First Report of Leaf Blight of Camellia japonica Caused by Diaporthe fukushii in Tennessee and the United States

Japanese camellia (Camellia japonica), is an important ornamental species that has an increasing economic value in China, Japan, Australia and the USA (Vela et al. 2013). Leaf blight symptoms were observed on 20-year-old C. japonica 'April Tryst' leaves collected from a research plot in McMinnville, TN in March 2022. Leaf blight first appeared in the leaf tips and was irregular in shape (2 to 3 cm in diameter). Affected areas displayed gray color discoloration with a deep black margin and gradually expanded in size along the leaf margin, eventually causing leaf death and defoliation. Dark brown globose to subglobose conidiomata (pycnidia) were observed abundantly on the infected leaves (Fig. 1a). Disease severity was 25 to 50% of leaf area and incidence was 10% out of 60 plants. Three leaves were collected from each symptomatic plant and the surface disinfected with 10% NaOCl for 60 s, washed thrice with distilled water, and plated on potato dextrose agar (PDA). Colony growth of the isolates FBG4744 and FBG6184 on PDA, 15 days after incubation at 25°C (light/dark: 12/12h) were white to pale grey with dense and felted mycelium with concentric zonation. Spherical black pycnidia were observed on the concentric rings 2-3 weeks after incubation. Alpha conidia were on average 7.15 × 4.82 µm (4.89 to 9.37 µm × 2.91 to 6.74 µm) in size and were aseptate, hyaline, smooth, and ellipsoidal (n=50). Beta conidia were not observed. Pathogen identity was confirmed by extracting total DNA using the DNeasy PowerLyzer Microbial Kit from 7-day-old cultures. Primer pairs ITS1/ITS4 (White et al. 1990), T1/T222 and EF1/EF2 (Stefańczyk et al. 2016) were used to amplify and sequence the ribosomal internal transcribed spacer (ITS), beta-tubulin (BT), and translation elongation factors 1-α (EF1-α) genetic markers, respectively. The sequences (GenBank accession nos. OR607729, ITS; OR608485, BT; OR608487, EF1-α) were 100% similar to Diaporthe fukushii (=Phomopsis fukushii) in the NCBI nr/nt database (JQ807450: ITS; MG812590: BT, and MG281573: EF1-α). A phylogenetic analysis was performed using concatenated sequences of ITS, BT, and EF1-α of D. fukushii and other closely related taxa retrieved from GenBank (Fig. 2). Pathogenicity tests were performed on 1-year-old 10 healthy potted plants of C. japonica 'April Tryst' per isolate (Mathew et al. 2015; Yang et al. 2019). One leaf per plant was wounded with a sterilized 0.2-mm needle. PDA plugs (5 mm) taken from 7-day old cultures of FBG4744 and FBG6184 isolates were deposited on the wounded leaves and covered with moist cotton (Yang et al. 2019; Zhao et al. 2020). Ten additional plants were used as control and sterile PDA plugs were placed on the wounded leaves. Plants were covered with clear plastic bags and kept inside a greenhouse at 21 to 23°C, 70% RH, 16 h photoperiod. All inoculated leaves exhibited blight symptoms 14 days after inoculation (Fig. 1b) while control plants remained asymptomatic (Fig. 1c). The pathogen was reisolated from all the inoculated leaves and was confirmed as D. fukushii using morphological and molecular tools. Diaporthe species (D. tulliensis, D. passiflorae and D. perseae) have been previously reported to cause leaf spot on Camellia sinensis in Taiwan (Ariyawansa et al. 2021), but to our knowledge, this is the first report of leaf blight of C. japonica caused by Diaporthe fukushii in Tennessee and the United States. Identification of this novel disease is important in developing necessary management approaches.