First Report of Gray Mold Caused by Botrytis cinerea on Hazelnut ( Corylus heterophylla ) in South Korea

Corylus heterophylla Fisch. ex Trautv., known as hazelnut is a minor crop in South Korea, however, interest in its cultivation has been gradually increasing in recent years. In July 2023, hazelnut fruits exhibiting distinct gray mold symptoms were observed on approximately 10-year-old hazelnut trees in Bonghwa, South Korea (36°57'46.8"N 128°43'48.4"E). This disease first appeared in May and persisted until August, with the incidence rate of 20 to 30%. Small, round, and brown lesions initially appeared on the husks surrounding the hazelnut fruits. As the lesions gradually expanded, they ultimately led to the complete decay of both the husks and the fruits. In order to identify the pathogen, two diseased fruits were randomly selected. The surface of diseased fruit samples was sequentially sterilized with 2% sodium hypochlorite and 70% ethanol for 30 seconds each. Subsequently, the samples were rinsed 3 times with sterile distilled water and air-dried on a sterile paper towel in a clean bench. Small tissue fragments (5 × 5 mm) from the internal parts of infected fruits were placed on potato dextrose agar (PDA) plates, with four pieces per plate, and incubated at 25 °C for 3 days. The growing mycelium was transferred on new PDA. After 10 days of incubation at 25 °C, the colony diameter of the isolate on PDA ranged from 75 to 90 mm. The colony exhibited a woolly texture, dark gray at both the center and the margins, and had a circular shape. Sclerotia formed on PDA after 2 weeks, with a size range of 0.41 - 1.20 × 0.40 - 1.09 mm (average = 0.83 × 0.75, n = 30), were hard, black, round or irregular shapes within the hyphae. The conidia were one-celled, hyaline and round or elliptical, with a size range of 6.55 - 11.32 × 8.52 - 12.53 μm (average = 8.97 × 10.61, n = 50) and were attached on tips of brown conidiophores. Genomic DNA was extracted from two isolates (nifos03 and nifos04). The internal transcribed spacer (ITS) region of rDNA, the RNA polymerase II (RPB2), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using the primer pairs ITS1F/ITS4 (Gardes and Bruns, 1993, White et al., 1990), RPB2-5F/RPB2-7cR (Sung et al. 2007) and GDF1/GDR1 (Guerber et al. 2003), respectively. Sequences were deposited in GenBank (Accession numbers described in eXtra file). BLAST search results revealed 100% identity with B. cinerea for ITS, RPB2, GAPDH sequences (ITS: KP255842, RPB2: MT787340, GAPDH: KF015583). Phylogenetic analysis was performed by constructing an ML tree using the RAxML-NG (Alexey et al., 2019), with the concatenated sequences of 3 genetic regions. Both isolates were identified as B. cinerea. For pathogenicity tests, conidial suspensions (10 ml, 1×106 spores/ml) were sprayed on 8 healthy, 10-year-old hazelnut trees in experimental forest, and other 8 healthy, 10-year old plants were sprayed with sterile water as a control. After 7 days of incubation in plastic bags, gray mold symptoms developed on all the B. cinerea inoculated fruits. In contrast, no symptoms were found on control group, confirming that disease development was associated with the inoculation. The same fungus was re-isolated from the inoculated fruits and identified as B. cinerea in both molecular and morphological characterization, thereby fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of B. cinerea causing gray mold on hazelnut fruits in South Korea. Our results highlight the need for additional research into disease management of hazelnut cultivation.