First Report of Colletotrichum nymphaeae Causing Leaf Spot of Lithocarpus litseifolius (Hance) Chun in China.

Lithocarpus litseifolius (Hance) Chun has been used as tea for 1500 years in China since the Southern and Northern Dynasties (AD 423). In mid-May 2021, a leaf spot disease of L. litseifolius (Hance) Chun was observed in Dexing County in Jiangxi Province, China (28°56'54.1N 117°45'08.0E). The disease incidence was estimated to be above 30%. The symptom of natural damage to plant leaves is early necrotic lesions, with a grayish-white center and a brown halo (<5mm) around it. Later, the lesion expanded to the edge of the leaf, with a yellowish-brown center and brown surrounding. Samples were collected from the field, and small tissues around the lesion were cut off and sterilized in 75% ethanol for 3 min, then washed with sterile water 3 times. The sterilized tissues were placed on PDA plates and incubated at 28°C in darkness. After 3 days of incubation, the hyphal tips from the edges of growing colonies were transferred to fresh PDA plates for further purification. Finally, five isolates showing the same morphology were obtained, and one pure single-colony fungal strain JM7 was selected for further analysis. After 7 days of incubation on PDA at 28°C, the fungal colonies showed white aerial mycelium and gelatinous orange conidial masses. The conidia were unicellular, smooth-walled, hyaline, cylindrical with obtuse to rounded ends, and their size was from 8.44 to 15.48 µm (mean = 10.33 µm, n = 60) × 2.95 to 3.42 µm (mean = 3.14 µm, n = 60). These morphological characteristics correspond to those of Colletotrichum nymphaeae. For molecular identification, the internal transcribed spacer, partial actin, β-tubulin 2, glyceraldehyde-3-phosphate dehydrogenase, chitin synthase, and histone H3 regions of JM7 were amplified by PCR using primer pairs ITS1/ITS4, ACT-512F/ACT-783R, T1/Bt2b, GDF/GDR, CHS-79F/CHS-354R and CYLH3F/CYLH3R, respectively (Weir et al. 2012；Crous et al. 2004). The sequences of the amplified fragments were deposited in GenBank (MZ496772, MZ561462-MZ561466), and showed 98 to 100% identities with the corresponding sequences of C. nymphaeae. A phylogenetic tree based on the above six genes of type specimens or ex-type of Colletotrichum (Damm et al. 2012) was generated using the maximum likelihood method by MEGA X, in which the strain JM7 and other C. nymphaeae strains clustered in the same branch. Thus, the strain JM7 was identified as C. nymphaeae based on its morphological and molecular characteristics. Pathogenicity tests were conducted on the leaf of L. litseifolius (Hance) Chun. Six leaflets were superficially disinfected with 75% ethanol and wound-inoculated with 6-mm plugs of PDA medium containing C. nymphaeae acervuli and conidia, while the control treatments received only PDA discs. The inoculated leaves were wrapped with a plastic bag containing moistened cotton and incubated in a greenhouse at 26°C 60% humidity with a photoperiod of 12 h. Symptoms similar to the original observations were observed on leaflets inoculated with C. nymphaeae after 5 days. No symptoms were observed in the control treatments. The pathogen was re-isolated from symptomatic tissues, and was identified as C. nymphaeae, which full-filled the Koch's postulates. To our knowledge, this is the first report of C. nymphaeae infection on L. litseifolius (Hance) Chun in China. These findings will help develop better preventive measures in accordance with the emergence of this new pathogen.