First Report of Bark Cracking of Koelreuteria bipinnata var integrifoliola Caused by Lasiodiplodia theobromae in China.

Koelreuteria bipinnata var integrifoliola is becoming a popular urban green tree in Ningbo City, Zhejiang Province, China, because of its adaptation ability to local conditions, fast growth, and beautiful appearance. A survey conducted from 2007 to 2010 revealed serious bark cracking on greenbelt trees approximately 15 to 16 years old that had been transplanted 5 to 6 years ago. Bark cracks increased in size over time, extending into the phloem and leading to extensive areas of bark loss with discoloration of the underlying xylem. Symptomatic trees had fewer new shoots in spring; many wilted and died in summer. Root rot was not observed in the withered trees but large light brown lesions were observed on cross sections of the main stem, each with a dark brown outer margin. In a September 2009 survey, 95% of symptomatic trees had stem lesions more than 50 cm long. Pieces of xylem (2 × 2 × 1 mm thick) were obtained from the margin of lesions surface sterilized using 0.1% mercuric chloride for 30 s, washed in sterile distilled water, and placed on 2% potato dextrose agar (PDA) at 28°C for 2 days. The fungus was then isolated and 12 colonies were obtrained. Three isolates KL-1-2, KL-3-2, and KL-4-3 were incubated on 2% PDA at 28°C for 30 days to produce spores. On PDA, the colonies were circular or near circular with irregular gray edges turning black green or black. The fungus also produced abundant aerial hyphae that were villous, septate, and irregular branched. Conidia were elliptical (or rounded) and hyaline when immature, becoming dark brown and septate longitudinally when mature and ranged from 23.2 to 27.0 × 10.8 to 16.2 μm (average 25.3 × 13.6 μm), similar to Lasiodiplodia theobromae (Patouillard) Griffon =Botryodiplodia theobromae Pa.t, Botryosphaeria rhodina (Berkeley & Curtis) von Arx (2). DNA extraction directly from the mycelium of KL-1-2, KL-3-2, and KL-4-3 was performed after 10 days' growth on PDA (1). The identities of the three isolates were confirmed by ITS1-5.8S-ITS2 rDNA sequence (GenBank Accession Nos. JN681172, JQ894322, and JQ894323, respectively) analysis that showed 99%, 100%, and 100% sequence similarity to L. theobromae xsd08006 (Accession No. FJ478102), L. theobromae PD20 (Accession No. GU251120), and L. theobromae xsd08008 (Accession No. EU918707), respectively. Pathogenicity tests were performed on 20 five-year-old K. bipinnata var integrifoliola plants by placing mycelia plugs of isolate KL-1-2 (10 × 10 mm) on the main trunk after wounding with a metal needle. Control plants received PDA plugs without mycelium. After inoculation, humidity was maintained using wet absorbent cotton and PE wrap film. Stem bark and phloem cracking was observed after 60 days on 85% of inoculated plants; 30% of those trees also had xylem discoloration. Symptoms were similar to those with natural infection. Control plants remained symptomless. The same fungus was reisolated from the brown xylem of inoculated plants. To our knowledge, this is the first report of bark cracking of K. bipinnata var integrifoliola caused by L. theobromae in China. References: (1) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (2) G. Fu et al. Australas. Plant Dis. Notes 2:75, 2007.