First Report of Black Spot in Dendrobium officinale Caused by A. alternata in Zhejiang Province, China

Abstract

HomePlant DiseaseVol. 102, No. 4First Report of Black Spot in Dendrobium officinale Caused by A. alternata in Zhejiang Province, China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Black Spot in Dendrobium officinale Caused by A. alternata in Zhejiang Province, ChinaC. Q. Zhang, J. X. Zhang, Y. H. Liu, and D. J. DaiC. Q. Zhang†Corresponding author: C. Q. Zhang; E-mail: E-mail Address: cqzhang@zafu.edu.cnSearch for more papers by this author, J. X. ZhangSearch for more papers by this author, Y. H. LiuSearch for more papers by this author, and D. J. DaiSearch for more papers by this authorAffiliationsAuthors and Affiliations C. Q. Zhang † J. X. Zhang Y. H. Liu , Department of Crop Protection, Zhejiang Agriculture & Forest University, Lin’an 311300, China D. J. Dai , Institute for the Control of Agrochemicals of Zhejiang Province, Hangzhou 310020, China (This research was supported by Project 2016C32002, Zhejiang Public-Interest Technology Application Study). Published Online:12 Feb 2018https://doi.org/10.1094/PDIS-07-17-1090-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Dendrobium officinale is one of the most valuable medicinal herbs in China. Zhejiang Province produces 80% of the world’s crop of D. officinale, with 2,749 t and $57 million per year (Pan et al. 2016). In 2001, growers in Lin’an city, Zhejiang, reported sporadic occurrence of black spot symptoms on D. officinale plants. The same symptoms occurred in about 60% of D. officinale greenhouses in Zhejiang in 2014. Symptoms on leaves initially appeared as small lesions (1 to 7 mm diameter), circular, light to dark brown. The spots gradually enlarged in size with brownish-red borders. Severely affected plants defoliated earlier. Small pieces of symptomatic leaf tissues were surface sterilized with 1.5% sodium hypochlorite for 2 min, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 7 days. A total of 13 single-spore isolates, namely ZJA-1 to ZJA-13 from 15 leaf samples, were transferred onto potato carrot agar (PCA) plates (Simmons 2007). After 5 days, olive-green colored colonies with smooth edges developed. Eleven of the 13 isolates were identified as Alternaria tenuissima based on morphology of conidia (Simmons 2007). The remaining two isolates were identified as A. alternata (Simmons 2007): conidia were 18.2 to 35.3 × 9.7 to 17.6 μm (average of 200 conidia for each isolate), typically obclavate to obpyriform or ellipsoid, with short conical beaks. Conidia exhibited 2 to 7 horizontal septa, and 0 to 5 vertical septa. Identification was further confirmed by amplification the endoPG, OPA1-3, and OPA2-1 gene fragments from DNA extracted from six single-spore isolates including the two A. alternata isolates using the primers EPG-f/EPG-3b, OPA1-3R/OPA1-3 L, and OPA2-1 L/OPA2-1R (Peever et al. 2005), respectively. The endoPG (GenBank accession no. KY952745–750), OPA1-3 (KY952757–762), and OPA2-1(KY952751–756) amplicons were 100% identical to A. tenuissima (JQ811977, AY631463, AY631438) for the isolates identified as A. tenuissima or 100% identical to A. alternata (AY629225, AY631468, AY631443) for the two isolates identified as A. alternata. All the isolates were adopted for pathogenicity tests, and four seedlings of D. officinale were inoculated with suspensions of 1 × 105 conidia per ml for each isolate. After inoculation, the seedlings were covered with plastic bags to maintain high relative humidity at 25°C and were monitored daily for symptom development. Eight days following inoculations, symptoms similar to those in the greenhouses were observed, while control leaves remained symptomless. Cultures reisolated from lesions had the same morphological characteristics as those previously observed in greenhouse plants. Although A. tenuissima has been reported to cause black spot disease in D. officinale in other regions of China (Wang et al. 2016), to our knowledge, this is the first report of black spot in D. officinale caused by A. alternata.