First Report of Colletotrichum siamense causing Anthracnose on Ficus microcarpa in China.

Abstract

Chinese banyan, Ficus microcarpaLinn. f. (Moraceae), is a native and popular landscape tree with highornamentalandeconomicvalue in South China. In May 2018, a foliar disease occurred on F. Microcarpa in Zhanjiang city (21˚17'51''N, 110˚18'16''E), Guangdong Province, China. Theincidenceof thediseasewas 10%andseveritywas 20% (n = 100 investigated plants). Symptoms first appeared on leaves as small black spots (up to 5 mm diam) with yellow haloes. The spots gradually enlarged in size and coalesced, resulting in necrosis and early shedding of the leaves. Ten symptomatic leaves from 10 plants were collected. The margins of the samples were cut into small pieces (5 × 5 mm), surface disinfected (75% ethanol 30 s, 0.1% HgCl2 45 s, rinsed 3 times with sterile distilled water), and placed on potato dextrose agar (PDA) at 28°C with a photoperiod of 12 h. After 5 days, a total of four isolates with 100% isolation frequency were obtained and three representative strains (T6, T6-1, and T6-2) were used for morphological and molecular characterization. Colonies on PDA were white to gray with cottony mycelia, a few bright orange conidial masses developed near the inoculum point. Conidia were single-celled, smooth-walled, straight, colorless, fusiform with obtuse to slightly rounded ends, and measured 13.2 to 17.5 (avg. 15.3) × 4.3 to 6.0 (avg. 5.2) μm in size (n = 50). Appressoria were oval to irregular in shape, dark brown, and ranged from 5.8 to 9.1 (avg. 7.5) × 4.8 to 6.5 (avg. 5.6) µm (n = 50). Morphological characteristics of the isolates agreed with the description of Colletotrichum siamense (Prihastuti et al. 2009). The ITS (Nos. MK225610, ON979519, ON979520), TUB2 (Nos. MK348533, OQ130408, OQ130409), ACT (Nos. MZ852755, OQ116933, OQ116934) andGAPDH (Nos. MZ852756, OQ130406, OQ130407) sequences were 99.35%, 98.76%, 98.92% and 97.60% identical to the type C. siamensestrain MFLU 090230 (accession nos. NR_144784, FJ907438, FJ907423 and FJ972575) through BLAST analysis, respectively. A phylogenetic tree was generated using the concatenated sequences of ITS, ACT, GAPDH and TUB2. The result showed that the 3 isolates were clustered with C. siamense strains including the type MFLU 090230. To perform pathogenicity tests, One leaf (wounded and/or unwounded) per seedling (n = 20, 2-year-old) was inoculated by dropping a 10 ul droplet of the conidial suspension (1 × 105) of isolate T6 or sterile distilled water (as control) at one point, respectively. These plants were wrapped in polyethylene bags for 2 days and incubated in a greenhouse at 28± 2°C with 80-90% relative humidity. Initial symptoms appeared within 5 days on all wounded and unwounded inoculated leaves, while the controls remained symptomless. Colletotrichum siamense was reisolated from the lesions in terms of morphology and molecular characterization, whereas no fungus was isolated from control leaves. The pathogenicity test was repeated three times under the same conditions. The diseases caused by C. siamense have been reported to cause anthracnose in a wide range of hosts (Weir et al. 2012), but not in F. microcarpa. In China, C. gloeosporioides has been reported to cause anthracnose on potted banyan F. microcarpa (Yao et al. 2016), but not C. siamense. This is the first report of C. siamense causing anthracnose on F. microcarpa in China.This study provides valuable information for the identification and control of the anthracnose on F. microcarpa.