Ixeridium dentatum Tzvelev (family Asteraceae) is a perennial herb native to and widely distributed in Korea, Japan, and China [1]. The plant is well known for its anti-cancer, anti-oxidative and anti-allergenic activity [2, 3]. It is cultured in some areas, including Seosan, Dangjin, and Jinan. Sclerotinia rot symptoms were occasionally observed on Ixeridium dentatum cultivated as a succeeding crop in garlic fields in Seosan-si during the growing season of 2016 and 2017. Infected plants formed initially irregular, water-soaked spots on the base of the stems, wilted, then blighted and eventually died. Spherical or irregular sclerotia (2 to 7 mm in size) formed on the basal stem and on the soil surface (Fig. 1A, 1B). Cucumber mosaic virus (CMV disease), tomato spotted wilt virus (TSWV) and Puccinia lactucae-debilis (Rust) have been reported to be associated with diseases of Ixeridium dentatum in Korea [4]. The aim of the present study was to identify the causal agent of sclerotinia rot on Ixeridium dentatum, based on morphological features, sequence analysis of the internal transcribed spacer (ITS) region and pathogenicity test.
Sampling and Isolation
Infected tissues and sclerotia on the basal stem of Ixeridium dentatum were collected from several sites, where it was cultivated as succeeding crop in garlic fields at Seosan-si during 2016 and 2017. The tissues and sclerotia were surface-sterilized by dipping in 1% NaOCl solution for 1 min, rinsed three times with sterilized distilled water, and placed on water agar at 25°C for two days. The margins of each fungal hyphae growing from the tissue and sclerotia were transferred to potato dextrose agar (PDA; Difco, Detroit, MI, USA) plates. The isolated strains were maintained in a sterilized distilled water stock with 30% glycerol at −70°C for identification and pathogenicity tests in Herbal Crop Research Division (HCRD).
DNA extraction, PCR, sequencing, and phylogenetic analysis
Genomic DNA extraction and PCR amplification of the ITS region were performed using previously described methods [5]. DNA sequencing was performed at Macrogen (Seoul, Korea), using an ABI Prism 3700 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were assembled, proofread, edited using MEGA ver. 5.0 [6]. The ITS sequences of the present isolates were aligned with reference sequences downloaded from GenBank using the default settings of MAFFT v7 [7]. Neighbor-joining (NJ) trees were constructed with MEGA 5 using Kimura 2-parameter model [8] and 1,000 bootstrap replicates. All the sequences were deposited in GenBank (accession nos. MF270175~ MF270177). HCRD 16078, HCRD 16080, and HCRD 16084 isolated from infected tissues formed a monophyletic group with Sclerotinia sclerotiorum with 56% bootstrap value. The sequences of the three strains were identical to those of strains previously identified as Sclerotinia sclerotiorum (Fig. 2).
Culture and morphological characteristics
Pure cultures on PDA grew rapidly and formed aerial, white mycelia and turned gray to chocolate after 3 to 4 days. Fungal cultures formed spherical or irregular, dark-black sclerotia on the peripheral edges of the PDA, which ranged from 3 to 8 mm in size (Table 1). The optimal temperature for hyphal growth and sclerotia formation was 20°C, whereas that for sclerotia germination was 25°C. The morphological characteristics of the strains from sclerotinia rot of Ixeridium dentatum were similar to those of previously reported S. sclerotiorum [9].
Pathogenicity test
Pathogenicity tests were conducted by placing five agar plugs (6 mm) of mycelium near the base of the stems of three healthy plants at soil line level. Non-inoculated plants were used as controls. All plants were kept in a dew chamber at 25°C and over 95% relative humidity. After three days, all inoculated plants showed symptoms, such as water soaked spots on the stem, covered with white mycelia, and then rotting, followed by wilting, blighting and eventually, death (Fig. 1C, 1D), whereas non-inoculated plants remained symptomless. Sclerotinia sclerotiorum was consistently re-isolated from the symptomatic tissue in full compliance with Koch’s postulates.
Three Korean strains (HCRD 16078, HCRD 16080 and HCRD 16084) related to sclerotinia rot on Ixeridium dentatum were identified S. sclerotiorum, based on these morphological characteristics, sequence analysis, and pathogenicity test. This is the first report of S. sclerotiorum causing sclerotinia rot on Ixeridium dentatum in Korea.
