Tubakia subglobosa Causes Leaf Spots on Ring-Cupped Oak Trees in Korea

Abstract

Ring-cupped oak, Quercus glauca Thunb., is an evergreen broadleaf tree growing up to 15 to 20 m tall. It is native to eastern Asia (China, Japan, Taiwan, and Korea). In Korea, it is distributed in the islands of the southern part of the peninsula, mostly in Jeju Island, South Korea. This species is planted as a windbreak and as an ornamental tree in urban forest areas and its fruit, leaves, and bark have been used in medicine. Small, dark-brownish leaf spots with a tiny yellowish center were observed on ring-cupped oak leaves in Jeju Island from May to July 2015, most extensively at an altitude of 203 to 228 m above sea level (33°28.087′N, 126°29.589′E). Symptoms enlarged with time, becoming distinctive epiphyllous lesions with a necrotic margin. Tiny, brownish, disc-shaped, crustose conidiomata composed of scutella, columella, and conidia (typical of the genus Tubakia), resembling grains of dust, were produced around and over the lesions. From these, a fungus was consistently isolated and cultured on malt extract agar (MEA). Specimens from Jeju Island and their cultures were deposited at the Herbarium of the Korean Forest Research Institute, Seoul, South Korea (HKFRI) and the Korean Collection for Type Cultures, Jeonbuk, South Korea (KCTC), with accession numbers HKRFI4093 and HY4604 (KCTC46432), and HKRFI4094 and HY4702 (KCTC46433). On MEA, fungal cultures formed creamy, cinnamon-colored colonies in the center, with concentric rings of aerial mycelium with a scalloped margin. Scutella on leaves were septate, leaves have radiating, septate hyphae, irregular at margin, usually tufted, brown to dark-brown, 80 to 150 μm in diameter; columellae were parenchymatous, 12 to 18 × 6 to 9 μm in size; conidia were subglobose, hyaline to reddish brown, 10 to 13 × 9 to 11 μm in size. Microconidia were not observed. These macro- and micromorphological traits closely fitted to those of Tubakia subglobosa (Diaporthales), which was described from Japan (Yokoyama and Tubaki 1971). ITS rDNA was PCR-amplied using primers (ITS1F/ITS4) and sequences (612 bp each) of the two fungi were analyzed and compared using BLAST search with sequence data from a Japanese T. subglobosa culture (CBS193.71, KY952636) from CBS-KNAW culture collection, and with sequences from our two isolates of T. subglobosa deposited in GenBank (accession nos. KY934453 and KY934454). Our fungus showed complete sequence homology to these T. subglobosa isolates, confirming its identity as T. subglobosa. In order to carry out the pathogenicity test, fungal spores produced on sterilized ring-cupped oak leaves were wound-inoculated on the main veins of the tree leaves as in Harrington et al. (2012). Small dark-brownish spots, with a yellowish center, matching initial natural symptoms, were produced 13 days after inoculation. Brownish disc-shaped conidiomata were formed on or around the necrotic spots, from which the fungus was reisolated, thus fulfilling Koch’s postulates. To our knowledge, this is the first report on the occurrence of T. subglobosa on Q. glauca in Korea.