First Report of Grovesinia moricola Causing Zonate Leaf Spot on Hernandia nymphaeifolia in Taiwan.

Hernandia nymphaeifolia (C. Presl) Kubitzki, a native tree of Taiwan, is a sea drift plant (Yang and Lu 1996). It is a salt- and wind-tolerant tree (Bezona et al. 2009) and was selected for the afforestation of badlands in coastal areas of Taiwan. In December 2022, all H. nymphaeifolia seedlings at a nursery in Wu-Lai, Taiwan were diseased and wilted with a similar progression. The initial symptom was small zonate white or gray lesions with water-soaked periphery on leaves. Then, expansion and fusion of leaf spots which caused leaf blight and defoliation were observed. Seedlings eventually wilted. Sporophores found on the host were generally hypophyllous, solitary, erect, and easily detachable. The upper portion of the sporophore was considered an individual conidium and consisted of a pyramidal head that was fusiform to ventricose, 206.3 to 501.8 μm (average: 378.0 ± 75.3 μm) long, and 63.6 to 104.5 μm (average: 85.0 ± 16.2 μm) wide at the broadest point (n=30). Branches within the pyramidal head were short, compact, and di- or trichotomously branched. The central stipe was hyaline, broad, septate, tapering toward an acute apex, and sometimes constricted at the basal septum. Sclerotia were gray or black, spherical, and 1.0 to 2.5 mm (n=10) in diameter and observed on older lesions. The fungus was isolated from infected tissue and sporophores and maintained on potato dextrose agar (PDA) at 20°C in darkness. Sclerotia were produced on PDA after 4 to 5 weeks and were irregular or spherical, but no sporophore was developed. The fungus was identified as Grovesinia moricola (I. Hino) Redhead based on morphological characteristics (Tomoko et al. 2006). Three DNA samples was obtained from the cultures isolated from the diseased leaf, sporophores and sclerotia. They were then amplified by PCR with primers for the internal transcribed spacer region (ITS; primers ITS5/ITS4) and the large subunit nuclear ribosomal RNA gene (LSU; primers LR0R/LR5) (Cho et al. 2017), and then sequenced respectively. The sequences were deposited into GenBank with accession nos. PP727191 to PP727193 and PP748518 to PP748520. BLAST analysis of the three isolates showed 100% identity to the sequences of G. moricola from Taiwan (OP550202, OP550203) for the ITS region and 99.9% identity to the sequence of G. moricola from the USA (MW013804) for the LSU rRNA gene. The specimens (FS2022-140) and the culture (Asco-0109) in this study were deposited into the herbarium of Taiwan Forestry Research Institute in Taiwan. Koch's postulates were performed by inoculating four 8-month-old, asymptomatic, potted H. nymphaeifolia plants; every plant was inoculated with sporophores from infected leaves on the upper surface of each of five leaves. Four uninoculated plants were kept in separate pots and served as controls. All plants were covered with transparent plastic bags individually and incubated in a growth chamber at 18 to 20°C with 8 h of light. Similar leaf spots and sporophores were observed after 2 to 4 days and 10 days on every inoculated plant but not on uninoculated plants. The pathogen with a similar colony on PDA was reisolated from the leaf spots of the inoculated plants. Molecular identification of the reisolated pathogen by the above method was carried out. The sequences showed 99.9% identity to the sequence of G. moricola, and were deposited into GenBank with accession nos. PQ157896 to PQ157897 (ITS region) and PQ157701 to PQ157702 (LSU rRNA gene). The pathogenicity test was repeated once. G. moricola is known to cause severe defoliation on woody and annual plants, including at least 73 host species and 36 families distributed in the eastern United States and Japan (Trolinger et al. 1978). This is the first report of G. moricola on H. nymphaeifolia in the world. Control of the disease would play an important role in maintaining healthy seedlings for the afforestation in Taiwan.