First Report of Colletotrichum siamense Causing Anthracnose on Partridge tea (Mallotus oblongifolius) in China

Abstract

Partridge tea (Mallotus oblongifolius [Miq.] Mull.Arg.) is an important medicinal plant in Hainan province of China used in tea making. During April 2017, circular necrotic lesions were observed on partridge tea plants with a disease incidence of 25% in an experimental station of Hainan University (110.19° E, 20.03° N) in Haikou, Hainan province, China. The lesions began as small circular spots, which later developed into larger spots (diameter of 1.2 to 2.9 mm) with a grayish white center and a dark brown banding pattern on the edges. To identify the pathogen, 10 symptomatic leaves from five plants were collected. Diseased tissue (2 × 2 mm) was cut from the margins of lesions, surface disinfected with 70% ethanol for 5 s, and rinsed four to five times with sterile distilled water. The excised sections were transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 3 to 4 days in the dark. A total of six isolates of Colletotrichum sp. were obtained from diseased tissue, and three strains were randomly selected (Z1, Z4, and Z6) for further analysis. The colonies appeared white to gray on the upper surface of PDA medium at 28°C. Conidia were cylindrical to subcylindrical, rarely clavate, hyaline, and guttulate with 3.96 to 4.21 µm (width) and 12.30 to 18.90 µm (length). These morphological characteristics were compatible with those of species from the Colletotrichum gloeosporioides complex (Weir et al. 2012). For accurate identification, fungal DNA of the three isolates was extracted. Fragments of internal transcribed spacer (ITS) and genomic regions including chitin synthase (CHS-1), actin (ACT), glutamine synthetase (GS), the Apn2–Mat1–2 intergenic spacer, and the mating type (Mat1–2) gene (ApMAT) were amplified and sequenced. These nucleotide sequences were deposited in GenBank (A70814043, ITS; A7110043, CHS-1; A7110049, ACT; A7110052, GS; and A7110046, ApMAT). A BLAST search of the amplified sequences of ITS resulted in 99% similarity with those of C. siamense strain CCL8. ACT sequences resulted in 100% similarity with those of C. siamense strain ICK. CHS-1 sequences showed 99% similarity with those of C. siamense strain WC-OILPALM4. GS gene sequences showed 99% similarity with those of C. siamense strain LQ22, and the ApMAT sequences showed 100% similarity with those of C. siamense strain PAS. Phylogenetic analysis based on the ITS, ACT, CHS-1, GS, and ApMAT amplified sequences was undertaken using MEGA6 software. This analysis clustered the three isolates with the C. siamense strain CBS130417 with high bootstrap support (99%). Therefore, based on their morphological characteristics and sequence similarity data, these isolates were identified as C. siamense. Pathogenicity was confirmed by spraying 10 45-day-old partridge tea plants with a 1 × 10⁶ CFU/ml monosporic suspension. Three strains (Z1, Z4, and Z6) were used to pathogenicity test in a growth greenhouse. Three control plants were treated with sterile water only. After 6 days at 25°C and 85% relative humidity, sunken necrotic spots similar to those observed in the field developed on the inoculated plants. The control plants remained symptomless. C. siamense was successfully reisolated from lesions. C. siamense has been reported on many crops in China (Cheng et al. 2013; Liu et al. 2017; Niu et al. 2016). To our knowledge, this is the first report of anthracnose caused by C. siamense on partridge tea in China.