Development and application of multiplex targeted-sequencing approaches to identify Phytophthora species associated with root rot and wilting complex of red raspberry

Phytophthora species are primary causal agents of raspberry root rot and wilting complex (RRWC), a disease complex that is of major concern to raspberry producers worldwide. Accurate identification of the causal agents is a first step for effective disease management. Advancements in molecular diagnostics can facilitate the detection of multiple pathogen species associated with this disease complex. We developed multiplex targeted-sequencing methods using degenerate primers for heat shock protein 90, elongation factor 1α and β-tubulin genes to identify Phytophthora species causing RRWC. One hundred and twenty-eight isolates recovered during 2018 to 2020 from diverse fields in major raspberry growing areas of British Columbia (BC) were sequenced and identified by comparing with known reference sequences of 142 Phytophthora species, 111 Pythium species, and nine Phytopythium species in the NCBI database. This multiplex targeted-sequencing method was highly specific and identified two species of Phytophthora associated with RRWC. These were P. rubi (85% of isolates) and P. gonapodyides (15% of isolates). Phytophthora rubi was predominantly isolated from the cultivars ‘Chemainus’ (51%), ‘Rudi’ (27%) and ‘Meeker’ (15%), whereas P. gonapodyides was predominately isolated from the moderately resistant cultivar ‘Cascade Bounty’. Pathogenicity studies on intact plants and detached leaves confirmed that P. rubi and P. gonapodyides can cause symptoms of RRWC on raspberry, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of P. gonapodyides as a causal agent of RRWC on raspberry in BC. This study provides novel insights into the identification and species composition of Phytophthora associated with RRWC in raspberry production systems.