Soft rot Enterobacteriaceae-molecular detection and biocontrol under in vitro and in vivo conditions

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Destructive soft rot Pectobacteriaceae are pathogenic to a number of vegetables, fruits and ornamentals crops and cause high economic losses in the field and during storage. An increasing number of reports in recent years illustrate the losses caused by Pectobacterium and Dickeya in Europe, and a lack of effective control strategies. These indicate a need for the formulation of commercially available and effective biocontrol measures to counteract soft rot pathogens.

The first aim of this study was to describe the diversity of Pectobacterium and Dickeya spp. detected in vegetables from several locations in Northern Ireland in the years 2015 - 2017. From 184 isolates confirmed by real-time PCR, 33 strains were selected for further phylogenetic analysis based on the recA gene region. Results from the analysis of over 3456 potato samples for plant health statutory purposes in the years 2005 - 2017 demonstrated that Dickeya spp. is not the major pathogen causing soft rot or blackleg in Northern Ireland. The most dominant species were Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum. Pectobacterium atrosepticum was also detected on hosts other than potato. Testing of bacteria isolated from carrots led to the detection of P. carotovorum and Dickeya sp. This is the first study to provide knowledge about Pectobacterium and Dickeya spp. diversity causing soft rot of vegetables in Northern Ireland confirmed by real-time PCR and DNA sequencing.

The second aim was to isolate and characterise bacteriophages active against the most diverse Pectobacteriaceae detected in Northern Ireland, and formulate a phage-based cocktail which would be tested in vitro and in vivo in planta. Twenty bacteriophages were isolated and characterised using transmission electron microscopy and tested for their activity against P. atrosepticum and P. carotovorum. Isolated bacteriophages in Northern Ireland belonged to three families Podoviridae, Myoviridae and Siphoviridae and had the broad host range active against both species of Pectobacterium. All twenty bacteriophages were subjected to DNA sequencing using next-generation sequencing. Ten bacteriophages were successfully confirmed as a members of Podiviridae of the Autographivirinae subfamily and two bacteriophages belong to Siphoviridae familiy of the Chivirus subfamily. Six bacteriophages were selected for a phage-based cocktail formulation for potato treatment and for further tests including lytic activity, stability and suppression of bacterial growth on potato tubers. Four different from the six used for potato bacteriophages were used for the formulation of phage-based cocktail for application on onions.

Isolated bacteriophages were tested singly and as multiple formulations as a cocktail on potato tubers. Both formulations show significant activity in vitro to suppress Pectobacterium growth in bioassays, however host specificity of single bacteriophages against several Pectobacterium strains was shown, in contrast to the phage-based cocktail which significantly inhibited growth of the Pectobacterium cocktail consisting of P. atrosepticum and P. carotovorum subsp. carotovorum.

Testing the phage-based cocktail over three years of field trials indicated the potential for use as a microbial pest control agent (MPCA) against P. atrosepticum and P. carotovorum subsp. carotovorum under natural conditions; however inconsistency of results between years was observed. The phage-based cocktail significantly inhibited Pectobacterium on potato in three replicated experiments performed in 2018 via two methods of application (vacuum-infiltration and soil drench). No negative effect of the formulated phage-based cocktail was observed on plants, and treated potato tubers showed significantly better yield than untreated potatoes. The phage-based cocktail tested on onions significantly suppressed soft rot disease in the field, though there was some inconsistency in terms of effects different application methods between years. The phage-based cocktail was shown to be stable under a range of changing environmental conditions and efficacious against SRP species present on Northern Ireland potatoes and onions.

Given the reduction in commercially available agrochemicals and need for the development of safe and effective biological control agents in Europe, this study suggest that MPCA based on bacteriophage formulations holds great promise for preventing losses in the agricultural supply chain from ‘farm to shelf’.
Date of AwardJul 2019
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SponsorsDepartment of Agriculture, Environment and Rural Affairs
SupervisorKatrina Campbell (Supervisor) & Richard O'Hanlon (Supervisor)

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