The Invasive Fungal Pathogen Neopestalotiopsis in North Carolina: Molecular Characterization, Virulence, and Host Susceptibility

A Neopestalotiopsis spp. has recently emerged as an invasive fungal pathogen threatening strawberry (Fragaria × ananassa) production in North Carolina by causing leaf spots, fruit rot, and crown rot, resulting in significant production challenges. Understanding pathogen diversity and virulence variability is vital for developing effective disease management strategies and improving host resistance. Sixty-five Neopestalotiopsis isolates were collected from multiple strawberry cultivars across diverse production regions in North Carolina and characterized using a high-resolution melting quantitative real-time PCR (HRM-qPCR) assay targeting a partial β-tubulin (β-tub) gene sequence. HRM-qPCR analysis differentiated the isolates into two distinct genetic groups, comprising 28 and 37 isolates, respectively, indicating substantial genetic diversity within the pathogen population. To establish reliable disease screening methods, four inoculation techniques were evaluated: foliar spray inoculation with spore suspensions and inoculation with Neopestalotiopsis-infested oatmeal grain, both of which consistently produced disease symptoms and were selected for virulence assessments. To further investigate pathogenic variability, 10 arbitrarily selected isolates were evaluated by spray inoculatingon across 15 strawberry cultivars. Significant effects of isolate, cultivar, and isolate × cultivar interactions were observed, suggesting potential pathogenic specialization among isolates. Although all isolates were pathogenic across the cultivars tested, notable variability in virulence was observed among isolate-cultivar combinations. Among the cultivars evaluated, AC Valley Sunset, Jewel, and Malwina consistently exhibited lower disease susceptibility, suggesting greater tolerance to Neopestalotiopsis. These findings improve understanding of the pathogen diversity and host-pathogen interactions and provide valuable information for cultivar selection, breeding programs, and integrated disease management strategies against this emerging pathogen.