First Report of Sclerotinia Stem Rot of Anemone Caused by Sclerotinia sclerotiorum in Korea.

In Korea, anemones (Anemone coronaria L.) are mostly grown during winter, in polyethylene tunnels that may have high humidity and poor ventilation, to meet the high demand of cut flowers in February and March for school ceremonies. During the winter of 2011 to 2012, symptoms typical of Sclerotinia stem rot were observed in commercial crops of anemone (cv. Rosso Mistral Plus) in Hwaseong City, Korea. About 40% of anemones withered or died before harvest due to the disease, causing considerable economic losses. In one farmer's polyethylene tunnel with a good ventilation system in Icheon City, Korea, less than 1% of the anemones showed Sclerotinia stem rot during the same season. Symptoms included stem necrosis and withering of leaves, followed by crown rot and wilt a few days afterward. White cottony growth of the fungus was observed on dead plants and the nearby soil surface, especially under high relative humidity. Black sclerotia 2 to 7 mm in diameter developed in the mycelium. Isolations from surface-disinfested stem pieces onto potato dextrose agar (PDA) consistently yielded white, fluffy colonies. Two-week-old colonies produced plentiful numbers of sclerotia on PDA. The isolated fungus was morphologically identified as Sclerotinia sclerotiorum (Lib.) de Bary (1,3). Voucher specimens (n = 4) were deposited in the Korea University herbarium (KUS). Two isolates were deposited in the Korean Agricultural Culture Collection with accession numbers KACC46708 (ex KUS-F26433) and KACC46834 (ex KUS-F26437), respectively. Fungal DNA was extracted with a DNeasy Plant Mini DNA Extraction Kit (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 (4) and sequenced using an ABI Prism 337 automatic DNA sequencer (Applied Biosystems, Foster, CA). The resulting sequences of 501 bp were deposited in GenBank (Accession Nos. KC412065 and KC412066). A BLAST search revealed that sequences of the two Korean isolates showed 100% identity with those of S. sclerotiorum (e.g., GenBank Accession No. JN012606). A pathogenicity test was achieved by placing agar segments (9 mm²) from a 7-day-old culture grown on PDA on the stems of healthy anemones (cv. Rosso Mistral Plus) near the soil line. Three plants inoculated with agar blocks served as controls. Plants were maintained in a greenhouse at 16 to 20°C and relative humidity >90%. After 2 days, all inoculated stems became discolored, soft, watery, and covered with white mycelia, whereas control plants remained symptomless. S. sclerotiorum was consistently reisolated from the symptomatic tissue, fulfilling Koch's postulates. Sclerotinia stem rot of anemones caused by S. sclerotiorum has previously been recorded from the United States and New Zealand (2), and to our knowledge, this is the first report of S. sclerotiorum on anemone in Korea. According to our observations in damaged plots, low temperature, high humidity, poor ventilation, and continuous cultivation would accelerate the incidence of Sclerotinia stem rot in polyethylene tunnel cultivation systems in Korea. References: (1) M. D. Bolton et al. Mol. Plant Pathol. 7:1, 2006. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA. Retrieved December 3, 2012. (3) S. Umemoto et al. J. Gen. Plant Pathol. 73:290, 2007. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.