First Host Record of <i>Paramyrothecium vignicola</i> Causing Leaf Spot on Banyan Tree ( <i>Ficus benghalensis</i> ) Discovered in Florida, U.S.A.

The Banyan tree, Ficus benghalensis L. (Moraceae, Rosales) is the national tree of India and is sacred for Hinduism and Buddhism (Chakraborty et al. 2022). In December 2023, Banyan tree liners (25%) in a nursery business in Lake County, Florida (FDACS-DPI LIST 12052023-12324 and 12182023-12759) exhibited several 1-cm diameter dark-brown leaf spots and after 2 weeks, concentric rings were developed with sporodochia at the point of infection. Isolation of the fungal pathogen followed the methods of Urbina et al. (2024). Fungal mycelia were consistently recovered from symptomatic plant tissue onto acidified potato dextrose agar (APDA) plates. In culture, the fungus produced round sporodochia that fused at maturity, 120-500 µm in diameter, with dark-green spore mass and white setose fringe > 150 µm in length, containing hyaline to yellowish conidia, cylindrical in shape, 5‒8 ×1.5‒2 µm (n=20). These structures are characteristic of Paramyrothecium vignicola Withee & Cheew. (Stachybotryaceae, Sordariomycetes) (Withee et al. 2022). Molecular identification was performed by the PCR amplification and sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of the rRNA operon (White et al. 1990) from sporodochia on leaf lesions and culture (GenBank acc. nos. PP599366 and PP599367), both with identical ITS sequences. Additional molecular markers were also sequenced (GenBank acc. no. in parenthesis), actin (act, PV699695), calmodulin (cmdA, PV699693), the small subunit (SSU, PP599384) of rRNA, translation elongation factor 1-α (tef1, PV699694), and β-tubulin (tub, PP620693) (White et al. 1990; Myllys et al. 2002, Withee et al. 2022). High similarity (>99.4%) was obtained after BLAST comparisons of the markers ITS (MZ373242), cmdA (OM810412) and tub (ON009015) against the SDBR-CMU376 ex-type culture of P. vignicola. Molecular identification aligned with phylogenetic analysis and morphological characterization; a finding also confirmed by the US Department of Agriculture. Dried symptomatic leaves were deposited in the Plant Industry Herbarium Gainesville (PIHG 18287) and culture at the Agricultural Research Service Culture Collection (NRRL 64847). Pathogenicity tests were conducted on 4-month-old Banyan seedlings (4 treated plants and 1 control). One leaf per seedling was wounded with Carborundum 320 grit (Fisher Scientific) followed by inoculation with a 6-mm mycelium plug (Mmbaga et al. 2010; Zhang et al. 2011) and covered with Parafilm® M. APDA media alone was used for the wounded controls. After, seedlings were sprayed with sterile tap water covered with a plastic bag for the first 3 days to maintain moisture. All inoculated plants exhibited symptoms consistent with those observed in the nursery (after 6 days), while control plants remained asymptomatic. The experiment was conducted in a climate-controlled greenhouse (12h/12h daylight, ~25 °C and >55 relative humidity) and performed twice. The presence of the pathogen was confirmed by subsequent isolation and identification by morphology and sequencing. In forest settings, this disease may hinder seedling establishment, while in nurseries, it could significantly diminish the aesthetic appeal of affected plants. To our knowledge this is the first documented case of P. vignicola infecting F. benghalensis and the first record of P. vignicola in the Americas. To manage the disease, it is recommended to discard affected plants followed by sanitation of the facility.