First Report of Colletotrichum siamense Causing Anthracnose in Osmanthus fragrans in China.

Abstract

Osmanthus fragrans Lin. is widely cultivated in China. Its flower is precious spices. It is also a garden ornamental plant. In March 2021, anthracnose-type lesions were observed on the leaves of O. fragrans in a public garden in Zhanjiang, Guangdong Province, China (21˚17'47''N, 110˚18'58''E). Disease incidence was around 50% (n = 100 investigated plants from about 30 hectares).The early symptoms were yellow spots on the edge or tip of the leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec . Thereafter, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 ℃. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Thirty-two isolates of Colletotrichum ssp. were obtained (isolation frequency = 32/4×10 = 80%). Three representative single-spore isolates (OFC-1, OFC-2, and OFC-3) were used for further study. Colonies on PDA were white to gray with cottony mycelia in 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 10.5 to 17.5 µm × 3.5 to 5.0 µm (n = 50). Appressoria were oval to irregular in shape and dark brown in color, and they measured 6.5 to 8.5 µm × 4.5 to 7.5 µm (n = 20). Morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009;Sharma et al. 2013). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences of them deposited in GenBank under nos. MZ047368-MZ047370 (ITS), MZ126925-MZ126927 (GAPDH), MZ126895-MZ126897 (CHS-1), and MZ126835-MZ126837 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, CHS-1, and ACT that clustered the isolates with C. siamense (the type strain MFLU 090230), while distanced the isolates with C. gloeosporioides (the type strain CBS 112999). The pathogenicity was tested through in vivo experiments. In group 1, the inoculation and control plants (n = 5, 3-month-old) were sprayed with a spore suspension (1 × 105 per mL) of the isolates and sterile distilled water, respectively, until run-off. In group 2, the unwounded leaflets were inoculated with mycelial plugs of the isolates or agar plugs (as control). Three plugs were for each leaflet ( n = 5). The plants were grown in pots in a greenhouse at 25°C to 28°C, with relativehumidities approximately80%. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy. The pathogen re-isolated from all the inoculated leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C. gloeosporioides has been reported to cause leaf spot on O. fragrans in Jiangxi Province of China (Tanget al., 2018), but not by C. siamense. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on O. fragrans. Thus, this work provides a foundation for controlling anthracnose in O. fragrans in the future.