First report of <i>Fusarium cymbidii</i> Causing Black Stem of <i>Dendrobium nobile</i> in China

Dendrobium is one of the largest genera within Orchidaceae, and D. nobile is a prominent species with significant medicinal and ornamental value in China. In July 2022, black stems were detected on D. nobile in a commercial plantation in Shantou, Guangdong Province, China. The affected area covered around 1 ha, with a disease incidence of about 20%. Typical symptoms showed dark brown leaf sheaths, leaf defoliation and plant wilt. Lesions with adjacent healthy tissues were cut into 5 × 5 mm pieces, disinfected with 75% alcohol for 10 s, followed by a 2% NaClO for 15 s, and rinsed three times with sterile distilled water. Tissue pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. All isolated colonies (&gt;20) were similar in macromorphology. After single-hyphal tip purification, two strains, ZHKUCC 25-0668 and ZHKUCC 25-0669, were obtained. The average growth rate on PDA was 6.4 mm/d at 25°C. Colonies were white to pale grey, raised, woolly, circular, with filiform margin, and reverse pale orange with a white margin. Purified strains were incubated on carnation leaf agar (CLA) for micro-morphological observation (Fisher et al., 1982). After 20 days, conidia were observed. Sporodochial conidiogenous cells were doliform to subcylindrical, hyaline, 6–14 × 2–4 µm. Macroconidia had curved apical cells and basal cells, or sometimes foot-shaped basal cells, 3–5-septate, 24–38 × 3–6 µm (x̅ = 31 × 4 µm, n = 50). Aerial conidiogenous cells were monophialidic and subcylindrical, 4–40 × 2–4 µm. Microconidia formed small false heads, mostly oval, obovoid, or fusoid, 0–1-septate, 7–24 × 2–5 µm (x̅ = 14 × 3 µm, n = 50). Chlamydospores were globose to subglobose, single or in pairs, 5–12 × 4–9 µm. The genomic DNA was extracted from a 5-day-old culture grown on PDA, and the partial sequences of translation elongation factor1-alpha (tef1-α), RNA polymerase largest subunit 1 (rpb1), rpb2, and beta-tubulin (tub2), were amplified using EF1/EF2, RPB1-Fa/RPB1-G2R, RPB2-5f2/RPB2-11ar, and T1/T2, respectively (Crous et al. 2021; Wang et al. 2022). BLASTn searches showed that our two strains had very high sequence similarity with those of Fusarium cymbidii. In a Maximum-Likelihood tree with various Fusarium species, our two strains grouped with F. cymbidii with 100% bootstrap support. All sequences were deposited in GenBank (tef1-α: PX060465, PX060466; tub2: PX088765, PX088766; rpb1: PX088768, PX088769; rpb2: PX088771, PX088772). A pathogenicity test was conducted with five replicate plants per isolate in a growth chamber at 28 °C with around 80% relative humidity and day/night alternation of 12 h/12 h. Conidial suspensions (1 mL of 1.2 × 107 conidia/mL) were inoculated into each leaf axil; sterile water was used as a control. The treated areas were covered with sterile wet cotton to promote moisture. After 6 days, leaf axils became brown, gradually turning dark brown which spread to leaf sheaths by 12 days, finally resulting in leaf drop, whereas controls remained asymptomatic. The same fungus was re-isolated from symptomatic tissues and identified by morphological and molecular analyses, fulfilling Koch’s postulates. Fusarium cymbidii has been reported from diseased leaves of Cymbidium sinense (Zhang et al. 2024), however, this is the first report of F. cymbidii causing black stem on D. nobile in China. This finding expands the known host range of F. cymbidii and provides valuable information for the diagnosis and management of diseases in Dendrobium orchids.