First report of Diaporthe eres causing leaf spot on Platanus acerifolia in China

Platanus acerifolia Willd. is one of the world famous urban greening trees, known as "the king of street trees" (Loretta et al. 2020). In August 2021, severe leaf spot disease was observed on P. acerifolia with 15% incidence on a street of Haidian district (116°29'E, 39°95'N), Beijing municipality, China. Typical symptoms were small and irregular brown spots with or without yellow haloes, which gradually expanded, coalesced and became necrotic lesions. For pathogen isolation, the leaf lesions were cut into small tissue pieces, disinfected by 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed in sterile distilled water, and then incubated on potato dextrose agar (PDA) plates. After incubation at 28°C for 4 days, three fungal isolates (FTDX2, FTDX3, and FTDX6) with similar colony characteristics were obtained after single spore isolation. Colonies were white with fluffy aerial mycelia, abundant pycnidia with black stomata appeared and cream-white liquid oozed after 20 days. Alpha conidia were 7.9 ± 0.6 × 2.5 ± 0.3 μm (n = 30), aseptate, hyaline, fusiform to ellipsoidal, and often biguttulate, while beta conidia were 22.7 ± 1.3 × 1.1 ± 0.1 μm (n = 30), aseptate, hyaline, linear, curved or hamate. The morphological characteristics were consistent with those of Diaporthe sp. (Udayanga et al. 2014). For further identification, total DNA was extracted from the three isolates. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1-α), beta-tubulin (TUB), calmodulin (CAL) and histone (HIS) genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), BT2a/BT2b (Glass and Donaldson 1995), CL1/CL2A (O'Donnell et al. 2000) and CYLH3F/H3-1b (Crous et al. 2014), respectively. The sequences were all deposited in GenBank (accession nos. OL870615 - OL870617 for ITS, OL870618 - OL870620 for EF1-α, OL870621 - OL870623 for TUB, OL870624 - OL870626 for CAL, and OL870627 - OL870629 for HIS) and aligned using BLASTn, obtaining 99-100% homology with the corresponding sequences of known Diaporthe eres strains in NCBI. Phylogenetic analysis of the combined sequences attributed the three isolates to the Diaporthe eres clade. Pathogenicity tests were performed on three healthy one-year-old P. acerifolia plants using the randomly selected isolate FTDX2. The leaves were inoculated with 20 µl of spore suspension (106 conidia/ml), with or without wound pretreatment, sterilized water inoculation under the same condition was used as control. All the treated plants were incubated in the greenhouse at 25°C and 90% RH with a 12-h photoperiod. After 8 days, the inoculated plants showed spot symptoms on leaves similar to those previously observed, whereas the control leaves remained symptomless. Lesions on the wounded leaves were much larger in size compared with those unwounded. The same pathogen was re-isolated and identified based on morphological characteristics and gene sequencing data, fulfilling Koch's postulates. Diaporthe eres has been reported to cause leaf spot on many horticultural plants, such as Photinia fraseri (Song et al. 2019) and Podocarpus macrophyllus (Zheng et al. 2020). To our knowledge, this is the first report of D. eres causing leaf spot on Platanus acerifolia in China. This finding is a valuable contribution to the knowledge on leaf spot disease development in horticultural plants.