First Report of Leaf Spot on Clematis brevicaudata Caused by Alternaria alternata in China

HomePlant DiseaseVol. 107, No. 3First Report of Leaf Spot on Clematis brevicaudata Caused by Alternaria alternata in China PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Leaf Spot on Clematis brevicaudata Caused by Alternaria alternata in ChinaH. F. Sun, H. Wang, H. Y. Yang, N. Li, M. Y. Wei, and Y. YanH. F. Sun†Corresponding author: H. F. Sun; E-mail Address: [email protected]https://orcid.org/0000-0001-6412-5687College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, ChinaSearch for more papers by this author, H. WangCollege of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, ChinaSearch for more papers by this author, H. Y. YangCollege of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, ChinaSearch for more papers by this author, N. LiCollege of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, ChinaSearch for more papers by this author, M. Y. WeiCollege of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, ChinaSearch for more papers by this author, and Y. YanCollege of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations H. F. Sun † H. Wang H. Y. Yang N. Li M. Y. Wei Y. Yan College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China Published Online:14 Feb 2023https://doi.org/10.1094/PDIS-04-22-0902-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleClematis brevicaudata DC. is distributed in China, Korea, Mongolia, Russia, and Japan. This plant is both ornamental and medical, used in the treatment of nervous disease, dyskinesia, and other diseases. In September 2019, a leaf spot on C. brevicaudata was first found in a 5 ha C. brevicaudata plantation in Harbin, Heilongjiang Province, China. The incidence was about 80%. The symptoms were elliptical, circular, or irregular brown to black necrotic lesions in the leaf apex and leaf margin. Ten fresh sample leaves with typical symptoms were collected from 10 C brevicaudata plants. The tissues (5 × 5 mm) between symptomatic and healthy areas were cut and surface disinfected in 75% ethanol and with 7% NaClO for 1 min, then rinsed three times with sterilized water for 30 s each time. The sterilized tissues were inoculated on potato dextrose agar (PDA) plates for 7 days at 25°C. The colonies were obtained and transferred to new PDA and potato carrot agar (PCA) plates by the single spore method to further purify. After 7 days, the colonies on PDA were 50 to 63 mm in diameter, circular, grayish brown, with white aerial hyphae. A total of 150 conidia on PCA were single or in chains, ovoid, inverted pear shaped, two to seven transverse septa, zero to three longitudinal or oblique septa, 17.5 to 57.5 × 7.5 to 17.5 μm. Beaks and false beak are mostly columnar, rarely conical, 2.5 to 6.0 × 2.0 to 3.0 μm. Conidiophores were solitary or clustered, pale brown, erect or bent, branched or unbranched, separated, 112.0 to 151.0 × 5.1 to 14.7 μm. Ten isolates purified on PDA were obtained. Morphological identification showed the 10 isolates were similar and appeared to be Alternaria alternata (Simmons 2007). Two strains from 10 isolates were selected for molecular identification. Genomic DNA was extracted from mycelia of two isolates (LD2020520 and LD2020521) on PDA using a modified CTAB method. Internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit gene (RPB2), Alternaria major allergen (Alt a 1), endopolygalacturonase (endoPG), and glyceraldehyde 3-phosphate dehydrogenase (gpd) were amplified and sequenced using two directional sequencing with the priers ITS1/ITS4, RPB2-F/RPB2-R, Alt-F/Alt-R, end-F/end-R, and gpd-F/gpd-R (Woudenberg et al. 2015). The sequences obtained were deposited in GenBank (ITS: MT501762, OK571395; RPB2: MT506027, OK631891; Alt a 1: MT506026, OK631890; endoPG: ON054189, ON054188; gpd: ON054191, ON054190). The phylogenetic analysis of a maximum-likelihood tree by MEGA 7 software showed that the two isolates had 99% identity with the A. alternata CBS 916.96. For pathogenicity testing, 18 leaves of six 5-week-old plants were sprayed with spore suspensions (1 × 106 spores/ml) of the 7 days-old isolates LD2020521 and LD2020520 (Each isolate infected three plants and each infected three leaves). Three plants were sprayed with sterile distilled water as a control group. The plants were incubated at 25°C. After 15 days, taupe irregular spots appeared on the leaves. The pathogenicity test was repeated three times. The same fungi were reisolated from the inoculated leaves and with the same morphological and molecular characteristics as LD2020520 and LD 2020521, fulfilling Koch’s postulates. No fungi were isolated from the control group. This is the first report of leaf spot on C. brevicaudata caused by A. alternata. Leaf spot can reduce the yields of C. brevicaudata. This study provides a reference for the prevention and treatment to the leaf spot of C. brevicaudata.The author(s) declare no conflict of interest.References:Simmons, E. G. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands. Google ScholarWoudenberg, J. H. C., et al. 2015. Stud. Mycol. 82:1. https://doi.org/10.1016/j.simyco.2015.07.001 Crossref, ISI, Google ScholarFunding: Funding was supported by the National Key Research and Development Program of the 13th Five-year Plan in China - R & D and Demonstration of Ecological Forest Exploitation and Utilization Technology of Forest Drugs in Northeast Forest Region (2016YFC0500303-03) and the national project survey of Traditional Chinese medicine resources from the State Administration of Traditional Chinese Medicine (SATCM) (2018hljzyzypc-19).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 107, No. 3 March 2023SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Article History Issue Date: 3 Apr 2023Published: 14 Feb 2023First Look: 28 Jul 2022Accepted: 11 Aug 2022 Page: 947 Information© 2023 The American Phytopathological SocietyFundingNational Key Research and Development Program of the 13th Five-year Plan in China - R & D and Demonstration of Ecological Forest Exploitation and Utilization Technology of Forest Drugs in Northeast Forest RegionGrant/Award Number: 2016YFC0500303-03State Administration of Traditional Chinese Medicine (SATCM)Grant/Award Number: 2018hljzyzypc-19KeywordsAlternaria alternataClematis brevicaudataleaf spotThe author(s) declare no conflict of interest.PDF download