First Report of Clonostachys rosea Causing Root Rot of Gastrodia elata in Korea

Gastrodia elata is an obligate mycoheterotrophic orchid that is symbiotically associated with at least two fungal partners: a broad range of Mycena spp. for seed germination and Armillaria mellea for plant growth. This plant has been widely cultivated throughout Korea as a traditional medicine (Taylor et al. 2002). Several fungal pathogens have caused root rot of G. elata in Korea (Han et al. 2017a, 2017b), resulting in approximately 70% reduction in gross annual revenue since 2014 (Jung et al. 2019). We observed necrotic spots (disease incidence 70.5% and severity 25%, n = 296) on tubers of G. elata from nine commercial fields (approximately 7 ha) in Chuncheon (37°52′N, 127°44′E) during July 2017 and August 2018. Diseased tubers showed round and grayish brown spots, which eventually coalesced into larger black lesions on tubers. A total of 74 symptomatic tubers were surface sterilized with 2% NaOCl for 2 min and then washed twice with sterile distilled water for 1 min. Symptomatic tissues (5 mm in diameter) in the border regions between necrotic and healthy tissues were transferred onto potato dextrose agar (PDA) and cultured at 25°C in the dark for 2 weeks. Thirty pure cultures were recovered from necrotic lesions from all fields and collected by single-spore isolations. All colonies were yellowish-white, and the surface was tomentose with thick aerial hyphae. Conidia often formed on both verticillate and penicillate conidiophores as described previously (Afshari and Hemmati 2017). Primary conidiophores were verticillium-like with two to three levels, and secondary conidiophores were mainly penicillate. Conidia were 5.7 to 10.3 × 5.1 to 8.6 µm (mean = 7.4 × 6.2 µm, 30 spores), and chlamydospores were 6.4 to 10.3 × 4.2 to 6.7 µm (mean = 8.0 × 5.9 µm, 30 chlamydospores). The cultural morphology of the isolates was consistent with those of Clonostachys rosea (Moreira et al. 2016; Sun et al. 2018). Three isolates were further confirmed two times by sequencing the internal transcribed spacer (ITS) locus using the primers ITS5/ITS4 (White et al. 1990). This primer set generated a 515-bp amplicon (GenBank accession no. MN548399), and the subsequent BLASTn analysis revealed a 99.81% match for C. rosea (e.g., MN486561). For pathogenicity tests, 15 tubers that were 2 years old and disease-free from the fields in Chuncheon for three isolates (five tubers/isolate) were surface sterilized and then dipped into 500 ml of a conidial suspension (6 × 10⁵ conidia/ml) in potato dextrose broth (PDB) for 30 min, and another 10 healthy tubers (control) were dipped into PDB medium. Each tuber (<40 mm in length) was then transferred into a Petri dish (90 × 10 mm) and incubated in the growth chamber at 25°C and 80% relative humidity in the dark for 2 weeks. Tuber necrosis was observed from all inoculated plants, similar to that observed in the field, whereas control plants showed no symptoms. Isolates developed grayish brown lesions on tubers with variable severity (tuber necrotic area from 28 to 60%). Subsequently, we reisolated the fungal pathogen from the symptomatic tissues, and the isolates were identified as C. rosea based on both morphology and ITS sequences. Three cultures of C. rosea were deposited to the Forest Bio Resources Bank, the National Institute of Forest Science (accession nos. FBRB-19-04251 to FBRB-19-04253). To our knowledge, this is the first report of C. rosea causing root rot on G. elata in Korea. Our result may assist to make the proper measures to control this disease in the commercial fields of G. elata in Korea.