Disease Notes
First Report of Black Spot Caused by Phyllosticta capitalensis on Persimmon Fruits in Taiwan
Chung-hang Duan, Che-ming Chang, and Chiou-chu Su, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung41358, Taiwan
In August 2014, an un-recorded disease of persimmon fruits (Diospyros kaki L.) of the cv. Hana Gosho (non-astringent) was found in an orchard located along ca. 2,500 feet hillside at Xinshe in central Taiwan. The affected persimmon fruits were at or near maturity and symptoms resembled black spot. Each circular sunken black spot was 1 to 2 mm in diameter and randomly distributed on the fruit surface. To isolate the causal agent, the symptomatic fruits were surface-sterilized with 70% ethanol and the lesions were cut from the fruit surface and placed on 2% water agar plates. The agar plates were incubated in room temperature (24~28°C)with diffuse light until the fungal hyphae of the pathogen grew out from the diseased tissue after 5 days. The hyphae were cut and transferred onto potato dextrose agar plate and incubated in the above conditions for the production of ascocarps and pycnidia within two weeks. The ascocarp was an ascostroma with asci erected in a locule. Eight single-cell ascospores (15.0 to 17.5 ×6.3 to 7.5μm, avg. 15.6 × 6.8 μm, n=30) biseriately arranged in each ascus. Ascosporeswere hyalineand mainly limoniform with a large central guttule. Occasionally, ellipsoid and elongated ascospores were also found. Single-cell pycnidiospores (8.5 to 12.5 × 6.3 to 7.5 μm, avg. 9.5 × 7.2 μm, n=30) were hyaline, ovoid to ellipsoid, and bearing a short and thin apical appendage. For molecular analyses, genomic DNA was extracted from the isolate P-1 using Tissue and Cell Genomic DNA Purification Kit (GeneMark, Taiwan).The nuclear ribosomal internal transcribed spacer (ITS) region (GenBank Accession numberKP998485), a partial sequence of the actin gene (ACT) (KP998487) and the translation elongation factor 1-alpha gene (TEF1) (KP998486) were amplified with the primer pairs of ITS1 and ITS4 (White et al. 1993), ACT-512F and ACT-783R (Carbone and Kohn 1999), and EF1-728F and EF2 (Carbone and Kohn 1999,O’Donnell et al. 1998), respectively. Phylogenetic analysis of the concatenate sequence of the three genes indicated that among 11 distinct species of Phyllosticta and Guignardia, the type strain of Phyllosticta capitalensis(CBS 128856) was the closest species to theisolate P-1 in a clad with 100 bootstrap value. Pathogenicity of the isolate P-1 was confirmed by inoculating, respectively, each four mature persimmon fruits of the cv. Hana Gosho, Fuyu, and Jirou (all non-astringent) with its spore suspension (1 to 2 × 104 spores/ml). The spore suspension was sprayed on the surface of the ethanol-sterilized persimmon fruits and the control fruits received sterile water without spores. The treated fruits were then incubated at 24-28°C, 100% humidity,and dark for 10 days. Inoculated fruits developed black spot symptom as described above. The fungus was re-isolated and confirmed as P1 isolate. All control fruits remained healthy. P. capitalensis was indicated as an endophyte and weak plant pathogen with extensive host range (Okane et al. 2003). Its occurrence in a commercial persimmon orchard and capability of causing black spot symptom may become a menace to the production quality of persimmon fruits because of its ubiquitous distribution and wide host range. To the best of our knowledge, this is the first report of P. capitalensis causing black spot disease on D. kakiin Taiwan.
References: (1) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (2) K. O’Donnell et al. PNAS USA 95:2044. 1998.(3) I. Okane et al. Mycoscience 44:353, 2003.(4) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Application. Academic Press, NY, 1993.