First report of Phyllosticta capitalensis causing leaf spot of Mahonia fortunei in China

Mahonia fortunei, belonging to the Berberidaceae family, is widely cultivated in fields, parks, courtyards, and roadsides for its excellent ornamental characteristics and medicinal values in southern China (Yu and Chung 2017). In May 2021, leaf spots were observed on nearly 60~80% of M. fortunei plants growing in Chongqing Normal University campus (29°36'42″N; 106°17'59″E) from Chongqing City, China. The typical symptoms on leaves were irregular spots with gray centers, brown edges, and chlorotic halos, about 1 to 7 mm in diameter, and eventually coalesced forming larger necrotic areas. Twenty symptomatic leaves were randomly sampled from five diseased plants. Tissues were cut from the lesion margins and surface sterilized in 75% ethanol for 1 min, rinsed thrice with sterile water, dried on sterilized paper, plated on potato dextrose agar (PDA) plates, and incubated at 25°C for 7 days in the dark. A total of 20 isolates were obtained from the infected leaves. Pure colonies of all fungal isolates had similar characteristics, and three isolates were randomly selected (SD11, SD18, SD19) for further study. Colonies of this fungus were olivaceous greenish to olivaceous black with a granular surface, and irregular light olive edges, finally turning black on PDA. Pycnidia were black, globose, granular, and in clusters. Conidia (n=30) were hyaline, aseptate, unicellular, obovoid to ellipsoid, narrow end with single apical appendage, and 7.5~11.2 × 4.5 ~6.5 μm. The DNA of three isolates were extracted and the internal transcribed spacer (ITS) region, actin (ACT), and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), ACT512F/ACT783R, and ER728F/EF986R (Carbone and Kohn 1999), respectively. The sequences of three isolates were 100% identical, and one representative isolate SD18 were deposited in GenBank (ON231754, ITS; ON246259, ACT; and ON246258, TEF1). Sequence analysis revealed that the consensus sequences of ITS, ACT, and TEF1 of isolate SD18 was 99 to 100% identical to each sequence of an Indonesian strain (CBS 117118) of P. capitalensis from Musa acuminate (FJ538339 for ITS, FJ538455 for ACT, FJ538397 for TEF1). Phylogenetic analysis using Maximum Likelihood and concatenated sequences (ITS+ACT+TEF1) with MEGA7 placed isolate SD18 in P. capitalensis with 100% bootstrap support. Based on these morphological and molecular characteristics, the isolates were identified as P. capitalensis (Wikee et al. 2013). To fulfill Koch's postulates, 8 healthy potted plants were inoculated with 106 conidia/ml suspension of isolate SD18 by spraying the leaves, and another 8 plants were sprayed with sterile distilled water as control. All plants were covered with plastic bags for two days and then arranged in a greenhouse with 80% relative humidity at 25°C. The pathogenicity test was repeated thrice. After 18 days inoculation, the similar symptoms were observed on the inoculated plants, whereas control plants remained healthy. The pathogen was reisolated from symptomatic tissue and identified as P. capitalensis by the methods described above. P. capitalensis has been reported causing leaf spot on various host plants around the world (Wikee et al. 2013), recently found on tea plant, castor, and oil palm (Cheng et al. 2019; Tang et al. 2020; Nasehi et al. 2020). This is the first report of P. capitalensis causing leaf spot on M. fortune in China, and will establish a foundation for controlling the disease.