Interactions and Population Dynamics Between Erwinia amylovora, Pantoea agglomerans, and their Bacteriophages for Effective Phage Therapy

Abstract

Fire blight is a globally devastating disease of apples, pears, and other rosaceous plants caused by the bacterial phytopathogen Erwinia amylovora. Our lab is developing a dual-action biological control product using the epiphytic bacterial antagonist Pantoea agglomerans and a cocktail of bacterial viruses called bacteriophages. P. agglomerans act as host cells for a cocktail of phages targeting E. amylovora in planta and also exclude the pathogen from the blossom pistil and hypanthia by natural antagonism and antibiotic production. The objective of this research is to understand the dynamics between the pathogen, the carrier, and their phages and use this information to develop an effective phage-carrier system for the control of E. amylovora. A novel DNA plasmid was created which allows simultaneous quantification of E. amylovora, P. agglomerans, and four Erwinia phage species using quantitative real-time PCR (qPCR). This plasmid standard-based qPCR was used to study the host range of 10 phages against a global collection of Erwinia strains (n=106) and potential carriers (n=30), and to study the population dynamics of Erwinia phages in different host and cocktail combinations in unprecedented detail. The host range on E. amylovora revealed global phage resistance is located largely in western North America, and this resistance is due in part to a greater effect of exopolysaccharide production by these hosts. Also three phages, ɸEa21-4, ɸEa46-1-A1, and φEa35-70, were chosen to investigate for potential biocontrol efficacy. Phage ɸEa35-70, while ineffective alone, synergized with both ɸEa21-4 and ɸEa46-1-A1 for enhanced reduction of E. amylovora growth over 24 h. The competition between ɸEa21-4 and ɸEa35-70 was exploited which maximized carrier survival and lead to the design of an effective phage-carrier combination which will be formulated and further investigated for the control of E. amylovora in planta. Finally, the quantitative host range data and host genomic sequences were used to identify 10 host genes that are potentially associated with phage infection using a novel, k-mer based, genome wide association study (GWAS). These genes are indicative of a potential phage receptor, two novel phage resistance mechanisms, and other metabolic and biological functions which may affect phage infection.