Development of a bacteriophage-based biopesticide for fire blight.

摘要

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 primary blossom 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 of Pantoea agglomerans, a non-pathogenic relative of E. 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 performance 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 reducing 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 significant 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 incidence of fire blight, the average blossom population of E. 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 treatments was statistically similar to that of streptomycin, which is the most effective bactericide currently available for fire blight prevention.; The in planta behaviour of E. amylovora was compared to that of Erwinia pyrifoliae, a closely related species that causes fire blight-like symptoms on pears in southeast Asia. Duplex real-time PCR was used to monitor the population dynamics of both species on single blossoms. E. amylovora exhibited a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae.; The genome of Enwinia phage &phis;Ea21-4 was sequenced and annotated. Most of the 84.7 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.