In the early summer of 2009, grapevine (Vitis vinifera), an important fruit crop in China, declined in most of the vineyards in Hunan, Hubei, and Zhejiang provinces. Characteristic symptoms of Botryosphaeria canker were apparent, including trunk cankers (visible in cross-section), leaf drop, shriveling and drying of fruit clusters, and berry rot (1). To identify the causal pathogen, we tested 126 samples by attempting to culture the pathogen from a small piece of tissue from the canker margin between the necrotic and apparently healthy tissue. Plant tissue was surface sterilized by placing it in 75% ethanol for 1 min and rinsed with sterilized water three times before culturing to potato dextrose agar (PDA) at 28°C. Five days later, the cultures were hyphal-tip purified and then single-spore isolates were used for identification. On the basis of colony characteristics in PDA, these colonies were identified as Botryosphaeria spp. (2). They were grayish white, becoming dark brown with age, and pycnidia were formed after incubation for approximately 9 days. Conidia measured 11 to 15 × 22 to 28 μm. A subset of isolates were used for rDNA ITS (internal transcribed spacer) sequence analysis with primers ITS1 and ITS4 (3). PCR products were separated by electrophoresis and bands were purified (Qiagen Plasmid Mini Kit; Qiagen, Valencia, CA) for sequencing (Sunbiotech Company, Beijing). BLAST searches of three ITS sequences (Accession Nos. GU226851, GU226853, and GU226856) had 100% identity to B. rhodina. EF1-α and β-tubulin sequence analysis gave similar results. Koch's postulates were completed in the laboratory on grape shoots inoculated with two isolates of B. rhodina, originally isolated from plants in the field with symptoms of Botryosphaeria canker. Isolates were incubated on PDA at 25°C for 1 week. Inoculations were made on green shoots of V. vinifera cvs. Muscat Hamburg and Crimson Seedless. Five shoots per cultivar were inoculated per isolate by wounding with a 4-mm cork borer (2 mm deep), placing a colonized agar plug on the wound, and wrapping the wound with Parafilm. Controls were mock inoculated with an agar plug from sterile PDA. Inoculated shoots were incubated in the laboratory in the dark under moist conditions for 10 days at 25°C. Inoculated shoots had necrotic cankers after 10 days and B. rhodina was recovered from each canker margin. The results suggest that some grapevines in China with symptoms of Botryosphaeria canker were indeed infected by B. rhodina. To our knowledge, this is the first report of this pathogen on grapevine in China.
References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) J. M. Niekerk et al. Mycologia 96:781, 2004. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.
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