First Report of <i>Apiospora guangdongensis</i> Causing Leaf Spot Disease on <i>Rhododendron pulchrum</i> in China

Rhododendron pulchrum Sweet is an ornamental plant with high horticultural value. In September 2024, a leaf spot disease was discovered on the R. pulchrum Sweet plants located on the campus of Guizhou University in Guiyang City, Guizhou Province, China (26°27 '7 "N, 106°39' 17" E). After on-site investigation of 2000 plants, approximately 45% of the plants exhibited the characteristics of this disease. The disease caused premature leaf aging and defoliation, significantly reducing the plant's horticultural value. The symptoms are manifested as irregular dark brown leaf spots. To identify the causal agent, Sixty diseased leaves from twenty plants (three leaves from each diseased plant) in the same growth period were collected randomly and cut into small pieces (5 mm × 5 mm), surface-disinfected with 1% NaClO for 30 s, 75% ethanol for 30 s, rinsed three time with sterile water, dried, and placed on potato dextrose agar (PDA) medium and incubated at 28°C for 5 d. 54 pure cultures were obtained through tissue isolation and purification. After ITS-BLAST, the results indicated that among the cultures, 49 were Apiospora. From 49 obtained isolates with same morphological characters, three representative strains (GUCC24-0107, GUCC24-0108 and GUCC24-0109) were selected. Colonies on PDA grew rapidly, floccose, sparse, no pigment. Conidia are pale brown to dark brown, oval or round, smooth and size range was 6.03-9.04×5.5-8.96 μm (n=50) in diameter. For molecular identification, the ITS, TEF1-α, LSU and TUB2 genes were amplified using primers ITS1/ITS4, EF1-728F/EF-2, LROR/LR5 and Bt2a/Bt2b (Liao et al. 2023). Sequences were deposited in GenBank (accession numbers PV910651 - PV910649 (ITS); PV640933, PV911113, PV911114 (LSU); PV878564 - PV878566 (TEF1-α) and PV640440 - PV640442 (TUB2)). BLAST searches of the sequences revealed 100% (543/543 bp), 99% (858/859 bp), 98.67% (445/451 bp), and 99.39% (487/490 bp) homology with those of A. guangdongensis ZHKUCC 23-0004T from GenBank (OQ587994.1, OQ587982.1, OQ586060.1, and OQ586073.1), respectively. A multilocus phylogenetic tree was constructed using the maximum likelihood method (ML) and Bayesian inference (BI) methods. The results showed that the isolates were clustered with A. guangdongensis ZHKUCC 23-0004T with high confidence (Liao et al. 2023). Therefore, the morphology and molecular analysis indicated that the pathogen was A. guangdongensis. To confirm pathogenicity, a conidial suspension of isolate (1×106 spores/mL) was sprayed onto three healthy R. pulchrum Sweet (five leaves per plant). Three additional plants were treated with sterilized distilled water as controls. All plants were incubated at 28°C with 85% relative humidity and a 12-hour light/dark cycle. After15 days, inoculated leaves developed symptoms identical to those in the field, while controls remained symptomless. The pathogen was re-isolated and confirmed as A. guangdongensis through morphological and molecular analyses, fulfilling Koch’s postulates. This study reports for the first time that A. guangdongensis can cause Rhododendron leaf spot disease. This discovery breaks through the traditional understanding that Rhododendron leaf spot disease is mainly caused by fungi of the genus Pestalotiopsis (Zhu et al. 2023, Li et al. 2024) or Diaporthe (Wu et al. 2024, Lin et al. 2025), providing a new pathogen identification index for disease diagnosis It also provides a scientific basis for growers to understand this disease.