Molecular profile of phage infection: A novel approach for the characterization of Erwinia phages through qPCR

Bacteriophage-mediated biocontrol has become an attractive alternative for control of pathogens in agriculture due to the emergence of antibiotic resistance and the consumers demand for pesticide-free food. Bacteriophages, or phages, are viruses that specifically attack bacteria. Historically, phage infection characteristics and productivity was described using plaque assays on artificial media and optical density decrease in bacterial suspensions. Data generated using phages that infect the apple pathogen Erwinia amylovora have a low reproducibility with these techniques. Phage and pathogen populations were determined by quantitative PCR (qPCR) using a standardized plasmid which detects specific phage and the pathogen populations. The system measures copies of DNA/ml instead of the traditional PFU/ml. The stage of the phage genome in the lytic cycle was determined through a combination of chloroform-based sampling, centrifugation, and DNase treatments. The phage genomes can then be monitored through the entire lytic cycle from adsorption, the phage attachment, to release of fully formed phage particles from lysed bacterial cells. The qPCR data provides a molecular profile from which phage infection characteristics such as adsorption rate, burst size, and the latent period can be determined. To our knowledge, this is the first report of a molecular technique being used for simultaneous determination of infection parameters. The characteristics of four different genera of Erwinia phages were determined when infecting their preferred hosts. The phage from the Sp6virus genus was able to adsorb to its host at a rate up to 6.6 times faster than Ea92virus and Agrican357virus. The low maximum enrichment titre of Ea92virus was shown to be due to the delay or absence of lytic burst within most E. amylovora hosts. The Sp6virus and Ea214virus phages had the highest production rate with a burst size of 57 virions in 38 min and 185 virions in 98 min respectively, suggesting these genera would make stronger candidates for phage-mediated biocontrol against E. amylovora. The information from this study will provide researchers critical data on the combination of phage genera and specific phages that should be included in the biocontrol phage mixtures or cocktails in order to obtain maximum control of the pathogen under field conditions.