Open Collections

Abstract

The emergence of stem gall disease caused by Agrobacterium tumefaciens (syn. Rhizobium radiobacter) has been reported by blueberry farmers in British Columbia (BC), Canada, since 2017. This disease prevents water and nutrient transportation, leading to the death of blueberry plants. The only currently available biocontrol agent, Dygall, is effective when applied during the pre-planting stage as a root dip treatment. Bacteriophages (phages), viruses that specifically target bacteria, have gained interest as environmentally friendly, species-specific antimicrobials in agriculture. This study aimed to isolate and characterize A. tumefaciens using whole-genome sequencing (WGS) and to develop a phage cocktail biocontrol system to control A. tumefaciens as a proactive prevention method. A total of 77 bacterial isolates were isolated from four stem gall-infected blueberry farms in BC. Preliminary identification using MicroLog classified 8 isolates as R. radiobacter and 17 as R. rhizogenes. However, the full length of 16S rRNA gene analysis of these isolates revealed that they belonged to A. tumefaciens. Average nucleotide identity (ANI) analysis showed that 17 isolates had high similarity (>96%) to A. tumefaciens, while only one isolate matched R. radiobacter ATCC 33970 (syn. A. fabrum). Phylogenetic analysis using core genome data and antimicrobial resistance patterns further confirmed the similarity of isolate PL3 to A. fabrum ATCC 33970. The virB operon, associated with gall disease, was detected in 18 isolates of Agrobacterium spp. A total of 76 phages were recovered from sewage and screened for their ability to inhibit A. tumefaciens growth. Phage diversity was characterized using WGS, with the phage lytic cycle genetically confirmed. Five potential phage candidates were chosen based on host range, temperature and pH stability, and lytic efficacy in a broth-based system. A three-phage cocktail comprising IC12, IG49, and LG08 was formulated and tested against A. tumefaciens in an artificially inoculated peat-based medium, resulting in a significant reduction (p < 0.05) of approximately 3 log10 CFU/g of bacteria after 24 h. These findings highlight A. tumefaciens as the potential causative agent of stem gall disease in blueberries through WGS and demonstrate the development of a phage-based control system as a potential proactive measure against this pathogen.