First Report of Stem Rot of Stevia Caused by Sclerotinia sclerotiorum in North Carolina.

Stevia (Stevia rebaundiana Bertoni) is an emerging perennial crop in the United States. The crop is grown for 3 to 5 years with two harvests per growing season. Stevia contains numerous glycosides that are used as a natural noncaloric sweetener, and in 2008 was approved by the USDA as a sugar substitute. In commercial plantings of second-year stevia in North Carolina, diseased plants were observed in April and May of 2013. Diseased plants were observed in several counties in the state in fields that had been planted primarily in a corn-soybean rotation prior to stevia planting. Symptoms included wilting, chlorotic leaves, necrotic leaves at the base of the stem, bleached stem lesions, and dead plants. Symptomatic plants often also had tufts of white hyphae present on stems and large, irregularly shaped 2- to 8-mm black sclerotia frequently were present on the base of the stem. Isolations from infected stem tissue were made on potato dextrose agar amended with 50 μg/ml of streptomycin sulfate and penicillin G. Based on hyphal and sclerotial characteristics, isolates were tentatively identified as Sclerotinia sclerotiorum (Lib.) de Bary (4). Koch's postulates were confirmed on 10-week-old Stevia plants cv. G3 grown in the greenhouse in 10-cm-diameter pots containing a sterile 1:1:1 sand, loam, media mix. Oat grains infested with one isolate obtained from diseased field plants served as the inoculum. Oats were sterilized on three consecutive days, inoculated with colonized agar plugs of S. sclerotiorum, and then incubated at room temperature until they were thoroughly colonized. Three infested oat grains were buried 1 cm deep approximately 2 cm from the base of the plant in each of the six test pots and plants were observed over a 3-week period for symptoms. Symptoms developed on all plants within 5 days of inoculation. Leaves began to wilt, then turned chlorotic and necrotic, with stem lesions and sclerotia present at the base of the plant. Isolations were taken from infected stem tissue and pure cultures were prepared for molecular identification. Uninoculated control plants did not develop symptoms. Pathogen identification was confirmed using universal primers ITS 4,5 and β-tubulin (2,3). Mycelium from the cultured greenhouse stem isolations were grown in potato dextrose broth. Mycelium samples were aspirated and lyophilized prior to DNA extraction. Extracted DNA was amplified through PCR with ITS and β-tubulin primers and sent for sequencing. Sequences were aligned using CLC Workbench. Sequences from ITS45 had 100% identity to S. sclerotiorum GenBank Accession No. KF859933.1, confirming S. sclerotiorum as the causal organism. The β-tubulin sequence was compared against the Broad Institute S. sclerotiorum whole genome shotgun sequence and was confirmed to have 100% identity to the beta tubulin chain (5). This is the first report of S. sclerotiorum on stevia in the United States. Chang et al. (2) reported a stem rot of stevia in Canada and confirmed S. sclerotiorum as the causal organism. References: (1) K. Chang et al. Plant Dis. 81:311, 1997. (2) J. Freeman et al. Eur. J. Plant Pathol. 108:877, 2002. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) J. E. M. Mordue and P. Holliday. CMI No. 513, 1976. (5) Sclerotinia sclerotiorum Sequencing Project, Broad Institute of Harvard and MIT. Online: http://www.broadinstitute.org/ , accessed July 16, 2014.