Starting from the May to August 2020 (average humidity 76.6% and temperature 25.2°C in Taipei), Boston ivy (Parthenocissus tricuspidata) plants on the campus of National Taiwan University (25°01'05.4"N 121°32'36.6"E) exhibited leaf rusts caused by Phakopsora ampelopsidis (Tzean et al., 2019) and leaf spots caused by an unknown pathogen. The leaf spots appeared reddish to brown color and mostly irregular to round shape on the simple and trifoliate leaflets (Supplemental Figure 1A-C). The leaf spots were surface-disinfected with 1% NaOCl for 30 seconds, and the margin of healthy and infected tissues was cut and placed onto water agar, which were incubated at room temperature. Hyphae grown out from leaf spots were sub-cultured on potato dextrose agar (PDA), and the majority of isolates exhibited white colony with black pycnidial conidiomata embedded in PDA. The pycnidial conidiomata of two-week-old has an average diameter of 463±193 μm (n=30) and the sizes of α-conidia were 5.71±0.49 μm in length and 2.42±0.32 μm in width (n=50) similar to the previous records (Crous et al. 2015). The α-conidium was one-celled, hyaline, and ovoid with two droplets (Supplemental Figure 1D-G). This putative pathogen was re-inoculated to confirm its pathogenicity on the leaves of Boston ivy plants. A PDA block with actively growing fungal edge was placed on the tiny needle-wounded leaves of detached branches (Supplemental Figure H-I) and the whole plants in pots (Supplemental Figure 1J-M) in a moist chamber at 28°C in dark. Reddish to brown leaf spots were observed by 2 days post-inoculation (dpi) and the leaf spots expanded by 5 dpi. To complete the Koch’s postulates, the pathogen was re-isolated from inoculated leaves and the re-isolated pathogen exhibited identical morphology to the original isolate. The internal transcribed spacer (ITS), translational elongation factor subunit 1-α gene (EF1α), β-tubulin (BT), and calmodulin (CAL) was amplified using the primers ITS1/ITS4 (Martin and Rygiewicz. 2005), EF1-728F/EF1-986R, Bt2a/Bt2b, and CAL-228F/CAL-737R, respectively (Manawasinghe et al. 2019). Using BLAST in the NCBI database, the ITS (MT974186), EF1α (MT982963), and β-tubulin (MT982962) sequences showed 98.57% (NR_147574.1, 553 out of 561 bp), 98.04% (KR936133.1, 350 out of 357 bp), and 99.23% (KR936132.1, 518 out of 522 bp) identity to the Diaporthe tulliensis ex-type BRIP 62248a, respectively (Dissanayake et al. 2017). Phylogenetic analysis using concatenated sequences of ITS, EF1α, and β-tubulin grouped the D. tulliensis isolated from Boston ivy leaf spots with the D. tulliensis ex-type (Supplemental Figure 1N). In summary, the morphological and molecular characterizations supported the causal pathogen of Boston ivy leaf spot as D. tulliensis. While Diaporthe ampelopsidis was reported to infect Parthenocissus quinquefolia and P. tricuspidata (Anonymous, 1960; Wehmeyer, 1933), there is no record for D. tulliensis infecting Boston ivy according to the USDA National Fungus Collections (Farr and Rossman. 2020). Because pathogens of Boston ivy such as P. ampelopsidis may also infect close-related crops like grape (Vitis vinifera L.) and D. tulliensis has been known to infect kiwifruits (Actinidia chinensis) and cocoa (Theobroma cacao) (Bai et al. 2016; Yang et al. 2018), the emergence of D. tulliensis should be aware to avoid potential damage to economic crops.
First Report of Leaf Spot Caused by Diaporthe tulliensis on Boston Ivy (Parthenocissus tricuspidata) in Taiwan
