First Report of Botryosphaeria dothidea Causing Stem Blight of Dioscorea oppositifolia in China.

Dioscorea oppositifolia Thunb. (Chinese yam) is one of the traditional foods and medicinal plants in China. It has nutritional and medicinal value and plays an important role in treatment of diabetes and hypertension. In 2018, stem blight was first observed on the stalks of D. oppositifolia in fields of Anguo City (115°27' N; 38°46' E), Hebei Province, China. Over 400 plants were surveyed in four fields, and nearly 30% of the plants were infected. At the initial stage of the disease, there were dark brown spots on the stems and in later stages the leaves and stems withered. To identify the pathogen, 10 symptomatic stalks were collected, and one diseased area was taken out from each sample. Small square stalk pieces (3 to 5 mm) were obtained with sterile scissors from the junction of infected and healthy tissues, sterilized with sodium hypochlorite (10%) for 1 min, followed by washing in sterile water three times, then pieces were transferred to potato dextrose agar (PDA) plates for 7 days at 25°C. The fungal isolates were purified by single-spore isolation. A total of three species of fungi were isolated, and initial pathogenicity tests found that one fungal species could cause the disease symptoms on D. oppositifolia stems. This pathogen was grown on PDA plates in the dark at 25 °C for 10 days. In the beginning, the colonies were white, and as the culture time was extended, the color of the colonies became darker and then became black. Conidia forming on pycnidia were one-celled, hyaline, aseptate, and ovoid, with dimensions of 4.6 to 7.6 × 2.6 to 4.8 µm (n=100). Mycelial DNA was extracted from a 7-day-old culture, and PCR amplifications were performed using primers ITS1/ITS4 and ß-tubF/ß-tubR (Glass and Donaldson 1995; White et al. 1990). BLAST searches at GenBank showed 100.00% nucleotide sequence identity for the ITS sequence with Botryosphaeria dothidea strain sdxf6 (MG282093; 545/545 bp) and for ß-tubulin 99.76% identity with B. dothidea strain SD-B8 (KP183131; 411/412 bp). Sequences from these regions were deposited in GenBank (ITS: OP104323; ß-tubulin: OK669147). Morphological and molecular results confirmed this species as B. dothidea (Angelica et al. 2017; Bernard et al. 2004). To inoculate plants, pathogen was grown on PDA at 25°C in the dark for 15 days, after which a spore suspension (3×105 spores/mL) was prepared by flooding the agar surface with sterilized double-distilled water. Pathogenicity tests were conducted by stem inoculation of 6-month-old healthy D. oppositifolia plants. The stems were wounded by lightly rubbing with a steel sponge, and the wounded stem was wrapped in sterile cotton treated with 1 mL of the spore suspension, then the plants were covered with plastic to maintain a moist environment for 72 h. Control plants were inoculated with sterile water. Inoculated and control plants (ten each) were kept in a moist chamber (25°C, 16-h light and 8-h dark period, 75% relative humidity). After 15 days, all of the inoculated plants showed dark brown spots on the stems, and the symptoms were the same as those in the field, while the controls were healthy. After 30 days, all of the inoculated but none of the control D. oppositifolia plants showed leaf wilting or leaf withering. Isolates from the inoculated and infected leaves were identified as B. dothidea by DNA sequencing with primers ITS1/ITS4 and ß-tubF/ß-tubR, fulfilling Koch's postulates. To our knowledge, this is the first report of B. dothidea causing stem blight on D. oppositifolia. The disease poses a threat to the production of D. oppositifolia, and management strategies need to be developed.