First Report of Leaf Spot on Hydrangea macrophylla Caused by Boeremia exigua in Korea.

Abstract

Hydragea (Hydrangea macrophylla) is a garden plant commonly used in parks and landscapes globally. In late May 2023, brown necrotic spots surrounded by chlorotic halos were observed on the leaves of H. macrophylla (cv. Renate Steiniger) at the Suncheonman National Garden in Suncheon (34°55'45.6" N 127°30'26.5" E), South Korea. The affected area covered approximately 20 m2, with the disease incidence ranging from 2% to 5%. Concentric rings with light brown centers measuring 0.5 to 6 cm were observed on the upper sides of the leaves. To isolate the pathogens, each infected leaf from three different plants was cut into 5 × 5 mm pieces, surface-sterilized with 70% ethanol for 1 min, and rinsed three times with sterile distilled water. The samples were cultured on 1.5% water agar amended with 100 μg/mL of streptomycin in the dark at 25 °C. Single-spore isolation from the grown hyphae was performed on the obtained isolates (SYP-1229 to 1231). The fungal colony on potato dextrose agar was olivaceous gray with floccose aerial mycelia, and black pycnidia developed on the medium after 14 days. The conidia observed were hyaline, cylindrical to ellipsoidal-oblong, and aseptate, measuring 4.9 to 9.1 μm × 1.9 to 4.2 μm (avg. 6.3 × 2.7 μm, n = 100) (length × width). The isolates were identified as Boeremia sp. based on their morphological characteristics (Boerema, 1976; Boerema, 2004). For molecular identification, the internal transcribed spacer (GenBank accession nos.: OR682193-OR682195), actin (OR689851- OR689853), β-tubulin (OR689854- OR689856) and translation elongation factor-1 alpha (OR689857- OR689859) sequences from the isolates SYP-1229 to 1231 exhibited 100% (558/558 bp), 100% (263/263), 100% (314/314), and 100% (316/316) similarity, respectively, with Boeremia exigua (EU167567, EU880846, OP611549, and KY484684, respectively) in GenBank BLAST search. Phylogenetics based on concatenated ITS, ACT, TUB2, and TEF sequences, using the maximum likelihood method of MEGA X (Kumar et al., 2018), showed that sequences of SYP-1229 to 1231 were clustered in the same clade as B. exigua. For pathogenicity tests conducted in pots, 10 mL conidial suspension (1 × 106 conidia/mL) derived from isolate SYP-1229, which had been cultured on PDA for 14 days, was sprayed onto two leaves of each of three independent healthy 1-year-old hydrangeas (cv. Renate Steiniger). The leaves of the control plants were inoculated with sterile distilled water, which was also applied to two leaves of each of the three independent healthy plants. The inoculated plants were placed in clear plastic boxes to maintain high humidity and incubated at 25 °C under 18 h of light. Brown spot lesions were observed on the inoculated two leaves seven days after inoculation, whereas the control were asymptomatic. Similar pathogenicity results were obtained for all three independent biological replicates. The fungus isolated from the lesion was identified as B. exigua through morphological characterization and ITS sequencing, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of B. exigua causing brown-spot disease on H. macrophylla in South Korea. The pathogen has been previously reported H. macrophylla in Italy and the United States (Farr and Rossman 2017; Garibaldi et al. 2006), and H. paniculata in Italy (Garibaldi et al. 2018). This study will help in future detection and control to enhance the ornamental value of hydrangeas in Korea.