First report of Diaporthe eres causing leaf spot disease on Machilus thunbergii in Korea.

Abstract

Machilus thunbergii (Japanese bay tree) is native to warm temperate and subtropical regions in East Asia such as China, Japan, Korea, Taiwan, and Vietnam (Wu et al., 2006). This tree is used for landscape trees, windbreaks, and furniture because the wood is hard and dense (Hong et al., 2016). In May 2020, a leaf spot disease was observed on M. thunbergii in an arboretum on Wando Island, Korea. Among 25 trees surveyed in the arboretum, 7 trees showed 5 to 30% leaf spot disease. Symptoms consisted of gray and dry leaf spots up to approximately one to two centimeters in diameter, surrounded by a deep black margin. Leaf samples containing lesions were collected from the seven diseased trees. Pieces of leaf tissue (5mm × 5mm) were cut from the lesion margins and surface disinfected with 1% sodium hypochlorite (NaOCl) for 1 min and rinsed with sterile distilled water three times, patted dry on sterile paper towel and placed on Potato Dextrose Agar (PDA) in Petri dishes. From the cultures, ten fungal isolates were obtained and two representative isolates (CMML20-5 and CMML20-6) were stored at the Molecular Microbiology Laboratory, Chonnam National University, Gwangju, Korea. Colony morphology of the two isolates on PDA was observed after 7 days at 25°C in the dark. Conidiomata were induced after 7days in a 14h-10h light-dark condition using sufficiently grown mycelium in PDA, and both alpha and beta conidia were observed. Alpha conidia were 7.6 ± 0.9 × 2.8 ± 0.4 μm (n = 30), fusiform, aseptate, and hyaline. Beta conidia were 28.1 ± 3.6 × 2.7 ± 0.4 μm (n = 30), aseptate, hyaline, linear to hooked. Genomic DNA of the two isolates was extracted using the CTAB DNA extraction method (Cubero et al., 1999), followed by PCR using primer sets of the internal transcribed spacer (ITS1/ITS4) (White et al., 1990), elongation factor 1-α (EF1-728F/EF1-986R), calmodulin (CAL228F/CAL737R) (Carbone and Kohn, 1999), and TUB2 (Bt2a/Bt2b) (Glass and Donaldson 1995). PCR products were sequenced and analyzed to confirm species identity. The obtained sequences were deposited in GenBank (accession numbers OM049469, OM049470 for ITS, OM069429, OM069430 for EF1-α, OP130141, OP130142 for CAL, and OP130139, OP130140 for TUB2). BLASTn search analyses for ITS, EF1-α, CAL, and TUB2 sequences of two isolates selected resulted in near identical match (>97% for ITS, 100% for EF1-α, >99% for CAL, and >96% for TUB2) to sequences of Diaporthe eres strain AR4346 (=Phomopsis fukushii) (JQ807429 for ITS, JQ807355 for EF1-α, KJ435003 for CAL, and KJ420823 for TUB2). Phylogenetic analysis using maximum likelihood indicated that the two isolates grouped with reference strains (AR4346, AR4349, and AR4363) of D. eres with 76% bootstrap support. Based on morphological and phylogenetic analyses, the two isolates characterized in this study are members of the Diaporthe eres species complex as described by Udayanga et at. 2014. Pathogenicity tests were conducted using both detached leaf and whole plant assays. Mycelial PDA plugs (5-mm in diameter) or 10μl of 106 conidia suspensions were inoculated on detached leaves of M. thunbergii from 2-year-old trees and placed in 90 mm Petri-dishes containing wet filter papers or water agar medium. Mock inoculated controls used water in place of conidial suspensions. The plates were sealed with Parafilm and incubated at 25°C in the dark. Two year old M. thunbergii trees were inoculated with wet mycelia (1.5g) that was ground with a homogenizer and mixed with 50ml of sterile water and sprayed onto wounded leaves and stems with a needle. Mock inoculated controls were sprayed with water only. The inoculated seedlings were placed in plastic containers at 25 to 30°C to maintain high humidity. The pathogenicity tests were repeated three times with three replications. In detached leaves, symptoms of black spots were observed 6 days after mycelial plug inoculation and 20 days after conidia inoculation. In whole plants, typical symptoms were observed 9 days after inoculation. Symptoms were not observed on the control leaves and plants. Diaporthe eres was re-isolated from the inoculated leaf and whole plants and morphologically identified, fulfilling Koch's postulates. Diaporthe eres has been reported to cause a leaf spot on Photinia × fraseri 'Red Robin' in China (Song et al. 2019). To our knowledge, this is the first report of leaf spot disease caused by Diaporthe eres on Japanese bay tree (Machilus thunbergii) in Korea. It is expected that use of this tree will expand given its utility, however infection with D. eres can cause serious diseases to the leaves and stems. Therefore, further studies on disease management are needed.