First report of Botryosphaeria dothidea causing dieback of Neolitsea sericea in Korea

Neolitsea sericea (Blume) Koidz., is an important warm-temperate tree species, with an expanding ecological distribution (Yun et al., 2014). It is also economically valuable as a source of anti-inflammatory essential oils (Yoon et al., 2010), and for large-diameter timber production (Won et al., 2017). In May 2024, dieback symptoms were observed in N. sericea growing in a greenhouse at the Warm Temperate and Subtropical Forest Research Center, Jeju, Korea, with a disease incidence of 25% (12 of 48 plants). To identify the causal agent, five symptomatic stems were collected and transported to the laboratory. The stem samples were surface-sterilized with 1% sodium hypochlorite (NaOCl) for 1 min and rinsed three times with sterile distilled water. The dried samples were placed on PDA plates containing streptomycin and incubated at 25 °C. The emerging hyphal tips were transferred to fresh PDA plates, and seven morphologically similar fungal isolates were obtained. The fungal colony of representative isolate CSSB24 initially exhibited gray to olive-colored mycelia with white margins that gradually turned dark gray as they matured. The isolate was cultured for 3 weeks on pine needle agar (PNA) under UV light (12 h light/12 h dark), and produced hyaline, smooth, fusiform, and aseptate conidia with an average size of 24.39 ± 3.45 × 7.56 ± 0.90 μm (n = 30). These morphological characteristics were similar to those of Botryosphaeria dothidea (Slippers et al., 2004). DNA was extracted, and PCR amplification and sequencing were performed for the ITS, β-tubulin (tub2), and translation elongation factor 1-α (tef1) regions. The primer sets used were ITS1/ITS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995), and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. The obtained sequences were deposited in GenBank under the accession numbers PV839846, LC880238, and LC918506. BLASTn analysis revealed that each sequence was highly similar to that of B. dothidea ex-type isolate CMW8000 (AY236949, AY236927, and AY236898), with identities of 99% (513/516), 100% (244/244), and 98% (280/285), respectively. Phylogenetic analysis of concatenated sequences was performed using the maximum likelihood method in MEGA 11. The isolate was clustered with B. dothidea strains, including the ex-type, isolate CMW8000, confirming its identity as B. dothidea. To test the pathogenicity of the B. dothidea isolate, mycelial plugs were placed on the wounded stems of healthy N. sericea seedlings (n = 6), and the sites were sealed with parafilm (Back et al., 2024). Sterile PDA was used as the control in this study. After 10 days, the leaf veins and stems turned dark brown, and progressively became necrotic from the margin, resulting in complete seedling dieback, consistent with the symptoms observed in naturally infected seedlings. The infected stems were covered with sprouting pycnidia, and the conidia obtained from the pycnidia were morphologically consistent with those produced on PNA, confirming the symptoms were caused by B. dothidea. In contrast, the control seedlings were healthy and did not exhibit any symptoms. The pathogen re-isolated from symptomatic seedlings was identified as B. dothidea based on its morphological characteristics and ITS sequence analysis. This is the first report of B. dothidea causing dieback of N. sericea in Korea. Since dieback disease of N. sericea seedlings causes tremendous loss in nursery production under greenhouse conditions, this study underscores the importance of accurately identifying the causal agent, to enable precise control strategies and facilitate the production of healthy and high-quality seedlings.