First Report of <i>Alternaria alternata</i> Causing Leaf Blight on <i>Boehmeria nivea</i> in China

Ramie (Boehmeria nivea), native to China, is an economically important natural fiber-producing crop (Angelini and Tavarini 2013). In May 2025, leaf blight was observed on 20% cultivated ramie seedlings in a 5-ha field, Dazhou city (30°51′36″N, 107°19′48″E), Sichuan province, China. Leaf tissues adjacent to and including lesions were superficially disinfected with 70% ethanol for 20 s and 1% Sodium hypochlorite for 40 s, washed, dried, placed on PDA amended with streptomycin sulfate (50 mg/L) in dark at 25 ℃. Finally, three purified isolates, named B3, B6, and B26, showing similar morphology were obtained by transferring hyphal tips to fresh PDA plates. Cultured on PDA for 14 days, fungal colonies displayed gray aerial mycelia, with underside becoming slightly grayish-white. 28 days later, the circular colonies transitioned to grayish-white and soft texture with brownish-white underside. Colonies produced abundant solitary or catenate conidia. Conidia were pyriform, ovate or elliptical, with one to four transverse and zero to two longitudinal septa, and measured 20.6 to 48.7 × 19.0 to 8.5 µm (n = 50). The morphological characteristics of our isolates match those of Alternaria species (Li et al. 2023; Woudenberg et al. 2013). Our isolates were further identified by sequencing rDNA internal transcribed spacer regions (ITS) , anonymous region OPA10-2 genomic sequence (OPA10-2), and gene fragments of translation elongation factor 1-alpha (TEF1), endopolygalacturonase (endoPG), major allergen Alt a1 (Alt a1), RNA polymerase II second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and large subunit (LSU) and small subunit (SSU) ribosomal RNA, using primer pairs ITS1/ITS4, OPA 10-2R/OPA 10-2L, TEF1-728F/TEF1-986R, PG3/PG2b, Alt-F/Alt-R (Romain et al. 2021), RPB2-5F/RPB2-7cR, gpd1/gpd2 (Luo et al. 2022), LSU-F/LSU-R and SSU-F/SSU-R respectively. BLASTN searches indicated our ITS (PX069539 - PX069541), OPA10-2 (PX092332 - PX092334), TEF1 (PX092323 - PX092325), endoPG (PX092326 - PX092328), Alt a1 (PX092320 - PX092322), RPB2 (PX092335 - PX092337), GAPDH (PX092329 - PX092331), LSU (PX070146 - PX070148), and SSU (PX070153 - PX070155) sequences showed 99.34 ~ 100% and 99.68 ~ 100% identity to the corresponding sequences of A. alternata CBS 121456 (KP124369, KP124682, KP125147, KP124073, KP123917, KP124839, KP124221, KP124523, KP124993) and CBS 795.72 (KP124309, KP124616, KP125085, KP124009, KP123862, KP124778, KP124166, KP124461, KP124931), respectively. Based on combination of the nine DNA sequences, our phylogenetic tree of Alternaria species confirmed that three isolates were A. alternata. To test pathogenicity, 2 ~ 3 leaves of ramie seedlings were sprayed with conidial suspension (1 × 105 conidia/mL) of isolate B6, with controls treated using sterile dH2O. Each treatment, including 3 seedlings, was incubated in a greenhouse (at 25°C and 90% relative humidity, 12/12 h light/dark cycle). Leaf blight appeared on infected leaves after 15 days’ inoculation, while controls remained disease-free. The experiment was repeated five times, showing consistent disease symptoms. Koch's postulates were fulfilled by re-isolation of A. alternata from diseased leaves, based on morphology and molecular methods described above. To our knowledge, this is the first report of A. alternata causing leaf blight of ramie worldwide. Our study will provide a basis for developing targeted disease management strategies in ramie.