Role of species interactions in the ecology of Erwinia amylovora, the causal agent of apple fire blight

Fire blight represents a significant threat to apple and pear production worldwide. The causal agent, Erwinia amylovora, spreads rapidly within host plants, making this devastating disease particularly difficult to manage. Copper and antibiotics remain the most effective means of controlling fire blight; however, their application contributes to environmental pollution and promotes the emergence of antibiotic-resistant E. amylovora populations. For these reasons, there is an urgent need to find new alternatives to such plant protection products. Notably, the presence of E. amylovora alone does not necessarily result in the disease development. Instead, fire blight results from multiple interactions established between E. amylovora cells, flower microbiota, plant host, insect vectors and environmental factors. For instance, specific humidity and temperature create suitable conditions for E. amylovora to grow and reach the cell density needed for plant infection. Once the disease develops, insects act as potential vectors of E. amylovora, contributing to its spread. The host plant plays a crucial role, as susceptibility varies among different species within the Rosaceae family. Recent studies have shown that apple flower microbiota might either promote or hinder the infection, thus representing a possible source of new biocontrol agents effective in controlling E. amylovora. Therefore, studying the influence of the abovementioned factors is necessary to develop novel eco-friendly solutions to manage fire blight disease. The first objective of this thesis was to investigate the interaction between the plant pathogenic bacterium and the apple flower microbiota to select new potential biocontrol agents. Flowers of Malus domestica cv. Golden Delicious were collected from Trentino apple orchards at the ‘Balloon stage’ and surface-sterilized to isolate only endophytic bacteria. According to the 16S rRNA gene sequencing, the bacterial isolates mainly belonged to the Enterobacteriaceae, Pseudomonadaceae, and Microbacteriaceae families. All bacterial strains isolated from apple flowers were tested against E. amylovora on detached apple flowers and immature pear slices, which were first treated with bacterial suspensions before being inoculated with E. amylovora. Among them, Pantoea agglomerans AFF2001 showed the highest efficacy in controlling the plant pathogenic bacterium in both assays. We then conducted several in vitro tests to investigate the modes of action underlying the antagonistic activity observed. Based on the results of the experiments conducted on flowers and pears, the bacterial strains selected as potential biocontrol agents grown in a specific medium that mimics the apple stigma nutrient conditions, and the cultural filtrates were tested to evaluate their impact on the growth and virulence of E. amylovora, with virulence referring to factors such as motility and biofilm production. To achieve this, the bacterial culture filtrates were used as the medium for the experiments. To further explore the molecular mechanisms involved in its biocontrol activity, knockout mutants of P. agglomerans AFF2001 were generated. Mutants with reduced ability to inhibit the growth of E. amylovora were subsequently characterised in vitro and in vivo. The results indicated that differences in motility, biofilm production, and siderophore release distinguished the knockouts from the wildtype, suggesting these factors may contribute to the biocontrol ability of P. agglomerans AFF2001 to control E. amylovora on apple flowers and pear slices. Altogether, this study highlights the potential of P. agglomerans AFF2001 as a promising biopesticide for fire blight disease management. The second objective of this thesis was to explore the potential role of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) as a vector of E. amylovora. Pitfall and olfactometer choice assays were conducted to assess whether D. suzukii is attracted to bacterial ooze produced by E. amylovora. To do that, E. amylovora was cultured on Petri dishes with a specific medium that promotes bacterial ooze formation, and D. suzukii was given a choice between inoculated and non-inoculated plates. The main difference between the two experimental setups was the exposure time, which was longer in the pitfall assay, and the application of airflow in the olfactometer choice assay, which was absent in the pitfall assay. The results showed a tendency of D. suzukii flies to be attracted to the ooze produced by E. amylovora, suggesting this behaviour may support the dispersal of E. amylovora in the environment. The attractiveness was particularly evident for female individuals, and this difference observed between the two sexes might be related to biological factors, such as the reproductive role of females, which may influence their choice. Next, we improved the protocol to test the acquisition of E. amylovora by D. suzukii. Several parameters were evaluated: the fruit type to inoculate with E. amylovora to produce the ooze; the time of exposure of the inoculated fruits to D. suzukii; the time points of sampling; the molecular detection of E. amylovora in D. suzukii. Overall, the results suggested that D. suzukii is attracted to E. amylovora ooze and can acquire plant pathogenic bacteria. Future studies are needed to assess the ability of D. suzukii to spread and transmit E. amylovora in the environment, with a particular focus on apple orchards.