First Report of Leaf Blight Caused by <i>Neopestalotiopsis rosae</i> on <i>Carex wahuensis</i> subsp. <i>wahuensis</i> in Hawaii

Carex wahuensis subsp. wahuensis (Oahu sedge) is a perennial sedge endemic to Hawaii and serves as an important groundcover species in both ecological restoration and ornamental landscapes. In March 2025, leaf blight symptoms were observed on 19 variegated and 1 non-variegated C. wahuensis subsp. wahuensis plants cultivated at the Magoon Research Facility, University of Hawaii at Manoa campus. Affected plants exhibited irregular to oval brown lesions (3-15 mm in diameter) with distinctly darker margins and occasional chlorotic halos. As the disease progressed, the lesions developed reddish-brown discoloration. Under high humidity, lesions coalesced, resulting in extensive blighting and premature leaf senescence. Black, raised acervuli were consistently observed on older lesions. Symptomatic leaves were collected, surface-sterilized in 10% sodium hypochlorite for 2 min, rinsed in sterile distilled water, and plated on potato dextrose agar (PDA) amended with streptomycin (100 mg/L). Plates were incubated at 25°C under a 12 h light/dark cycle. Fungal colonies with similar morphology were consistently isolated from all samples. Colonies on PDA were initially white and cottony, becoming grayish-white with age, and developed black acervuli within 7 days. Conidia were fusiform, straight to slightly curved, typically five-celled (three median pigmented cells), and measured (18.5-) 20.5-25.0 (-27.5) × (5.5-) 6.0-7.5 (-8.0) μm (n=100). Apical appendages were 2-4, filiform, 12-18 μm long, and a single basal appendage 3-5 μm long was present. Genomic DNA was extracted from a representative isolate (CXWAH) using the DNeasy Plant Mini Kit (Qiagen). The internal transcribed spacer (ITS), β-tubulin (β-tub), and translation elongation factor 1-α (TEF1) regions were amplified and sequenced using primer pairs ITS5/ITS4 (White et al. 1990), T1/Bt2b (Weir et al., 2012), and EF1-526F/EF1-1567R (Rehner and Buckley 2005). Sequences were deposited in GenBank with accession numbers PX720595 (ITS), PX726138 (TEF1), and PX726139 (β-tub). NCBI BLAST analyses of individual locus showed 98-100% similarity as well as the highest number of hits to Neopestalotiopsis rosae. Multi-locus phylogenetic analysis using maximum likelihood (1,000 bootstrap replicates) grouped CXWAH with N. rosae ex-type strain CBS 101057. Pathogenicity tests were performed on healthy variegated and non-variegated C. wahuensis subsp. wahuensis plants to fulfill Koch's postulates using conidial suspensions (1 × 10⁶ conidia/mL) of CXWAH. Control plants were inoculated with sterile distilled water under the same conditions. Plants were covered with plastic bags for 72 h to maintain humidity and incubated at 25 ± 2°C. Typical leaf blight lesions developed within 7-15 days on inoculated plants, resembling natural symptoms, while control plants remained symptom-free. The pathogen was successfully re-isolated from symptomatic tissue, thus fulfilling Koch’s postulates. The assay was conducted twice. This is the first report of N. rosae causing leaf blight on C. wahuensis subsp. wahuensis plants in Hawaii. N. rosae has been associated with leaf blight and dieback of various tropical hosts, including Rosa sp., Paeonia suffruticosa, guava, strawberry, and rubber (Maharachchikumbura et al. 2014; Li et al. 2014; Mang et al. 2025). This discovery extends the known host range of N. rosae to Carex spp.