First Global Report of <i>Fusarium proliferatum</i> Causing Black Leaf Spots in Foxtail Orchid ( <i>Rhyncostylis retusa</i> ) in India

Rhynchostylis retusa is an epiphytic orchid in the family Orchidaceae, valued for its long, dense inflorescences known as ‘foxtails’. It is the state flower of Assam, India, and is used in traditional medicine for treating a wide range of illnesses. In May 2025, black necrotic spots were observed on R. retusa in Jorhat district, Assam, India (26°44′9″N, 94°9′24″E), with c. 10% disease incidence (Figure 1). Diseased leaves were collected, surface-sterilised and cultured on potato dextrose agar (PDA) at 25°C ± 2°C. The isolates produced whitish colonies with dense aerial mycelia and violet pigmentation on the reverse (Figure 2). Macroconidia were slender, thin-walled with tapered apical and poorly developed basal cells, 3–5 septate, and measured 17.6–35.7 × 2.8–4.0 µm. Microconidia were abundant, hyaline, ovoid, aseptate and measured 4.5–12.7 × 2.0–3.6 µm. Based on the morphological characteristics described by Leslie and Summerell (2006), the isolates were tentatively identified as Fusarium proliferatum. Total genomic DNA was isolated from 1-week-old fungal cultures using the cetyltrimethylammonium bromide protocol and amplified with ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O'Donnell et al. 1998) primer pairs targeting the ITS region and tef1-α gene, respectively. PCR products were Sanger sequenced and deposited in GenBank (ITS: Accession No. PV942219; tef1-α: PX098483). Phylogenetic tree analysis based on the ITS and tef sequences confirmed that the fungal isolate in this study belongs to F. proliferatum (Figure 3). Pathogenicity tests were conducted on 10 one-year-old R. retusa plants using two different methods (five plants for each method). In the first method, a 4-mm-diameter PDA disk colonised by the test isolate was transferred to needle-inflicted wounds on the leaves, while control plants received sterile agar plugs. In the second method, a conidial suspension (1 × 10⁶ spores/mL) of the isolate was applied to needle-inflicted leaf wounds, control plants were treated with sterile distilled water. The inoculated plants were covered with plastic bags and placed in a net house at 25°C ± 2°C for 3 days. At 5 days post-inoculation, control plants showed no symptoms, whereas inoculated plants developed foliar lesions similar to those observed in the field (Figure 4). F. proliferatum was consistently reisolated from symptomatic tissues. F. proliferatum is known to cause leaf spot and blight in orchids such as cymbidium and dendrobium (Ichikawa and Aoki 2000; Swett and Uchida 2015), but it has not previously been reported on R. retusa. This finding highlights a potential emerging threat to commercial cultivation. To the best of our knowledge, this is the first report of F. proliferatum causing leaf spot on R. retusa worldwide. The authors express their sincere gratitude to CSIR-NEIST, Jorhat, Assam, for providing essential resources and facilities, and to the Publication and Intellectual Property Rights Committee of CSIR-NEIST, Jorhat (Manuscript No: CSIR-NEIST/PUB/2025/144) for their support. This work was funded by the CSIR Floriculture Mission (HCP0037).