First Report of Colletotrichum siamense Causing Anthracnose in Allamanda cathartica in China.

Allamanda cathartica L. is an important ornamental plant (Wong et al. 2013). In August 2020, anthracnose-type lesions were observed on the leaves of A. cathartica in a garden in Zhanjiang, Guangdong Province, China (N21°17', E110°18'). Disease incidence and severity varied from 18 to 20% and 20 to 50% (n = 100 investigated plants), respectively.  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. Twenty-eight isolates of Colletotrichum ssp. were obtained (isolation frequency = 28/4×10 = 70%). Three representative single-spore isolates (ACC-1, ACC-2, and ACC-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 11.5 to 16.5 µm × 3.5 to 5.5 µm (n = 50). Appressoria were oval to irregular in shape and dark brown in color, and they measured 7.3 to 10.5 µm × 5.7 to 6.5 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 with MightyAmp DNA Polymerase (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loci of the isolates using primer pairs ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1, respectively (Weir et al. 2012). Sequences of the isolates deposited in GenBank under acc. nos. MZ061912 to MZ061914 (ITS), MZ126886 to MZ126888 (CAL), MZ126856 to MZ126858 (ACT), MZ126916 to MZ126918 (CHS-1), and MZ126946 to MZ126948 (GAPDH), respectively. They were 100% identical to ITS (JX010250), CAL (HM131507), ACT (JX009895), CHS-1 (HM131497), and GAPDH (JX009713) sequences of C. siamense respectively too. A phylogenetic tree was generated using the concatenated sequences of ITS, CAL, ACT, CHS-1, and GAPDH. The isolates were clustered with C. siamense strains including the type ICMP 19118. Pathogenicity tests were performed by in vivo. The inoculation and control groups (n = 5 plants each, 1-month-old) were sprayed with a spore suspension of isolates (1 × 105 per mL) and sterile distilled water, respectively, until run-off. The plants were grown in pots in a greenhouse at 25°C to 28°C, with relative humidities approximately 80%. Anthracnose lesions were observed on the 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. siamense has been reported to cause anthracnose in a broad range of hosts (Weir et al. 2012), but not in A. cathartica. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on A. cathartica. Thus, this work provides a foundation for controlling anthracnose in A. cathartica in the future.