First Report of Colletotrichum gloeosporioides sensu stricto Causing Anthracnose on Euonymus japonicas in Sichuan Province of China

Euonymus japonicus Thunb. is an evergreen tree that is widely used in landscaping in China (†). In August 2014, lesions were found on leaves in Chengdu of Sichuan Province. Approximately 20% of the leaves exhibited symptoms of anthracnose. Symptoms began as chlorotic spots that often appeared at or near the leaf margins and then coalesced into larger, irregular lesions. The centers of the lesions were gray-white with brown borders surrounded by a yellow halo. Lesions eventually dried and formed extensive areas that contained numerous acervuli in concentric rings. Samples taken from lesions were surface disinfected for 5 min in 5% sodium hypochlorite, rinsed in sterile water, and plated on potato dextrose agar (PDA). Single conidia collected from similar colonies from all samples were plated on PDA. After 5 days at 25 ± 2°C in the dark, the mycelium of a pure culture of named DYHY2 covered the entire plate surface (9 cm in diameter). Hyphae were white at first and thereafter they turned pale-gray with many orange conidial masses near the center of the colonies. The nonseptate conidia were hyaline, cylindrical with obtuse to slightly rounded ends, and ranged from 15.3 to 18.9 μm (mean 16.7 μm) × 5.4 to 7.3 μm (mean 6.3 μm) (n = 100). The appressoria were ovoid to slightly irregular, brown to dark black, and ranged from 9.2 to 10.5 μm (mean 9.8 μm) × 4.7 to 5.5 μm (mean 5.2 μm) (n = 100). Based on such characteristics, our isolates were tentatively identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc (†). Pathogenicity tests were conducted by spraying a conidial suspension of DYHY2 (1 × 107 conidia/ml) to 10 wounded and 10 nonwounded leaves in total from 10 3-year-old E. japonicus plants. Two areas of cuticle on either side of the midrib of each leaf were wounded by lightly scratching with a needle prior to inoculation (†). As a control, distilled water was sprayed onto an equal number of wounded and nonwounded leaves. All inoculated and control plants were incubated in greenhouse (about 25 ± 2°C). Sealed plastic bags were used to maintain high humidity until tiny chlorotic spots started to develop. Lesions similar to those observed in the field appeared on all wounded inoculated leaves within 7 days after inoculation, whereas the nonwounded inoculated leaves and the controls remained symptomless. Reisolations of the putative pathogen were confirmed through morphological and cultural characteristics. Further identification was completed with DYHY2 by PCR, sequencing, and BLASTn of the ITS rDNA region, actin, GAPDH, CHS-1, β-tubulin, and CAL genes (†), having the accession numbers KY454840, KY454841, KY454842, KY454843, KY454844, and KY454845, respectively. These sequences were highly identical to those of C. gloeosporioides (culture IMI 356878): JX010152 (99%), JX009531 (99%), JX010056 (98%), JX009818 (100%), JX010445 (99%), and JX009731 (100%), respectively. Anthracnose occurs on E. japonicus in Jiangsu in China (†), but the confirmation of its presence in Sichuan using only morphological characteristics is controversial (†). Based on the morphology and our multilocus approach, the pathogen was convincingly identified for the first time as C. gloeosporioides within the C. gloeosporioides species complex in Sichuan where it causes a common disease on E. japonicus. © 2017, American Phytopathological Society. All rights reserved.