Nonastringent persimmons (Diospyros kaki) were introduced to Taiwan from Japan in 1974 (Lin and Chang 2004). The planted acreage of astringent (60% of acreage) and nonastringent (40% of acreage) persimmons is more than 5500 ha island-wide and still expanding (Anonymous 2014). The major cultivated varieties of nonastringent persimmons are Hanagosho, Fuyu and Jiro (Song and Ou 2004). These fruit trees are mostly planted on hillsides at an elevation between 600 to 1500 m, and fruit is harvested from September to December each year. Plant pests, either microbial pathogens or pest insects, have caused losses to the production of persimmons on this subtropical island. The common pathogens of persimmons in Taiwan are Botrytis cinerea, Colletotrichum gloeosporioides, Phyllactinia guttata, Pseudocercospora kaki, and some root rot diseases caused by Phellinus noxius, Ganoderma lucidum and Rosellinia necatrix (Anonymous 2002). In addition to plant diseases, pest insects such as Bactrocera dorsalis, Spodoptera litura and Planococcus citri are also of a great impact to the production of persimmons (Kao et al. 2004).

In August 2014, a black spot disease of persimmon fruit (D. kaki cv. Hanagosho) was found in an orchard located along a circa 700-m-high 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. This disease occurred sporadically on various persimmon varieties in that orchard. It was observed that an affected tree usually had many diseased fruits, while unaffected trees had fewer diseased fruits. It was also found that the disease severity was more serious on the precocious varieties such as Hanagosho, Soshu and Shinsyuu, while the late varieties such as Fuju and Jiro were less infected in the same orchard. In this study, we conducted a series of experiments to identify the causal agent and determine its association with persimmon.

To isolate the causal agent, persimmon (cv. Hanagosho) fruits with typical black spot symptoms (Fig. 1a) were collected from the orchard in Xinshe in central Taiwan. The diseased fruits were surface-disinfested with 70% ethanol for 3 min and lesions were cut from the fruit surface and placed on 2% water agar dishes. The agar dishes were incubated at room temperature (24–28 °C) with diffuse light until fungal hyphae grew out from the diseased tissue after 4 days. The hyphae were cut out and transferred onto potato dextrose agar (PDA, Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and incubated in the aforementioned conditions for four weeks.

Fig. 1
figure 1

Black spot caused by Phyllosticta capitalensis on persimmon fruit. a symptom of black spot on persimmon fruit. b pycnidia and ascocarps of P1 isolate forming on potato dextrose agar after 4 weeks growth. c ascus with ascospores. d conidia with an apical appendage

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These cultures were used to observe the morphology of ascospores and conidia. The dimensions and morphology of ascospores and conidia were measured from 30 spores each. The spores were recorded including their sizes and morphological characters. The initial colonies grown on PDA had grey or black mycelia, which produced pycnidia and then ascomata. Pycnidia with conidia were produced in 10 to 14 days (Fig. 1b). Single-cell conidia (8.5–12.5 × 6.3–7.5 μm, avg. 9.5 × 7.2 μm, n = 30) were hyaline, ovoid to ellipsoid, and bearing a short and thin apical appendage (Fig. 1d). The mature ascocarps and ascospores were produced in 3 to 4 weeks. The ascocarp was an ascostroma with asci erected in a locule. Eight single-cell ascospores (15.0–17.5 × 6.3–7.5 μm, avg. 15.6 × 6.8 μm, n = 30) were biseriately arranged in each ascus. Ascospores were hyaline and mainly limoniform with a large central guttule. Occasionally, ellipsoid and elongated ascospores were also found (Fig. 1c). The fungus was tentatively identified as Phyllosticta capitalensis.

Pathogenicity of the representative isolate P1 was confirmed by inoculating four mature persimmon fruits each of cv. Hanagosho, Fuyu and Jiro (all nonastringent) with a conidial suspension (1 to 2 × 104 spores/ml). The spore suspension was sprayed on the surface of the ethanol-sterilized persimmon fruit and the control fruit received sterile water without spores. The treated fruits were then incubated at 24–28 °C, >90% relative humidity, in dark conditions until symptoms appeared. The artificially inoculated persimmon fruits developed black spot symptoms 5 days after inoculation. These symptoms were similar to those of the naturally infected persimmon fruits. All control fruits remained healthy. The pathogen of the inoculated fruit was re-isolated, cultivated and confirmed as P1 isolate based on fungal morphology.

The morphological identification of the fungus was confirmed with a molecular barcoding and phylogenetic approach. Genomic DNA was extracted from P1 using a Tissue and Cell Genomic DNA Purification Kit (GeneMark, Taiwan). The nuclear ribosomal internal transcribed spacer (ITS) region, partial sequences of the actin (ACT) and translation elongation factor 1-alpha genes (TEF1) were amplified with the primer pairs ITS1 and ITS4 (White et al. 1990), ACT-512F and ACT-783R (Carbone and Kohn 1999) and EF1-728F and EF2 (Carbone and Kohn 1999; O’Donnell et al. 1998).

The DNA sequences of each gene were deposited in GenBank: ITS (KP998485), ACT (KP998487) and TEF1 (KP998486). These genes were used to obtain a molecular identification of the species. The genes were used in a nucleotide BLAST search and had high identity to species of Phyllosticta and Guignardia. The percent identity of ITS, ACT and TEF1 gene sequences of P1 were 99% (562/564 base pairs), 100% (232/232 base pairs) and 99% (209/210 base pairs), respectively, to those genes of ex-type of P. capitalensis, CBS 128856.

In order to investigate the relationships of the isolate P1 among the species of Phyllosticta, a total of 56 ITS sequences of the type or ex-type cultures of Phyllosticta and Guignardia obtained from GenBank database were used in a phylogenetic analysis. The sequences from type cultures of Phyllosticta and Guignardia were mostly published by Wikee et al. (2013). The ITS sequences were aligned with ClustalW and a phylogenetic tree was constructed by neighbor-joining method implemented in MegAlign module of the DNASTAR software package version 7.0.0 (Burland 1999). This analysis revealed that several species of Phyllosticta and Guignardia including P. capitalensis were closely related with P1 (data not shown). A phylogenetic species hypothesis was determined for the pathogen. The three gene regions of P1 were concatenated and aligned to the same genes of those closely related species of Phyllosticta and Guignardia obtained from GenBank. The sequences were aligned with ClustalW, and then phylogenetic analysis was performed by the Neighbor-Joining method with 1000 bootstrap replications in MEGA 7 (Kumar et al. 2016). The result indicated that P. capitalensis (CBS 128856) was conspecific with the isolate obtained from persimmon in Taiwan (Fig. 2). The representative isolate P1 was deposited at Taiwan Bioresource Collection and Research Center with the number BCRC FU 30762.

Fig. 2
figure 2

Phylogenetic analysis of the isolate P1 and its closely related species of Phyllosticta and Guignardia based on the concatenate sequences of ITS, ACT and TEF1 genes. This analysis was performed by the Neighbor-Joining method with 1000 bootstrap replications in MEGA 7. The numbers at the branch points indicate bootstrap values. Scale bar = 0.01 substitutions per site

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Phyllosticta capitalensis has been described as an endophyte and weak plant pathogen with extensive host range (Okane et al. 2003). Its occurrence in a commercial persimmon orchard and capability to cause black spot symptoms may threaten the production of persimmon fruit. To the best of our knowledge, this is the first report of P. capitalensis causing black spot disease on persimmon in Taiwan.