First Report of <i>Botryosphaeria dothidea</i> Causing Stem Canker on <i>Eucalyptus cinerea</i> in China

Eucalyptus cinerea (Myrtaceae), commonly known as Argyle apple or Eucalyptus, is an evergreen shrub or small tree native to Australia. It was introduced to China in the 1980s (Silva et al. 2011) and is valued for pulp production and as ornamental foliage. In April 2025, a stem canker disease was observed on 2- to 10-year-old E. cinerea in forests and nurseries in Kunming, Songming, and Pu’er City, Yunnan Province, China. Symptoms included dry, sunken, rough, and cracked tissues on the main stems. Cracks often developed between diseased and healthy tissues, with translucent gummy exudates, a condition exacerbated after rainfall. The disease incidence exceeded 41% across four surveyed forest areas. Total 40 symptomatic tissues were randomly collected from four tree nurseries. Single-spore isolates were obtained from necrotic cortical tissue at the canker margin following Choi et al. (1999), using a sterile microneedle to transfer individual conidia to PDA amended with kanamycin. Seven morphologically similar isolates (YAGF01-YAGF07) were obtained. On potato dextrose agar (PDA), colonies were initially white, turning grey, with short, thick aerial hyphae and irregular margins. Conidiogenous cells were hyaline, aseptate, and narrowly fusiform. Conidia were hyaline, aseptate, subcylindrical, measuring 11.2-15.3×3.4-5.1 μm (n=50), consistent with Botryosphaeria species (Crous et al. 2006). B. dothidea was successfully isolated from 37 of 40 symptomatic tissues (92.5%), with consistent morphology and sequence identity across all isolates. For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1- alpha (ef1-α) gene, and the beta-tubulin 2 (β-tub2) gene were amplified and sequenced (ITS accessions PX452877-PX452883; ef1-α accessions PX507525-PX507531; β-tub2 accessions PX507532-PX507538) using published primers (White et al. 1990; Ignazio and Linda, 1999; Glass and Donaldson 1995). BLASTn analysis revealed 99-100% identity to the ex-type strain of B. dothidea CWM8000. A phylogenetic tree constructed by Maximum Parsimony (MP) and Maximum Likelihood (ML) methods placed the seven isolates in a clade with B. dothidea with high bootstrap support (94%/95%) and were distinguished clearly from other species. Pathogenicity tests were performed using isolate YAGF01. Stems of six healthy 1-year-old E. cinerea saplings were surface sterilized with 75% ethanol and wounded with a sterile scalpel. A 3-mm mycelial plug of YAGF 01 from a 5-day-old PDA culture was applied to the wound. Control plants were inoculated with sterile agar plugs. All plants were incubated at 25°C with a 12 h photoperiod and 80% humidity. The entire assay was independently repeated three times. Within 5 days, all inoculated stems developed stem canker symptoms identical to those observed in the field, while controls remained asymptomatic. B. dothidea was re-isolated from all symptomatic tissues and confirmed by morphology and sequencing, thus fulfilling Koch's postulates. No fungus was isolated from controls. B. dothidea has been reported to cause stem canker on various hosts, including Cyclocarya paliurus, soybean, and apple(Ilyukhin et al. 2022; Chen et al. 2021; Zheng et al. 2020). To our knowledge, this is the first report of B. dothidea causing stem canker on E. cinerea in China. Given the high disease incidence observed, this pathogen poses a potential threat to the cultivation and economic value of E. cinerea in the region.