First Report of Botryosphaeria dothidea Causing Canker and Shoot Blight of Eucalyptus in China.

Abstract

Eucalyptus spp. are grown on approximately 2 million ha in China and is the most important genus in forest plantations worldwide. An outbreak of cankers and shoot blight was observed for the first time on Eucalyptus grandis in China during May of 2008. Symptoms included dieback of shoots and branches, lesions and canker formation on the stems, and clear or yellow-to-red exudates on stems and branches. Isolations were made from affected trees in Shilin County from May to July of 2008. Diseased samples were plated on potato dextrose agar (PDA) and incubated at 26°C. Fungal isolates developed copious, white, aerial mycelium that became dark gray after 5 to 6 days and formed black pycnidia after 14 days. Conidia were hyaline, aseptate, thin walled, fusiform, and measured 19 to 28 × 4 to 6 μm. Ascospores were hyaline, aseptate, and widest from the middle to upper third (17 to 28 × 6 to 13 μm). Identity was confirmed by analysis of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2) with primers ITS1 and ITS4. BLAST searches showed 99 to 100% identity with Botryosphaeria dothidea isolates from GenBank (Accession Nos. FJ358703 and EU080916). Representative sequences of B. dothidea from eucalyptus from China were deposited into GenBank (Accession Nos. FJ517657 and FJ517658). On the basis of morphological and molecular results, the fungus isolated from diseased eucalyptus wood was confirmed to be B. dothidea. Pathogenicity tests were conducted by stem inoculation of 10-month-old E. grandis seedlings. Two experiments were conducted using two inoculation techniques. In the first experiment, 2-mm-diameter actively growing mycelium plugs of B. dothidea were applied to 2-mm-long bark wounds on the middle point of the stems, and control seedlings were inoculated with sterile PDA plugs in a similar fashion as above. Inoculated and control seedlings were inoculated in a greenhouse and watered as needed. In the second experiment, segments of branches (averaging 18 mm in diameter and 30 cm long) were inoculated with 5-mm-diameter plugs of actively growing mycelium. Control segments of branches were inoculated as previously described. The branches were incubated at 26°C in moist chambers. There were five replicate seedlings per inoculation technique. After 20 days, all E. grandis seedlings showed leaf wilting, Dark, vascular stem tissue was observed. Symptoms were more abundant on the segment of branches. After 6 days, vascular necroses that developed on the inoculated plants were 5.2 ± 1.2 cm. B. dothidea was reisolated from all inoculated symptomatic tissue; no symptoms were visible in the control seedlings and no fungus was isolated from them. These results confirm previous reports of B. dothidea causing canker and dieback symptoms of Eucalyptus species in Australia (2), the United States (1), and South Africa (3). To our knowledge, this is the first report of B. dothidea causing canker disease on eucalyptus in China.