First Report of Penicillium olsonii Causing Strawberry Fruit Rot in China

Strawberry (Fragaria × ananassa) is an important crop in China and is highly susceptible to fungal diseases. In March 2025, fruit rot symptoms were observed in greenhouse-grown ‘Hongyan’ strawberries in Changping, Beijing (24 °C, 28% RH). Rotten fruits with dull-green sporulation were photographed, collected, labelled and taken into the laboratory. Single spore isolation was done using sporulated fruits to obtain pure cultures. The resulting cultures were incubated at 25 °C for 7 days on potato dextrose agar (PDA). Four isolates were obtained for subsequent analyses and deposited in the culture collection of the Beijing Academy of Agriculture and Forestry Sciences (JZB3740003–JZB3740006). All isolates were cultured on Czapek yeast autolysate (CYA), yeast extract sucrose (YES), and malt extract agar (MEA), and incubated at 25 °C for 48 h under a 12 h light/dark cycle to evaluate colony and microscopic characteristics. Colony diameters on CYA: 88–92 mm, x̄ = 90 mm; YES: 90–93 mm, x̄ = 91.7 mm; and MEA: 85–90 mm, x̄ = 87 mm, n = 3 at 25 °C, in 21 days. On all media, colonies showed characteristic dull-green conidial masses with dense, white aerial mycelium and entire, regular margins on the surface. On CYA and MEA, colonies are cottony-velutinous with concentric rings and flattened on the surface, while on YES, the aerial mycelium is dense and convex at the center. The reverse of the colonies is pale yellow on all media. Microscopic characters were observed from all isolates grown on PDA. Aerial hyphae produce erect conidiophores. Conidiophores 600–850 × 4–6.5 μm (x̄ = 771.23 × 5.45 μm, n = 35), hyaline, smooth-walled, branched or unbranched. Metulae 17 – 20 × 2 – 3 μm (x̄ = 19.81 × 2.7 μm, n = 50), hyaline, smooth-walled, septum or aseptate. Phialides 5–11 × 2–3 μm (x̄ = 7.87 × 2.53 μm, n = 58), flask-shaped, cylindrical basal part. Conidia 2–4 × 2.5–4.5 μm (x̄ = 2.44 × 2.9 μm, n = 50), hyaline, smooth-walled, globose, aseptate, unbranched basipetal chains. Morphologically, our isolates matched the description of Penicillium olsonii Amade et al. (1994) and Serra et al. (2007). Genomic DNA was extracted from four isolates using the Omega Fungal DNA Kit. Four gene regions were amplified and sequenced: internal transcribed spacer (ITS), calmodulin (CaM), beta-tubulin (tub2), and the second largest subunit of RNA polymerase II (rpb2) using primer pairs ITS1/ ITS4 (White et al. 1990), Ca1228F/Ca12R (Crous et al. 2004), BT2a/BT2b (O'Donnell et al. 1997), and RPB2-5F/ RPB2-7Cr (O'Donnell et al. 2010), respectively. BLASTn analysis showed 99–100% similarity of ITS, CaM, tub2, and rpb2 of all isolates with P. olsonii type strain (CBS 232.60). Sequences of four isolates were deposited in GenBank (ITS: PX632129, PX649955–PX649957; tub2: PX705450–PX705453; CaM: PX705447, PX645522–PX645524; rpb2: PX705455–PX705458). Phylogenetic relationships among four isolates were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI) approaches. All isolates from this study were clustered within the P. olsonii clade, including the ex-type strain (CBS 232.60), with 100% ML bootstrap and 1.00 Bayesian posterior probability value. Pathogenicity was confirmed for all isolates using inoculation assays on surface-sterilized fruits. Five replicates of wounded and non-wounded fruits were inoculated with 20 µL of 1.0 × 106 conidia/ml and sterilized water as the control. All inoculated fruits developed symptoms, with wounded fruits exhibiting larger lesions than non-wounded fruits. Symptoms included sunken lesions with white mycelia, which later turned dull green, resulting in soft rot, which is similar to the field symptoms. No symptoms were observed on wounded or non-wounded control fruits. The pathogen was re-isolated from wounded and non-wounded fruits for each isolate, and Koch’s postulates were fulfilled. The symptoms produced in this study were consistent with previous reports of P. olsonii in grapes and tomatoes (Anjum et al. 2018; Zou et al. 2022). This is the first report of P. olsonii causing strawberry fruit rot worldwide. This study expands the current knowledge of P. olsonii and highlights Penicillium as a field pathogen rather than solely a postharvest pathogen. Furthermore, our findings emphasise the importance of understanding the diversity and distribution of Penicillium species under controlled agricultural environments such as strawberry greenhouses to understand their potential role in disease development.