First Report of Grapevine Trunk Disease Caused by Botryosphaeria obtusa in China.

Abstract

In September 2010, grapevine (Vitis vinifera) trunk diseases were observed in several vineyards of Yantai District in Shandong Provinces and Changli County of Hebei Provinces of China. Characteristic symptoms of Botryosphaeria canker were apparent, including dark brown discoloration on the trunk (visible in cross-section), cob base shriveling, drying of fruit clusters, and berry falling (2). To identify the causal pathogen, culturing of fungi was attempted from 387 small pieces of tissue from the canker margins of 43 diseased plants. Samples were surface disinfected by placing them in 75% ethanol for 1 min and rinsing with sterilized water three times before culturing on potato dextrose agar (PDA) at 28°C for 7 to 10 days. Fungi isolated were single spored to obtain pure cultures. On the basis of colony characteristics on PDA, 18 isolates from the 387 tissue pieces were eventually identified as Botryosphaeria obtusa (1), Most of the other fungi isolated were B. dothidea. B. obtusa colonies were grayish white, becoming dark brown with age, and pycnidia were formed after incubation for approximately 7 days. Conidia measured 8 to 11 × 17 to 26 μm (n= 50). Two isolates were used for rDNA internal transcribed spacer (ITS) sequence analysis with primers ITS1 and ITS4 (3). PCR products were separated by electrophoresis and bands were purified for legation with PMD-18T (Takara Company, Dalian, China) vector for sequencing. BLAST searches of two ITS sequences had 99 to 100% identity to B. obtusa. EF1-α and β-tubulin sequence analysis gave similar results. Koch's postulates were completed in the greenhouse on grape shoots inoculated with two isolates of B. obtusa originally isolated from diseased plants in the field. Inoculations were made on green shoots of V. vinifera cv. Dunkelfelder T. Six shoots were inoculated per isolate by wounding with a 4-mm cork borer (2 mm deep) and placing a colonized agar plug from a 5-day-old culture on the wound and wrapping it with Parafilm. Controls were mock inoculated with an agar plug from sterile PDA. Inoculated shoots were incubated in the dark under moist conditions in the laboratory for 8 to 10 days at 25°C. Inoculated shoots had necrotic cankers after 8 to 10 days and B. obtusa was recovered from each canker margin. The results indicated that some grapevines in China with symptoms of Botryosphaeria canker were infected by B. obtusa. To our knowledge, this is the first report of this pathogen causing trunk disease on grapevine in China.