First report of gray mold caused by Botrytis cinerea on red raspberry (Rubus idaeus) in Korea

During May and June 2014, red raspberry (Rubus idaeus L., Rosaceae) plants with symptoms resembling gray mold were found in several plastic greenhouses with approximately 2 to 10% disease incidence (percentage of inflorescences infected) in Gangneung, Korea. Symptoms included extensive growth of mycelia with gray conidia, mostly on blossoms and fruits, rarely on leaves and twigs. Diseased fruit tissue was excised and surface sterilized by immersion in 0.1% sodium hypochlorite (NaOCl) for 1 min, rinsed three times with sterilized distilled water, placed on potato dextrose agar (PDA, Difco) plates, and incubated at 20 ± 2°C. Conidia (n = 50) were single-celled, ellipsoidal or ovoid, 6.3 to 8.2 × 5.7 to 9.7 μm on naturally infected fruits and 5.9 to 10.8 × 4.9 to 7.3 μm on PDA. Monoconidial isolates were obtained by plating conidial suspensions on PDA and selecting single colonies. After three weeks, the fungus formed several black sclerotia ranging from 1.1 to 4.2 × 1.3 to 3.2 mm (n = 20) near the edge of the Petri dish. A representative isolate (GRGM003) was deposited in Gangneung-Wonju National University and used for further studies. Morphological characteristics were consistent with those of Botrytis cinerea Pers.: Fr. (Ellis 1971). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS) region of rDNA of the isolate was amplified with universal primers ITS1/ITS4 and sequenced. BLAST analysis of the resulting 500-bp nucleotide ITS segment (GenBank Accession No. KP255842) showed 100% identity with the sequence of Botryotinia fuckeliana (teleomorph of Botrytis cinerea). For further confirmation, three nuclear protein-coding genes were sequenced: glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) (Staats et al. 2005). The G3PDH, HSP60, and RPB2 sequences (KT025631, LC020016, and KT025632) of the isolate were 100% identical to those of B. cinerea (e.g., KF015583, KJ018758, and KJ018756, respectively). For the pathogenicity test, inoculum was prepared by harvesting conidia from 2-week-old cultures on PDA. A suspension (1×10⁶ conidia/ml) amended with 0.5% glucose and 0.25% K₂HPO₄ was sprayed onto three potted red raspberry plants with fruits. Three plants of similar features were sprayed with sterilized water, serving as controls. Plants were covered with plastic bags for 2 days after inoculation to maintain high relative humidity (90 ± 10%) and were placed in a growth chamber at 20 ± 2°C. Gray mold symptoms on fruits and leaves developed 3 to 5 days after inoculation, whereas control plants remained symptomless. The pathogenicity test was performed twice with similar results. The pathogen was successfully reisolated from inoculated leaves, fulfilling Koch’s postulates. Red raspberry gray mold has been reported in many countries including the United States and China (Farr and Rossman 2015). To our knowledge, this is the first report of gray mold caused by B. cinerea on red raspberry in Korea. According to our observations, fruit infections occurred under high humidity of the greenhouse. We suppose that ventilation of the greenhouses may effectively reduce severity of gray mold on raspberry fruits.