First report of Curvularia geniculata causing leave spot disease of Bletilla striata in Zhejiang of China.

Abstract

Bletilla striata, a perennial herbaceous plant belonging to the family Orchidaceae, is native to China and is widely distributed in the Yangtze River basin. In China, B. striata is a popular medicinal plant that is typically used to reduce wound bleeding and inflammation. In September 2021, distinct leaf spot symptoms were observed in more than 50% of B. striata plants in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. Small, round, pale brown, necrotic spots were first observed on the leaves. Subsequently, these lesions became grayish brown in the center and dark brown with slight protuberances at the margins and eventually enlarged to 5-8 mm on the leaves. Over time, the small spots enlarged and coalesced into necrotic streaks (1-2 cm). Leaves with symptoms of disease were cut, surface-sterilized, and plated on potato dextrose agar (PDA). Fungal colonies (28×28 mm) with grayish-black mycelia from all tissues were produced after 3 days of incubation at 26 °C. The mature colonies eventually turned black in the center, with obvious rings appearing after 10 days of culture. Basal conidia ranged from pale to dark brown, whereas apical ones were pale brown, with central cells being larger and darker than basal cells. Conidia were smooth and either fusiform, cylindrical, or slightly curved with rounded tips. They ranged in length from 22.34 to 36.82 (mean = 28.63) μm with 2-4 septations and slight septal constrictions. Monospore isolation was performed to obtain a pure culture. Strain BJ2Y5 was subsequently stored in the strain Preservation Center of Wuhan University (Wuhan, China) and the strain preservation number CCTCC M 2023123 was obtained. Fresh mycelia and conidia that grew at 26 ℃ for 7 days were collected from PDA plates. DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Shanghai, China). The phylogenetic position of isolate BJ2-Y5 was clarified based on DNA sequence analysis of three loci, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Berbee et al. 1999), the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of the second largest subunit of RNA polymerase II (RPB2; O'Donnell et al. 2007). A BLAST search (GenBank accession nos. OP913168, OP743380, and OP913171) showed 99% homology to the reference isolate CBS 220.52. Based on the morphological and molecular information presented in this study these isolates were identified as C. geniculata (Hosokawa et al. 2003). Furthermore, we evaluated the pathogenicity of B. striata leaves by smearing a conidial suspension (106 conidia/mL) on both sides of leaves with and without wounds. Five inoculated and three non-inoculated leaves (smeared with sterile distilled water as a negative control) were kept in a greenhouse at 26 °C under natural sunlight and covered with plastic bags for 72 h to maintain humidity. After 7 days, small round spots appeared on the wounds. Fifteen days later, the symptoms of disease on the wounded inoculated leaves were similar to those observed in the original sample, whereas the control plants remained healthy. No symptoms of infection were observed in unwounded inoculated leaves. C. geniculata was successfully re-isolated from all five inoculated leaves and was confirmed based on Koch's postulates. To the best of our knowledge, C. geniculata infection has not been previously reported in B. striata.