Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Program Name/Specialization

Biological and Chemical Sciences

Faculty/School

Faculty of Science

First Advisor

Joel Weadge

Advisor Role

Supervisor-Mentor

Second Advisor

Hany Anany

Advisor Role

Supervisor-Mentor

Abstract

Fire blight is known as a disease of Rosaceae and it displays infection symptoms in about 200 species in 40 rosaceous genera. The most economically important crops that are affected by fire blight are apples and pears. Fire blight can cause severe outbreaks and a single fire blight outbreak can disrupt orchard production for years. The bacterium that causes this disease is indigenous in North America, and according to Statistics Canada, economic losses in apples and pears were valued at an estimated $4 million in 2005, which is approximately 5% of the total production. In addition, production costs can increase substantially during a fire blight spread because labor costs for diligent and regular pruning trips through the orchard are required.

Erwinia amylovora is the causal agent of the contagious fire blight disease. This bacterium can infect different parts of the tree, such as blossoms, shoots, vascular tissues, rootstock crowns, and fruits. The bacterial ooze that occurs during infection consists of a high population of E. amylovora found within self-produced exopolysaccharides and the ooze is the main source for the spread of infection between plants. Once on a host, the pathogen spreads systemically after entering through natural openings (nectarthodes, stomata) or wounds. Depending on the plant part affected, disease symptoms develop as blossom blight, shoot blight or rootstock blight.

Streptomycin is considered the most effective bactericide against E. amylovora. However, the emerging resistance of E. amylovora to streptomycin raises the need for novel management measures. In addition, using antibiotics in agriculture is under critical analysis due to concerns that spray-drifted antibiotics greatly impact the environment and soil, including microorganisms that act as reservoirs of genes for antibiotic resistance that could ultimately have an impact on clinical medicine. Alternatively, the application of antagonistic bacteria as biocontrol agents can significantly reduce the epiphytic growth of E. amylovora. The effectiveness of these agents is based on combined modes of action, e.g. site exclusion, nutrient competition, and antibiotic production. Pantoea agglomerans represents one of these antagonistic biocontrol agents due to antibiosis and its superior growth rate on blossoms as an epiphytic bacterium. Bacteriophages have also been used as a biocontrol agent on other plants to control bacterial pathogens and the first bacteriophage product was registered with the U.S. Environmental Protection Agency in 2005 by OmniLytics Inc.. A few phage formulations are now commercially available in the United States for agricultural applications, such as AgriPhage-Fire Blight for apple and pear tree treatment, as well as AgriPhage-Citrus Canker for orange and grapefruit treatment.

While successes have been noted for the use of antagonistic bacteria and bacteriophage as biocontrol agents, E. amylovora remains an agricultural problem, so further development and combination of alternative techniques are needed to mitigate the spread and destruction caused by this pathogen. With this in mind, the ultimate goal of this thesis was to develop an efficient E. amylovora phage therapy method that relies on a phage-carrier system (i.e., P. agglomerans infected with E. amylovora phages). The initial part of this research was to develop an economical production protocol for the phage-carrier system. Using spray drying, an improved survival method for carrier (P. agglomerans) and phage-carrier (P. agglomerans infected with E. amylovora phages) production was developed. The results showed good efficacy of the spray-dried carrier and phage-carrier products to reduce E. amylovora growth with about 4.5 log reductions in bacterial count on treated pear discs and ≥90% of P. agglomerans cell viability after the spray drying procedure.

Given that improvements in biocontrol formulations and effectiveness are always being sought, a second aim of this thesis research was to understand better the dynamic interactions between E. amylovora, P. agglomerans, and E. amylovora phages. With the use of various bacterial surface receptor isolations and phage trial analyses, the receptors for two E. amylovora phages, phiEa21-4 and phiEa46-1-A1, were identified. These cumulative results showed that lipopolysaccharide (LPS) and OmpA are the receptors for these phages on E. amylovora, but that only OmpA serves as a receptor on P. agglomerans. Previous work demonstrated that the pathogenicity of E. amylovora to host plants is strictly dependent on a functional type III secretion system, amylovoran, and LPS. While LPS was further highlighted as a phage receptor in our work, the importance of OmpA has now also been established as important for phages across bacterial species.

While understanding phage receptors was necessary, we also explored the development of phage resistance mechanism(s) in E. amylovora to ultimately provide a foundation for how our phage therapy product may be adapted/enhanced in the future. Studying phage resistance in E. amylovora was achieved by generating a bacteriophage-insensitive mutant (B6-2) against phiEa46-1-A1. The B6-2 strain was compared with the parent E. amylovora D7 strain on phenotypic, genomic, and transcriptomic levels. Analysis of this combined data revealed that an insertion mutation in the rcsB gene (confirmed by genetic sequencing) is the main driving force for phage resistance development. In the transcriptomic data, there was downregulation of amylovoran, LPS, and OmpA genes, suggesting that receptor adaptions have occurred as a phage resistance mechanism in strain B6-2. These changes were paralleled by transcriptomic changes in the levels of the rcsA and rcsB genes, which further reflects their roles as a part of the Rcs two-component phosphorelay system that regulates genes in the EPS, LPS and OmpA pathways.

Additionally, there was upregulation of putative retron Ec48, which may act as a second-line "abortive" antiphage mechanism. On the phenotypic level, strain B6-2 had an altered metabolism of nitrogen sources and an increased sensitivity to streptomycin. This decrease in antibiotic resistance when phage resistance increases is considered as significant given that streptomycin is the primary antibiotic used to treat fire blight. In combination, this data provides a solid foundation to have a better understanding of the development of phage resistance in E. amylovora and how this may impact its pathogenicity and antibiotic resistance.

In conclusion, this research has provided a number of substantial advancements with phage therapy involving E. amylovora. Essential improvements in the formulation of a biocontrol agent that takes advantage of two alternate treatments (i.e., antagonistic bacteria and phage) have been developed and can now be tested in field trials. Recognizing that target phytopathogenic bacteria can develop resistance to the phage treatments, this study has proactively identified key phage receptors on E. amylovora and carrier cells and provided a sound basis for understanding the role of the Rcs system in receptor alterations that can lead to phage resistance. Ultimately, this work will lead to the continued improvement and applicability of phage therapy using a phage-carrier biopesticide system to mitigate the spread and damage caused by E. amylovora and fire blight.

Convocation Year

2024

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