Host range of bacteriophages against a world-wide collection of erwinia amylovora determined using a quantitative PCR assay

Citation

Gayder, S., Parcey, M., Castle, A.J., Svircev, A.M. (2019). Host range of bacteriophages against a world-wide collection of erwinia amylovora determined using a quantitative PCR assay. Viruses, [online] 11(10), http://dx.doi.org/10.3390/v11100910

Plain language summary

Fire blight is the the common name given to a disease of apples and pears that is caused by the bacterial pathogen, Erwinia amylovora. Disease symptoms associated with fire blight in the orchard were first observed in North America in the 18th century. Subsequent migration of people and infected bud wood has led to a global spread of this pathogen from North America to 72 countries across 6 continents. Bacteriophages, or phages, are bacterial viruses that attack and destroy the pathogenic bacteria. The bacterial viruses are being developed as biological control products which act as supplements or alternatives to antibiotics during open bloom. The researchers developed a small circular piece of DNA, called a standardised plasmid, which enabled simultaneous quantification of phage and bacteria using real time PCR, or quantitative PCR (qPCR). The qPCR protocol can simultaneously quantify the populations of phages used in the biological control program and the pathogen, E. amylovora. The plasmid and qPCR were used to quantify the total number of phage produced by each of the 106 global isolates of E. amylovora. This novel method was used to establish the host range of the AAFC bacteriophages used in the biological control program. Nine of the 10 phages exhibited a broad host range having the ability to infect over 88% of the global bacterial hosts. Every bacterial host had the ability to increase the phage initial concentration 10 fold. Bacterial hosts from western North America were less susceptible to most phages, as the mean phage population per bacterium dropped by as much as a 100 fold. The ability to infect a bacterial host was strongly correlated to the amount of exopolysaccharide produced by the host. Exopolysaccharides are complex sugars produced by the bacteria on the exterior of the cell. The qPCR and the plasmid method allowed a highly efficient and quantitative establishment of host range. This analysis was faster and required less effort and materials than the traditional plaque assay techniques. The host data has demonstrated that the 10 AAFC phages have the ability to control the fire blight on the global scale. In addition, this qPCR based host range data may be used to determine which phages should be selected into a phage-mixture for geographic and global control of E. amylovora.

Abstract

Erwinia amylovora is a globally devastating pathogen of apple, pear, and other Rosaceous plants. The use of lytic bacteriophages for disease management continues to garner attention as a possible supplement or alternative to antibiotics. Aquantitative productive host range was established for 10 Erwinia phages using 106 wild type global isolates of E. amylovora, and the closely related Erwinia pyrifoliae, to investigate the potential regional efficacy of these phages within a biopesticide. Each host was individually infected with each of the 10 Erwinia phages and phage production after 8 h incubation was measured using quantitative real time PCR (qPCR) in conjunction with a standardized plasmid. PCR amplicons for all phages used in the study were incorporated into a single plasmid, allowing standardized quantification of the phage genome copy number after the infection process. Nine of the tested phages exhibited a broad host range, replicating their genomes by at least one log in over 88% of tested hosts. Also, every Amygdaloideae infecting E. amylovora host was able to increase at least one phage by three logs. Bacterial hosts isolated in western North America were less susceptible to most phages, as the mean genomic titre produced dropped by nearly two logs, and this phenomenon was strongly correlated to the amount of exopolysaccharide produced by the host. This method of host range analysis is faster and requires less effort than traditional plaque assay techniques, and the resulting quantitative data highlight subtle differences in phage host preference not observable with typical plaque-based host range assays. These quantitative host range data will be useful to determine which phages should be incorporated into a phage-mediated biocontrol formulation to be tested for regional and universal control of E. amylovora.

Publication date

2019-10-01

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