First report of Neopestalotiopsis rosae causing blueberry shoot blight in China

In September 2024, reddish-brown lesions were observed on shoots of blueberry (Vaccinium corymbosum cv. ‘Emerald’) in a commercial blueberry farm in Anqing, Anhui Province, China. Disease incidence was approximately 60%. Lesions rapidly coalesced, resulting in shoot desiccation, leaf wilting, and severe growth reduction. Symptomatic stems were collected from three randomly selected sites. Tissue segments (5 mm) excised from lesion margins were pooled, surface sterilized, and plated on PDA. After purification by single hyphal tipping, 7 morphologically similar isolates were obtained from 18 cultures. Representative isolate Dpj4 was selected for further characterization. After 7 days incubation at 25°C, colonies on PDA were circular (mean 58 mm, n = 5) with white cottony aerial mycelium and irregular margins; the reverse was light yellow. Black conidial pustules developed on the colony surface after 15 days. Conidia were fusiform, straight to slightly curved, five-celled, and measured 20.99–31.14 × 4.90–8.12 μm (mean 25.2 × 6.4 μm, n = 50). The three median cells were pigmented, with the upper two dark brown and separated by melanized septa and the third pale brown. The apical cell was hyaline and conical, bearing 2–4 filiform appendages, 18.62–46.12 μm long (mean = 31.3 μm, n = 50). The basal cell was hyaline with a central pedicel-like stalk. Based on morphological features consistent with those of Neopestalotiopsis spp. (Maharachchikumbura et al. 2014), the isolate was preliminarily identified as Neopestalotiopsis sp. The isolate showed optimal mycelial growth at 25 °C and pH 7.0, with glucose and peptone as the optimal carbon and nitrogen sources, respectively, under continuous light (24 h). Genomic DNA of isolate Dpj4 was amplified using primers ITS1/ITS4 (ITS), Bt2a/Bt2b (β-tubulin), and EF1-728F/EF1-986R (TEF1-α) (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999). The resulting sequences (528 bp, 432 bp, and 259 bp, respectively) were deposited in GenBank under accession numbers OQ607755, OQ658114, and OQ658115. BLASTn analysis showed 99.81%, 100%, and 100% identity with Neopestalotiopsis rosae sequences MN341543.1 (ITS), PV033951.1 (β-tubulin), and MN704868.1 (TEF1-α), respectively. Sequences were aligned in Geneious Prime 2022.2.1, and phylogenetic analysis of the concatenated ITS, β-tubulin, and TEF1-α dataset was performed using MrBayes (Huelsenbeck and Ronquist 2001) under the HKY+G substitution model (Hasegawa et al. 1985). The analysis placed isolate Dpj4 within the Neopestalotiopsis rosae clade, clustering with reference strains CBS 101057 and CBS 124745, with strong support. Pseudopestalotiopsis cocos CBS 272.29 was used as the outgroup. Pathogenicity tests were conducted on detached shoots (n = 8) and one-year-old potted plants (n = 4) of blueberry cv. ‘Emerald’. Mycelial plugs (7 mm diameter) from 7-day-old PDA cultures were placed on wounded stems; sterile PDA plugs served as controls. Seven days after inoculation, distinct necrotic lesions developed on shoots inoculated with isolate Dpj4, resembling symptoms observed in the field, whereas control plants remained symptomless. The pathogen was re-isolated from symptomatic tissues and showed morphological characteristics identical to the original isolate, fulfilling Koch’s postulates. To our knowledge, this is the first report of Neopestalotiopsis rosae causing shoot blight on blueberry in China.