First Report of Nothophoma quercina Causing Leaf Spot Disease of Phellodendron amurense in China

Abstract

HomePlant DiseaseVol. 101, No. 10First Report of Nothophoma quercina Causing Leaf Spot Disease of Phellodendron amurense in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Nothophoma quercina Causing Leaf Spot Disease of Phellodendron amurense in ChinaW. L. Jiao, R. J. Zhou, J. F. Fu, H. J. Xu, and N. HaoW. L. JiaoSearch for more papers by this author, R. J. Zhou†Corresponding author. E-mail: E-mail Address: zrj5823@163.comSearch for more papers by this author, J. F. FuSearch for more papers by this author, H. J. XuSearch for more papers by this author, and N. HaoSearch for more papers by this authorAffiliationsAuthors and Affiliations W. L. Jiao R. J. Zhou † J. F. Fu H. J. Xu , Department of Plant Pathology, College of Plant Protection, Shenyang Agricultural University, Shenyang, Liaoning, 110866, China N. Hao , Department of Chinese Herbal Medicine Cultivation and Identification, College of Horticulture, Shenyang Agricultural University, Shenyang, Liaoning, 110866, China. Published Online:20 Jul 2017https://doi.org/10.1094/PDIS-01-17-0079-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Phellodendron amurense Rupr (Rutaceae) is a perennial herb distributed in Liaoning, Jilin, Heilongjiang, and Inner Mongolia provinces of China, and was used as a traditional Chinese medicine to treat jaundice and gastrointestinal diseases (Zhang et al. 2012). At the end of August 2013, typical signs and symptoms of leaf spot disease on P. amurense were observed at the Medicinal Herb Garden in Shenyang Agricultural University (SYAU), Shenyang, Liaoning, China. The disease incidence ranged from 45 to 87% rated by the percentage of infected leaves among a total of 500 leaves (five replicates, each with 100 leaves). Symptoms initially appeared as small, oval-circular, yellow-brown lesions on lower leaves, which subsequently enlarged and were surrounded by a faded-green margin. Later, the leaves withered and dropped off the plant early. Symptomatic leaves were washed with sterile water, surface-sterilized in 75% ethanol for 30 s, 1% sodium hypochlorite for 3 to 5 min, and were then washed three times with sterile water. Leaf fragments (approximately 5 × 5 mm) were aseptically cut from the margins of the lesions placed onto potato dextrose agar (PDA), and incubated at 25°C. The aerial mycelium was white, and the mycelium was olivaceous near the colony center, 65 mm in diameter after 7 days on oatmeal agar (OA). Malt extract agar (MEA) colonies had regular margins, and white to pale gray aerial mycelium covered the entire colony, 70 mm in diameter after 7 days. Black pycnidia were scattered over the medium at 15 days. The pycnidia were globose, glabrous, ostiolate, and 85.7 to 130.5 × 97.4 to 210.8 μm. Conidia were subglobose to oval, thin-walled, smooth, and aseptate, measuring 5.2 to 8.5 × 3.1 to 5.2 μm, with 2 min guttules. To confirm pathogenicity, 10 healthy leaves were spray-inoculated with spore suspension (106 conidia/ml), while 10 control leaves were treated with sterile water. All leaves were covered with polyethylene bags to maintain humid conditions (>80% RH) and incubated in an incubator (25°C) under a 12-h photoperiod. Typical spots appeared on all the inoculated leaves after 14 days, whereas the control plants remained asymptomatic. The same fungus was reisolated from the inoculated leaves, satisfying Koch’s postulates. For molecular analysis, the large subunit (LSU) and internal transcribed spacers (ITS) of the ribosomal DNA were amplified with primers LR0R/LR7 and ITS4/ITS5, respectively, and the amplicons were sequenced and deposited in GenBank under accession numbers KY437156 and KY435896. Sequence queries on the BLAST search engine of NCBI GenBank (www.ncbi.nlm.nih.gov/genbank/) confirmed that the species belongs to the genus Nothophoma. Phylogenetic tree based on neighbor-joining (NJ) analyses with the combined datasets of ITS and LSU using MEGA 4.0 showed high similarity with the sequence of Nothophoma quercina (CBS 633.92). Therefore, on the basis of morphological, cultural characteristics, and phylogenetic analysis, the causal agent of leaf spot on P. amurense was identified as N. quercina (Aveskamp et al. 2010; Chen et al. 2015). To our knowledge, this is the first report of leaf spot on P. amurense caused by N. quercina in China.