Development of a bacteriophage-based biopesticide for fire blight

Fire blight is an economically important disease of apples and pears that is caused by the bacterium Erwinia amylovora. Control of the disease depends on limiting primaly blosson1 infection in the spring, and rapidly removing infected tissue. The possibility of using phages to control E.amylovora populations has been suggested, but previous studies have. failed to show high treatment efficacies. This work describes the development of a phage-based biopesticide that controls E. amylovora populations under field conditions, and significantly reduces the incidence of fire blight. This work reports the first use ofPantoea agglomerans, a non-pathogenic relative ofE. amylovora, as a carrier for E. amylovora.phages. Its role is to support a replicating population of these phages on blossom surfaces during the period when the flowers are most susceptible to infection. Seven phages and one carrier isolate were selected for field trials from existing collections of 56 E. amylovora phages and 249 epiphytic orchard bacteria. Selection of the . /' phages and carrier was based on characteristics relevant to the production and field perfonnance of a biopesticide: host range, genetic diversity, growth under the conditions of large-scale production, and the ability to prevent E. amylovora from infecting pear blossoms. In planta assays showed that both the phages and the carrier make significant contributions to reducirig the development of fire blight symptoms in pear blossoms. Field-scale phage production and purification methods were developed based on the growth characteristics of the phages and bacteria in liquid culture, and on the survival of phages in various liquid media. Six of twelve phage-carrier biopesticide treatments caused statistically signiflcant reductions in disease incidence during orchard trials. Multiplex real-time PCR was used to simultaneously monitor the phage, carrier, and pathogen populations over the course of selected treatments. In all cases. the observed population dynamics of the biocontrol agents and the pathogen were consistent with the success or failure of each treatment to control disease incidence. In treatments exhibiting a significantly reduced incidel1ce of fire blight, the average blossom population ofE.amylovora had been reduced to pre-experiment epiphytic levels. In successful treatments the phages grew on the P. agglomerans carrier for 2 to 3 d after treatment application. The phages then grew preferentially on the pathogen, once it was introduced into this blossom ecosystem. The efficacy of the successful phage-based treatnlents was statistically similar to that of streptomycin, which is the most effective bactericide currently available for fire blight prevention. The in planta behaviour ofE. amylovora was compared to that ofErwinia pyrifoliae, a closely related species that causes fire blight-like synlptoms on pears in southeast Asia. Duplex real-time PCR was used to monitor the population dynamics of both species on single blossonls. E. amylovora exhibited a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae. The genome ofErwinia phage Ea21-4 was sequenced and annotated. Most of the 8-4.7 kB genome is substantially different from previously described sequences, though some regions are notably similar to Salmonella phage Felix 01 . Putative functions were assigned to approximately 30% of the predicted open reading frames based on amino acid sequence comparisons and N-terminal sequencing of structural proteins.

Erwinia amylovora bacteriophage resistance

It has been proposed that phages can be used commercially as a biopesticide for the control of fire blight caused by the phytopathogen Erwinia amylovora. The aim of these studies was to investigate two common bacterial resistance mechanisms, lysogeny and exopolysaccharide production and their influence on phage pathogenesis. A multiplex real-time PCR protocol was designed to monitor and quantify Podoviridae and Myoviridae phages. This protocol is compatible with known E. amylovora and Pantoea agglomerans rtPCR primers/probes which allowed simultaneous study of both phage and bacterial targets. Using in vitro positive phage selection, bacteriophage insensitive derivatives were isolated within sensitive populations of E. amylovora. Prophage screening with real-time PCR and mitomycin C induction determined that the insensitive derivatives harboured the temperate Podoviridae phage ΦEaTlOO. Lysogenic conversion resulted in resistance to secondary homologous phage infections. Prophage screening of environmental samples of E. amylovora and P. agglomerans collected from various locations in Canada, United States and Europe did not demonstrate lysogeny. Therefore, lysogeny is rare or absent while these bacterial species reside on the plant. Recombineering was used to construct exopolysaccharide deficient E. amylovora mutants. The EPS amylovoran mutants became resistant to Podoviridae and certain Siphoviridae phages. Increasing amylovoran production increased phage population growth, presumably by increasing the total number of bacterial cell surface receptors which promoted increased phage infections. In contrast, amylovoran did not playa role in Myoviridae infections, nor did production of the EPS levan for any phage pathogenesis.

Role of Exopolysaccharides and Monosaccharides in Erwinia amylovora Resistance to Bacteriophages

Fire blight is a disease caused by the phytopathogenic bacterium Erwinia amylovora, an economically important pathogen in the commercial production of apples and pears. Bacteriophages have been proposed as a commercial biopesticide to relieve the pressures on apple and pear production and provide alternatives to existing biological control options. This work reports on the investigation of host resistance in the development of a phage biopesticide. Exopolysaccharide (EPS) deficient bacterial mutants were generated through recombineering to investigate the role of EPS in bacteriophage adsorption and infection. The mutants that were deficient in amylovoran production were avirulent and resistant to infection by phages of the Podoviridae and some of the Siphoviridae family. Levan deficient bacterial mutants resulted in reduced phage titers in some phages from the Myoviridae family. Exopolysaccharide mimetic monosaccharides were used to demonstrate that levan and amylovoran play an important role in phage attack of E. amylovora.