Pathogenicity and Phylogenetic Placement of Neopestalotiopsis clavispora Causing Crown Rot on Strawberry

Abstract

Crown rot symptoms characterized by tissue browning, vascular discoloration, reduced root development, wilting, and plant collapse were observed in strawberry plantations and nursery systems, leading to significant losses in transplant establishment and field productivity. Diseased crown tissues were collected for isolation and characterization of the associated fungal pathogen using morphological, pathogenicity, and molecular approaches. Fungal isolates consistently produced fast-growing colonies with white to gray mycelia and dark sporulating acervuli bearing fusiform, multi-septate conidia with characteristic apical and basal appendages, consistent with Neopestalotiopsis clavispora. Considerable variation in colony morphology, growth rate, and sporulation intensity was observed among isolates recovered from different production sites. Pathogenicity assays conducted on healthy strawberry transplants reproduced typical crown rot symptoms, including crown necrosis, root decay, and plant collapse, whereas control plants remained symptom-free. Re-isolation of the fungus from inoculated tissues fulfilled Koch’s postulates, confirming its pathogenic role. Molecular identification was performed using sequencing of the internal transcribed spacer (ITS) region along with additional loci including β-tubulin and translation elongation factor 1-α genes. Sequence analysis revealed high similarity with reference N. clavispora isolates in public databases. Phylogenetic analysis clustered the obtained isolates within the N. clavispora clade with strong bootstrap support, confirming species identity and indicating limited but detectable genetic variation among regional populations. Disease development was favored by warm temperatures and high humidity conditions typical of strawberry nursery environments, which facilitated rapid pathogen spread and infection. Infected plants exhibited reduced vigor, poor establishment, and significant losses in commercial transplant quality.