Pear is a popular fruit in the world market, and has been widely cultivated in China. Since 2008, a severe canker disease has consistently been observed on 20-year-old pear trees (Pyrus communis cv. Duchess de' Angouleme) grown in a nursery in Xingcheng, Liaoning Province, China. Observed symptoms include brown elongated ulcerative lesions (more than 20 cm in length in general), with red brown conidia produced on wet lesions. Reductions in tree vigor and yield were observed for infected trees. Tree mortality was observed for severe infections. To diagnose the pathogen, 15 canker samples were collected from five pear trees in April, 2012. Bark pieces (3 to 5 mm) taken from the border of healthy and diseased tissue were surface-disinfected with 0.1% mercury bichloride and 75% ethanol for 45 s, and placed on potato dextrose agar (PDA) medium at 25°C in darkness. Fungal colonies with a common colony morphology were consistently recovered from three samples. These fungal colonies were initially white, becoming olive green in 3 days. Conidia produced on colonies were hyaline, allantoid, and single-celled with average length × width of 6.04 (5.43 to 6.59) × 0.65 (0.51 to 0.73) μm, which were consistent with descriptions of Valsa leucostoma (1). Genomic DNA was extracted from a representative isolate F-LN-32b, and subjected to PCR amplification of the internal transcribed spacer region (ITS), β-tubulin gene, and EF1 gene using the primer pairs ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R (3), respectively. Sequence alignment of the amplified fragments with the deposited data in NCBI showed that sequences of EF1, ITS, and β-tubulin (GenBank Accession No. KF293296 to KF293298, respectively) of isolate F-LN-32b had the highest similarity of 99% to those of V. leucostoma strain 32-2w (JQ900340, JN584644, and JQ900374), and suggested that isolate F-LN-32b is a V. leucostoma strain. Pathogenicity tests was carried out by placing a 5-mm-diameter, 2-day-old mycelium agar plug of isolate F-LN-32b onto a punched bark hole of a detached 1-year-old pear shoot after it was surface disinfested with ethanol. Inoculated shoots were incubated at 25°C in plastic containers covered with plastic film. Pathogenicity assays were conducted on 18 pear varieties (cvs. Qiuyue, Jinshui 2, Hohsui, Huali 1, Cuiguan, Shinseiki, Xuehua, Dangshansu, Zaosu, Hongxiangsu, Yuluxiang, Nanguoli, Xizilv, Bartlett, Huanghua, Huashan, Duchess de' Angouleme, and Packham's) collected from a nursery in Wuhan, Hubei Province, China. Six shoots were inoculated for each variety and the assay was conducted three times. All inoculated shoots developed the typical canker symptoms after 6 days post inoculation (dpi) and sporulated at 25 dpi while the control shoots inoculated with non-colonized PDA plugs remained asymptomatic. Isolates recovered from inoculated samples were of the same morphology and ITS sequence as F-LN-32b. Based on these results, V. leucostoma was determined as the pathogen responsible for the Valsa canker disease on pear. Valsa mali var. pyri was identified as the only pathogen causing Valsa canker disease on pear in China (2). To our knowledge, this is the first report of V. leucostoma causing a canker disease on pear in China.
References: (1) G. C. Adams et al. Australas. Plant Pathol. 35:521, 2006. (2) X. L. Wang et al. Mycologia 103:317, 2011. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.