First Report of Anthracnose of Atractylodes chinensis Caused by Colletotrichum chlorophyti in China

Abstract

Atractylodes chinensis (DC.) Koidz. is a perennial medicinal plant in the Compositae family in China. The dried roots have medicinal functions of invigorating the spleen, dispelling wind, and improving eyesight. Anthracnose-like symptoms were observed on the leaves and stems of a 2-year-old A. chinensis landrace in Changli County, Hebei Province, from July to September in 2017. In the field, small, round dark-brown spots appeared on the leaves and stems at the beginning. Later, the lesions on the leaves expanded to irregular spots, slightly sunken, grayish brown to grayish white in the center, and dark brown on the edge. The lesions on the stems spread from the spot to circle around the whole stem. Eventually, the whole plant started to wither and die. The incidence was as high as 30% in 2017. Symptomatic tissues from leaves were cut into 0.2- to 0.5-mm² squares and stems were cut into 0.5- to 1-cm lengths; they were surface disinfested with 70% ethanol, rinsed with sterile distilled water, and then cultured on potato dextrose agar (PDA) at 25°C with a 12-h light regime. Seven Colletotrichum-like isolates were obtained with a frequency of 79%. Colonies on PDA were initially pale, gradually turning black after 3 days, with obvious ring patterns and less aerial mycelium (colonies were 3.25 cm in diameter). Conidia ranged from 15.6 to 23.2 μm long (mean 19.4 μm) × 2.5 to 4.8 μm wide (mean 3.6 μm). Conidia were aseptate, hyaline, smooth, scythe or crescent with oil droplets, curving with tapered ends and a truncated base. Conidial length/width ratio was 3.0 to 6.0. Chlamydospores were dark brown, globose to subglobose, in chains and clusters. Setae were straight, dark brown, and septate. The isolates were further characterized by sequencing of internal transcribed spacer (ITS), actin, β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes. The sequences of the isolates (GenBank accession nos. MH290362 for ITS, MH935512 for actin, MH935514 for β-tubulin, and MH935513 for GAPDH) showed 99 to 100% similarity with Colletotrichum chlorophyti in GenBank (JX126475 for ITS, GU227993 for actin, JX126477 for β-tubulin, and GU228286 for GAPDH). Phylogenetic analysis showed the fungal isolates were grouped into the clade with C. chlorophyti. The fungi were identified as C. chlorophyti on the basis of morphological characteristics and sequence analysis. A pathogenicity test was conducted on 2-year-old A. chinensis. Twenty leaves and 20 stems were inoculated with 5-mm-diameter plugs from a 7-day-old culture in moist Petri dishes for 7 days at 25°C with a 12-h photoperiod, with PDA plugs serving as the control. The inoculated leaves and stems exhibited similar symptoms to the original field plants. However, there were no symptoms on control plants. The pathogen was reisolated from infected leaves and stems, confirming Koch’s postulates. This fungus previously has been reported as the pathogen of soybean anthracnose, moringa anthracnose, and soybean seed anthracnose (Cai et al. 2016; Yang et al. 2012, 2013). To our knowledge, this is the first report of C. chlorophyti causing anthracnose of A. chinensis in China. The disease poses a threat to production of A. chinensis, and this new finding is important in the diagnosis and management in field production.