First report of leaf spot on Daphniphyllum macropodum caused by Neopestalotiopsis clavispora in China.

Daphniphyllum macropodum Miq., an evergreen arbor, is widely cultivated in southern China for its ornamental and medicinal value (Su et al. 2013). In October 2019, a severe leaf spot was observed on D. macropodum in Jinggangshan National Nature Reserve in Ji'an city, Jiangxi, China (114°06'23″E, 26°32'25″N). The plants were about 15 years old, and the disease incidence was estimated to be 15% (4/26 plants). The disease primarily appeared as small black spots on the leaves. At the late stage, the spots enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. We collected five samples per plant and total 20 samples were collected to isolated the pathogen strains. The margin of the diseased tissues was cut into 5 mm × 5 mm pieces; surface disinfected with 70% ethanol and 2% NaOCl for 30 s and 60 s, respectively; and rinsed thrice with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark. Pure cultures were obtained by single-spore isolation method, and the representative isolates, JRM3, JRM6, and JRM8 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface at 25 °C. Conidiomata was black conidial masses on PDA. Conidia were 5-celled, clavate to fusiform, smooth, 19.3 to 24.4 long × 6.1 to 8.6 μm wide (n = 50). The 3 median cells were dark brown to olivaceous, the central cell was darker than the other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.4 to 8.3 μm long; n = 50), and 2 to 3 apical appendages (18 to 32 μm long; n = 50), filiform. The morphological characteristics of the isolates are comparable with those of the genus Neopestalotiopsis (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. The sequences of the isolates were submitted to GenBank (ITS, OQ372202 to OQ372204; TUB2, OQ390129 to OQ390131; TEF1-α, OQ390126 to OQ390128). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed JRM3, JRM6, and JRM8 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. To confirm pathogenicity, eight healthy 10-year-old D. macropodum plants growing in the field were chosen, and 4 leaves per plant were wounded with a sterile needle and inoculated with 10 μL conidial suspension per leaf (106 conidia/ml). Eight plants inoculated with sterile water were used as control. All the inoculated leaves were covered with plastic bags to maintian a humidity environment for 2 days. The leaves inoculated with conidial suspension showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 10 days. The same fungus were re-isolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora was determined as the pathogen of a variety of plant diseases, including Kadsura coccinea (Xie et al. 2018), Dendrobium officinale (Cao et al. 2022), Macadamia integrifolia (Santos et al. 2019). However, this is the first report of N. clavispora infecting D. macropodum in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.