First Report of Fruit Rot Caused by Cladosporium anthropophilum on Strawberry in China

Strawberry (Fragaria × ananassa), a perennial herbaceous plant of the genus Fragaria in the family Rosaceae, produces an aggregate fruit characterized by high nutritional value and diverse modes of consumption. It is widely cultivated worldwide and represents an economically important horticultural crop. In March 2024, a new fruit rot was observed on strawberries fruits in Changchun, Jilin Province, China (43°49′N,125°23′E). During field surveys conducted in the affected greenhouse, fruit rot symptoms were observed on a substantial proportion of the fruits. The disease initially manifested as surface decay, and under favorable environmental conditions, a dark green mold layer developed on the fruit surface, leading the affected fruits to completely lose their commercial value. To isolate the pathogen, symptomatic tissues were collected from the junction between diseased and healthy areas and cut into 0.5 cm pieces. These pieces were surface-sterilized in 75% ethanol for 60 s, followed by immersion in 3% sodium hypochlorite (NaClO) for 3 min, rinsed three times with sterile water and air-dried under sterile conditions before being plated on potato dextrose agar (PDA) and incubated at 25±1℃. Ten isolates of Cladosporium were obtained and pure-cultured through single-sporing. All isolates exhibited similar colony morphology on PDA. Morphological observations and measurements were conducted using a representative isolate (CMGF-23). On PDA plates, after two days of incubation, dark green, velvety mycelia emerged. The mycelium was dense, and the colonies were dark green with a velvety surface, showing concentric rings and a white marginal zone. The conidiophores were hyaline, straight or slightly curved, and mostly unbranched. The conidia were ovoid, lemon-shaped or fusiform, unicellular, and measured 3.22 – 13.5 µm × 2.08 – 5.32 µm (n=100). Based on these morphological and culture characteristics, the causal agent was preliminarily identified as C. anthropophilum. Further identification was performed using molecular analysis based on the internal transcribed spacer regions (ITS region, primers ITS4 and ITS5; White et al., 1990) and the actin gene (ACT, amplified with primers ACT-512F and ACT-783R; Carbone and Kohn 1999). These two loci were selected because they are widely accepted as phylogenetically informative markers for species delimitation and identification within the genus Cladosporium, as recommended in authoritative taxonomic frameworks (Bensch et al., 2012, 2015；Sandoval-Denis et al.,2016). The ITS and ACT sequences obtained were identical among the ten isolates. The resulting sequences were deposited in GenBank under accession numbers PX634066 (ITS) and PX693205 (ACT). The ACT sequence (PX693205) has been assigned by GenBank and is currently under processing and will be released publicly upon completion of annotation. BLAST search results revealed that the ITS sequence showed 99.3% identity with C. anthropophilum (NCBI accession no. OM899885.1), while the ACT sequence exhibited 98.8% similarity to C. anthropophilum (NCBI accession no. OM953786.1) , corroborating the morphological identification. Phylogenetic analyses based on ITS and ACT sequences placed isolate CMGF-23 within the C. anthropophilum clade with support, further confirming its identity. Pathogenicity of ten isolates of C. anthropophilum obtained from strawberry was confirmed by artificial inoculation of healthy strawberry fruits with a spore suspension (1 × 106 spores/ml) produced on PDA. For each treatment, five fruits were inoculated with 5 mL of spore suspension. Control fruits (n=5) were inoculated with sterilized water. All fruits were enclosed in plastic bags for 48 h in a greenhouse at 25℃ and 12 h/d light cycle. After 3 days, inoculated fruits developed symptoms identical to those observed on naturally infected fruits, while control fruits remained symptomless. C. anthropophilum was successfully re-isolated from symptomatic plants to fulfill Koch's postulates. To our knowledge, C. anthropophilum has previously been reported as the causal agent of fruit rot on Rubus idaeus (Swett et al., 2019). However, this study represents the first report of C. anthropophilum causing fruit rot of strawberry.