First Report of Botryosphaeria dothidea Causing Canker and Dieback Disease of Helwingia chinensis in China

Helwingia chinensis Batal is distributed in the western and southern regions of China. The aerial part of this plant has long been used to treat dysentery, hematochezia, and swelling. An outbreak of cankers and dieback was observed for the first time on H. chinensis in China during June of 2010. Disease symptoms included dieback of shoots and branches, lesions, and canker formation on the stems. In order to identify the causal agent(s) of this canker disease, samples composed of inner bark and woody tissues were collected from the junction of healthy and diseased tissues of declining trees from Kunming and Wenshan districts of China during July to October of 2010. Pieces of surface-sterilized tissue samples were plated on potato dextrose agar (PDA) and incubated at 26°C. Fungal colonies developed copious, white, aerial mycelium that became dark green with age. Pycnidia started to develop after 20 days. Macroconidia, which were 20 to 29 × 4 to 6 μm, were hyaline, aseptate, and fusiform. No fungus was isolated from water-inoculated tissues of control plants and healthy trees. Identity was confirmed by analysis of the rDNA internal transcribed spacer region (ITS1-5.8S-ITS2) with primers ITS1 and ITS4. BLAST searches showed 99% identity with Botryosphaeria dothidea isolates from GenBank (Accession Nos. HQ660454 and FJ790846). Representative sequences of B. dothidea from H. chinensis from China have been deposited in GenBank (Accession No. JQ766122). On the basis of morphological and molecular results, the fungus isolated from diseased H. chinensis was confirmed to be B. dothidea. Pathogenicity tests were conducted by stem inoculation of 1-year-old H. chinensis seedlings. Mycelial plugs (3 to 4 mm in diameter) of B. dothidea from actively growing colonies were applied to same-sized bark wounds on the middle point of the stems. Control seedlings were inoculated with sterile PDA plugs. Inoculated and control seedlings (three each) were kept in a greenhouse and watered as needed. After 4 weeks, all H. chinensis seedlings developed vascular tissue discoloration and leaf wilting; no such symptoms were manifested by seedlings in the control treatment. B. dothidea was reisolated from all B. dothidea-inoculated, symptomatic tissues, fulfilling Koch's postulates. In China, B. dothidea has previously been reported to cause canker and dieback disease of Eucalyptus grandis (2) and gummosis of peach (1); however, to our knowledge, this is the first report of B. dothidea causing canker and dieback on H. chinensis. References: (1) Y. Ko et al. Plant Pathol. Bull. 1:70, 1992. (2) L. Yu et al. Plant Dis. 93:764, 2009.