens of Brassicas such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response, or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens indicating its potential for disease identification. This reporter phage displays promise for the rapid and specific diagnostic detection of cultivated isolates, and infected plant specimens.