Rhododendron pulchrum Sweet is a widely cultivated ornamental garden plant. In September 2023, a leaf spot disease was observed at Qujing Normal University (25.525°N, 103.748°E) and Qujing Maternal and Child Health-Care Hospital (25.708°N, 103.429°E), with an incidence rate of 40–60%. The disease caused premature leaf aging and defoliation, significantly reducing the plant's horticultural value. The symptoms manifested as elliptical leaf spots, initially brown with water-soaked margins, later turning dark brown. Microscopy revealed black, subglobose pycnidia (200–300 μm) clustered, immersed in host tissue. To identify the causal agent, 15 symptomatic leaves were collected for pathogen isolation. Black pycnidia were picked with a sterile needle, placed on water agar, and incubated at 25°C for 24 hours. Germinated spores were transferred onto potato dextrose agar (PDA) for further purification and morphological study. Three pure isolates (DJ01, DJ02 and DJ03) were obtained. Colonies on PDA grew rapidly with white fluffy aerial mycelium and yellowish pigmentation on the reverse side, darkening over time. Sporulation occurred after 2 months at 25°C in darkness. Alpha conidia were hyaline, aseptate, ellipsoid to clavate, tapering at both ends, often with two oil droplets, measuring 6.7–9.6 × 2.8–4.5 µm (n=30). Beta conidia were hyaline, aseptate, smooth, straight to slightly curved, and measured 12.1–21.1 × 0.9–2.2 µm (n=30). These morphological features matched descriptions of Diaporthe sojae (Dissanayake et al. 2015). For molecular identification, DNA was extracted from the isolates, and the ITS, β-TUB, CAL, TEF1-α, and HIS genes were amplified using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b (Udayanga et al. 2015). Sequences were deposited in GenBank (accession numbers PQ896484- PQ8964846 (ITS), PV031708-PV032710 (HIS), PV031711- PV031713 (TEF1), PV031714- PV031716 (β-TUB), and PV031717- PV031719 (CAL)). BLAST analysis showed 99.42% (ITS), 100% (TUB), 100% (CAL), 99.02% (TEF1-α), and 99.79% (HIS) identity with D. sojae FAU635. A multilocus phylogenetic tree was constructed using maximum likelihood (ML) analysis of concatenated ITS-TUB-CAL-TEF1-HIS sequences. The isolates (DJ01, DJ02, and DJ03) clustered with other D. sojae strains with a high probability of 98.5%, confirming their identity as D. sojae. Pathogenicity tests were conducted by inoculating 15 healthy mature leaves (one leaf per twig) with 0.2 ml of a spore suspension (1 × 10⁶ conidia/ml). To retain the suspension on the leaf surface, a sterile cotton ball was placed over each inoculation site. An additional 15 twigs were mock-inoculated with sterile water (Li et al. 2024). All twigs were enclosed in plastic bags for 72 hours in a garden setting. After 21 days, inoculated leaves developed symptoms identical to those in the field, while controls remained symptomless. The pathogen was re-isolated and confirmed as D. sojae through morphological and molecular analyses, fulfilling Koch’s postulates. D. sojae is a known pathogen causing pod and stem blight, as well as postharvest fruit brown rot (Dissanayake et al. 2015; Gomes et al. 2013; Xiao et al. 2022). This study represents the first report of D. sojae causing leaf spot disease on R. pulchrum. These findings provide crucial information for the diagnosis and management of this emerging disease.
Li et al. (Wed,) studied this question.