Native Hawaiian ferns play crucial ecological roles in forest ecosystems and have cultural significance in Hawaiian traditions. In April 2025, anthracnose symptoms were observed on four native Hawaiian fern species in a research greenhouse facility on Oahu: Reholttumia hudsoniana (Hudson's fern), Ctenitis latifrons (broad-leaved chain fern), Cibotium menziesii (Hawaiian tree fern), and Cibotium chamissoi (Hawaiian man fern). Symptoms included circular to irregular brown to black necrotic lesions with dark brown margins on fronds (2-15 mm diameter). On severely infected plants, lesions coalesced, causing extensive frond necrosis and premature senescence. Lesions occupied up to 70% of leaf surfaces of affected plants. Symptomatic frond tissue was collected (n=4 samples per fern species). Tissue sections (5 mm 2) from lesion margins were excised, surface-sterilized in 10% sodium hypochlorite for 1 min, rinsed three times in sterile distilled water, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C under 12 h light/dark photoperiod for 7 days. Fungal isolates with similar morphological characteristics were consistently recovered from the four fern species. Colonies on PDA were initially white, turning pale orange to salmon-color with age, showing abundant aerial mycelia and concentric ring patterns. Conidia were hyaline, cylindrical to fusiform, straight, and measured 10-20. 5 × 3. 5-5. 5 μm (n=50). Appressoria were brown to dark brown, oval to irregular, and measured 8. 5-12. 5 × 6. 5-9. 5 μm (n=20). These morphological characteristics were consistent with Colletotrichum species. Two representative isolates (FNii₁ and FNii₂) were obtained through single-spore isolation. DNA was extracted from both isolates using DNeasy Plant Mini Kit (Qiagen). The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and β-tubulin (TUB) gene were amplified using primers ITS1F/ITS4 (Gardes and Bruns, 1993), GDF1/GDR1 (Damm et al. 2012), and T1/BT2b (Weir et al. 2012). PCR products were sequenced and deposited in GenBank (accession numbers: ITS: PX242764, PX242765; GAPDH: PX246505, PX246506; TUB: PX246507, PX246508). BLAST searches showed 99-100% identity with C. tropicale sequences. Phylogenetic analysis using the maximum likelihood method with 1, 000 bootstrap replicates confirmed the identification as C. tropicale, clustering with reference strains from GenBank. Pathogenicity tests were conducted to fulfill Koch's postulates. Healthy fronds from the four fern species were inoculated with conidial suspensions (4×10⁶ conidia/mL) of isolate FNii₁. Control plants were inoculated with sterile distilled water. Inoculated fronds were covered with plastic bags for 72 h to maintain humidity and were further incubated for 3 weeks. Anthracnose symptoms developed 7-10 days post-inoculation on all inoculated plants, while control plants remained symptom-free. The pathogen was successfully re-isolated from symptomatic tissue, completing Koch's postulates. The assay was conducted twice. This is the first report of C. tropicale causing anthracnose on ferns globally. C. tropicale has been reported as a pathogen of various tropical and subtropical crops including avocado, mango, papaya, and banana (Damm et al. 2012; Lima et al. 2013), but this discovery extends its known host range to include pteridophytes.
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