Strawberry (Fragaria ananassa Duch.) is one of the most economically important fruits in Korea. In November 2015, a leaf spot symptom was found on strawberry seedlings in a farm located in Wanju, Korea. Tanned brown spots appeared on the leaves of strawberry seedlings left out in the field after planting (Fig. 1A, 1B). The severely affected leaves withered and finally died. Approximately 1%~3% of seedlings showed these symptoms.
A fungal isolate was obtained from the diseased leaves using the single hyphal tip method. One pure culture (Fig. 2I) was deposited in the Korean Agricultural Culture Collection under the accession number KACC48095. A colony of the fungus on potato dextrose agar (PDA) attained 90 mm diameter at 23°C (optimal temperature) in three weeks, and was beige to buff, slimy, concentric, and consisting of sparse aerial mycelia, with an entire margin. Morphological characteristics were observed and measured from the fungal structures that were produced on fruits inoculated with the isolate. Morphologically, conidiomata were disk-shaped, flattened, brown to dark brown, sometimes blackish, and 0.2~1 mm in size (Fig. 2A~2E). Conidiophores were hyaline, unbranched or branched, and up to 62.5~87.5 µm in length and 1.8~2.0 µm in width (Fig. 2F, 2G). Conidia were hyaline, aseptate, allantoid, and 5~9 µm in length and 1.7~2.5 µm in width (Fig. 2H). The morphological features of the causal fungus were consistent with the description of Pilidium concavum (Desm.) Hönh [1].
Genomic DNA was extracted from mycelia scraped from the surface of agar plates. The internal transcribed spacer (ITS) rDNA region was amplified using the primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) [2]. The PCR products were purified and directly sequenced by BIOFACT (Daejeon, Korea). The raw DNA sequence data for P. concavum was edited using SeqMan program (DNASTAR, Madison, WI, USA). The resulting sequence has been submitted to GenBank (accession number MG212519). Phylogenetic relationships between P. concavum and related species were inferred from ITS rDNA sequences, including five reference sequences of Pilidium spp. retrieved from GenBank. Chaetomella zambiensis (CPC 22465) was used as an outgroup. A phylogenetic tree was constructed by MEGA6 [3] using the neighbor-joining method with the Tajima-Nei model. A BLAST search of the resulting sequence showed 100% similarity with the sequences of P. concavum in GenBank. In the neighbor-joining tree (Fig. 3), the present isolate positioned into a clade comprising the P. concavum reference isolates. It was also confirmed that P. concavum is further related to P. pseudoconcavum [4], with a high value of 94%. The molecular phylogenetic analysis verified the identity of the present isolate.
To fulfill Koch’s postulates, the pathogenicity of the isolate was tested using leaves and fruits of strawberry. The inoculum was prepared by harvesting mycelial mats and conidia from seven-day-old cultures grown on PDA. Leaves of five plants, some wounded by a needle, and some left intact, were sprayed with hyphal and conidial suspensions. Five plants spray-inoculated with sterile water served as controls. The fungal suspensions were also sprayed on the surface of five non-wounded fruits. Five healthy fruits sprayed with sterile water served as controls. After inoculation, the plants were maintained in a growth chamber at 23°C for one week. Wounded leaves developed typical symptoms of tan-brown leaf spots after seven days of inoculation (Fig. 1C). On the surfaces of the inoculated fruits, conidiomata were abundantly produced (Fig. 1D, 1E). Control plants remained symptomless. The fungus was consistently re-isolated from the lesions after inoculation, which proved that the isolate of P. concavum is pathogenic to strawberry.
Pilidium concavum exhibits a broad host spectrum that infects diverse crops and wild plants worldwide [5]. It has been reported to occur on the leaves and fruits of strawberry in Poland [6], Brazil [7], Belgium [8], China [9], the USA [10], and Iran [11]. However, there has been no previous record of strawberry associated with P. concavum in Korea. To our knowledge, this is the first report on tan-brown leaf spot pathogen on strawberry seedlings in Korea.
