First Report of Colletotrichum siamense Causing Anthracnose on Bixa orellana in China

Annatto (Bixa orellana L.) is widely cultivated in China. Its seed is used as medicine and as an astringent antipyretic. Since 2019, anthracnose-type lesions have been observed on the annatto leaves in the field (about 30 hectares) in Zhanjiang (21˚18'12''N, 110˚17'22''E), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 3 ha). The early symptoms were yellow spots on the edge or tip of leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions (Supplemental Figure 1-A). Ten symptomatic leaves from 10 plants were sampled. The margins of the lesions were cut into 2 × 2 mm pieces and the surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec. After that, pieces were rinsed thrice in sterile water, placed on potato dextrose agar(PDA) medium, and incubated at 28 ℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (BOC-1, BOC-2, and BOC-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. Besides, the three isolates were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University. Colonies on PDA were white to gray with cottony mycelia after incubating in the dark for 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 9.6 to 18.5 µm × 3.5 to 5.5 µm (n = 50). Appressoria were oval to irregular in shape and dark brown, and they measured 6 to 9 µm × 4.5 to 8 µm (n = 30) (Supplemental Figure 1-D, E and F). These morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates were amplified using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences were deposited in GenBank under nos. MZ047377-MZ047379 (ITS), MZ126934-MZ1269346 (GAPDH), MZ126904-MZ1269046 (CHS-1), and MZ126844-MZ1268446 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from ITS, GAPDH, CHS-1, and ACT sequences that clustered the three isolates with C. siamense (the type strain MFLU 090230), (Supplemental Figure 2). The pathogenicity of the three isolates was tested respectively in a greenhouse maintained at 25 to 29℃ and 80% relative humidity. Annatto seeding ( n =5, 2-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water.. The experient was repeated three times. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy (Supplemental Figure 1-G, and H). The same pathogen was re-isolated from all the inoculated leaves based on morphology and ITS analysis. C. siamense has been reported to cause anthracnose in a broad range of hosts (Weir et al. 2012; Wang et al. 2017; Liu et al. 2017; Zhuo et al. 2017 ), but not in B. orellana. To our knowledge, this is the first report of C. siamense causing anthracnose on B. orellana in China. Our study provides important reference information for controlling this disease.