First Report of Curvularia lunata Causing Leaf Spot on Shatangju in China.

Shatangju (Citrus reticulata Blanco) is an economically important citrus cultivar in China. It has a unique flavor and is very popular among consumers. In May 2018, we observed leaf spots on ~20% of Shatangju trees in a 7 ha orchard in Huaiji country, Guangdong, China. Small maroon spots initially developed on the lower leaf surfaces of the symptomatic trees, which then expanded and coalesced into larger lesions. The lesions became visible from the upper leaves, with light gray centers and dark brown margins surrounded by yellow halos. The infected leaves would finally become wilted. To isolate the pathogen, we cut 0.5 × 0.5 cm2 squares from the lesion margins of the symptomatic leaves, disinfested them using 1% NaClO for 20 s, and then with 70% ethanol for 1 min, rinsed them in sterile distilled water three times, and inoculated them onto three potato dextrose agar (PDA) plates (5 squares/plate), which were kept under 25 °C in the dark. Colonies with similar appearance were observed on all the plates and subcultured via the single-spore method (Ho et al. 1997). The mycelia of the subcultures gradually turned from white to black. The colonies were morphologically close to the fungal species Curvularia lunata (Wakker) Boedijn (Ellis 1971). At 8 days after inoculation, the conidia were 18.9-25.1 μm × 8.1-11.6 μm in size (n=50), fusiform or geniculate, smooth-walled, dark-brown, 3-septate, and with a slightly curved second cell and an expanded third cell from the pore end. Three randomly selected isolates from different plates were applied in further analysis. The internal transcribed spacer (ITS: MZ026467) region, translation elongation factor (EF-1α: MZ042646), large subunit ribosomal RNA (LSU: MZ026469), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: OK086974) segments were amplified and sequenced using primers ITS1/ITS4, EF-1/EF-2, LR5F/ LROR, and gpd1/gpd2, respectively (White et al. 1990; O'Donnell 1998; Marin-Felix 2020). The four segments were identical among the three isolates, which shared the highest nucleotide identities (100% on ITS, LSU, and GAPDH, and 99.8% on EF-1α) with C. lunata strains in the GenBank database. Based on the ITS and GAPDH sequences, phylogenetic analysis using Maximum-likelihood and Bayesian inference methods by W-IQ-TREE (accessed on 09/01/2021) (Trifinopoulos et al., 2016) and MrBayes v3.2.7a (Ronquist et al., 2012) both supported that the isolates belong to C. lunata. For pathogenicity tests, we sprayed the conidial suspension (1×106 conidia/ml) of each of the three isolates on three healthy three-month-old Shatangju seedlings, which were kept under 27 °C and ~100% humidity in plastic bags in the greenhouse. Another three seedlings were sprayed with sterile water and kept under the same conditions as negative controls. Three days after inoculation, small maroon dots started appearing on the inoculated leaves, and the symptoms, same as those observed in the field, developed afterward. No symptoms appeared on the negative controls. C. lunata was reisolated from the infected leaves of all plants inoculated with the three isolates but not from the negative controls. This pathogen has been reported to cause diseases in Pennisetum hydridum and Capsicum frutescens in China (Xu et al. 2018; Pei et al. 2017). But to our knowledge, this is the first report of C. lunata causing leaf spots on citrus plants in the world.