Abstract/Summary

The most commonly cultivated mushroom in Europe and North America is the Agaricus bisporus, also known as the button mushroom or Portobello mushroom. Bacterial diseases of Agaricus bisporus caused by Pseudomonas species are a cause of significant crop loss and downgrading of produce, resulting in considerable economic cost. Bacteriophage have long been an attractive option for biocontrol of bacterial contamination of food products, however the precise genetic interactions between phage, bacterium and host are often inadequately explored. This project aims to explore the genetic interactions between the mushroom pathogenic bacterium Pseudomonas tolaasii and Pseudomonas agarici with the newly identified bacteriophage Pseudomonas phage NV1 and Pseudomonas phage ϕNV3. Full genome sequencing has been performed on the P. tolaasii strain 2192T and P. agarici NCPPB 2472, and the genomes of both mined for potential biosynthetic clusters involved in virulence as well as genes involved in phage resistance. Within the genome of P. tolaasii 2192T, putative non-ribosomal peptide synthases have been identified which are hypothesised to be involved in the production of the tolaasin toxin involved in disease symptom appearance on mushroom surfaces. Within the genome of P. agarici NCPPB 2472, a biosynthetic cluster was identified that is hypothesised to produce the siderophore achromobactin, an important virulence factor. P. agarici NCPPB 2472 was identified as possessing a single Type I-F CRISPR/Cas system, predicted to be involved in the development of phage resistance, as well as complete operon predicted to be involved in the production of the exopolysaccharide alginate. A third Pseudomonas species was identified on the surface of disease free mushrooms which was identified as a potentially new species of Pseudomonas, named Pseudomonas sp. NS1. The genome of P. sp. NS1 was likewise sequenced and mined for potential biosynthetic gene clusters which identified a cluster demonstrated to be involved in the production of White Line Inducing Principle. The Pseudomonas phages NV1 and ϕNV3 were isolated from environmental samples and identified to be narrow host range phage specific for P. tolaasii 2192T and P. agarici NCPPB 2472 respectively. Both phage NV1 and ϕNV3 were identified as new species of the Luz24likeviruses and phiKMVlikeviruses respectively. The genomes of both phages were isolated and sequenced, with phage ϕNV3 identified as containing a conserved Signal-Arrest-Release endolysin system, which was confirmed by in vitro protein expression. Likewise, the lysis cassette proteins of ϕNV3 were identified and investigated via protein complementation assay in vitro. The full growth characteristics and life cycle of phage ϕNV3 has been investigated and reported in this study and a broad-host range mutant of ϕNV3 identified which has allowed the T7-like tail protein of ϕNV3 to be identified as the host specificity determinant. Transcriptome analyses of non-infected P. agarici NCPPB 2472 and P. agarici NCPPB 2472 infected with phage ϕNV3 at a multiplicity of infection (MOI) of 1 at 40 min post infection, were performed in triplicate using RNA-seq. A reliable method has been established that will be useful in future studies, although comparative gene expression analysis revealed no significant differences in expression between the two treatments at the multiplicity of infection and time point chosen in this case a significant quantity of phage transcripts were detected, demonstrating active phage infection.