First report of Diaporthe hongkongensis causing leaf spot on Rhododendron latoucheae in China.

Abstract

During a field study in the Baili Azalea Forest Area in Guizhou Province, China (27°12'N, 105°48'E) between May and July 2023, symptoms of leaf spot were observed on Rhododendron latoucheae Franch. The incidence of leaf spot on R. latoucheae leaves was about 12% in a field of 1 hm2, significantly reducing their ornamental and economic value. The affected leaves bore irregular, grey-white lesions with distinct dark brown borders, accompanied by black conidiomata. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. Conidiomata present on the lesions were transferred onto water agar and incubated at 25℃ for 24 hours. Germinated spores were then transferred onto potato dextrose agar (PDA) to generate single spore cultures for morphological analysis. This process yielded three single-spore isolates: GZULJ 3-6.1, GZULJ 3-6.2, and GZULJ 3-6.3, all displaying identical morphological characteristics. Representative isolate GZULJ 3-6.1 was used for further study. The colonies, cultivated on PDA at 25°C in the dark, appeared pale gray with white aerial mycelium. Alpha conidia were single celled, translucent and fusiform, measuring 6.2 to 8.1 × 2.6 to 3.0 μm (n=50). Beta conidia were filamentous and hook-shaped, measuring 17.0 to 23.0 × 0.9 to 1.5 μm (n=50). The morphological features were consistent with the established description of Diaporthe hongkongensis (Zhang et al. 2021). For molecular identification, total genomic DNA was extracted. The complete internal transcribed spacer region (ITS), partial sequence of beta-tubulin (TUB), translation elongation factor 1-alpha (TEF1), and calmodulin (CAL) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson. 1995), EF1-728F/EF1-986R and CAL-228F/CAL-737R (Carbone and Kohn. 1999), respectively. The ITS, TUB, TEF1, and CAL sequences (GenBank accession nos. OR807761, OR825453, OR825450, and OR825447, respectively) were obtained, and BLASTn analysis against sequences in GenBank showed 97.37 to 99.30% identity with Diaporthe hongkongensis CBS 115448. Phylogenetic analysis reaffirmed the isolate's placement within a well-supported cluster alongside D. hongkongensis. The pathogenicity of GZULJ 3-6.1 was evaluated using three 2-year-old R. latoucheae plants inoculated with spore suspension (106 spores per ml). The inoculation targeted three leaves per plant, while an additional three plants, serving as controls, were sprayed with sterile water. The plants were maintained in the growth chamber at 25°C, with a 12-hour photoperiod and 75% relative humidity. The pathogenicity test was repeated three times. After 14 days, the inoculated leaves developed brown lesions similar to those observed in the field, whereas the control leaves remained symptom-free. The fungus was successfully reisolated from the infected leaves and identified through morphological characterization and molecular analyses, confirming its identity. D. eres, a closely related species to D. hongkongensis, has been reported to induce similar disease symptoms on R. latoucheae (Wu et al. 2024). However, to our knowledge, this is the first report of D. hongkongensis causing leaf spot disease on R. latoucheae in China. This finding will facilitate the development of effective management strategies to control the complex leaf spot disease affecting R. latoucheae and provide critical insights into its broader implications for the health of this species.