First Report of Purple Spot on Dandelion in China Caused by Alternaria tenuissima.

Abstract

Dandelion (Taraxacum mongolicum) is a perennial herb of the family Asteraceae, with a high edible and medicinal value and widely grown at medium and low altitudes in China. In July 2019, purple spot of dandelion was found in a field near Harbin City, Heilongjiang province, China. Thediseaseincidenceregionallyreached 95% in fields with yield losses between 10 and 20%, seriously reducing the economic and food value of dandelion. Multiple, irregular brown spots were first observed on the leaves of this plant- that later developed into circular or near-circular purple spots with raised centers, or purple lesions along the veins. When the leaf spots coalesced, the value of the commodity was lost. To isolate the pathogen, 5 × 5 mm pieces of leaf tissue from the margins of lesions were surface disinfected in 75% alcohol, rinsed in distilled water, and incubated on potato dextrose agar (PDA) plates at 28℃ until sporulation. Using single-spore isolation, a pure culture (YY-1) was obtained with abundant grayish white hyphae that later turned olive green. The underside of the colonies were brown. Conidia were typically obclavate, had a short beak with 1 to 6, but usually 3, transverse septa, and up to 3 longitudinal septa. The transverse septum was thicker and the wall of the conidium appeared brick-like. Conidia were pale brown, catenulate, and measured from 25 to 42 μm long by 6 to 10 μm wide. YY-1 was identified as Alternaria sp. based on morphological characteristics (Simmons 2007). Molecular identification was performed to detect the fungal species, and included the Internal Transcribed Spacer (ITS), translation elongation factor 1-alpha (EF1), actin gene (ACT), plasma membrane ATPase gene (ATP), and the calmodulin gene (CAL), which were respectively amplified with primers ITS4/ITS5 (Guo et al. 2012), EF1-728F/EF1-986R (Carbone and Kohn 1999), ACT-512F/ACT-783R (Carbone and Kohn 1999), and ATPDF1/ATPDR1 and CALDF1/CALDR1 (Lawrence 2013) (GenBank Accession Nos. MN746334, MT627208, MT627209, MT558864, MT558865). The species of Alternaria could not be confirmed by sequencing the above genes, as described previously (Zheng et al. 2015). Hence, a partial coding sequence of the histone 3 gene (GenBank Accession No. MN744235) was amplified using primers H3-1a/H3-1b (Zheng et al. 2015) and it shared 98.09% sequence identity with A. tenuissima (KP267543). The ITS sequence (MN746334) was 99.81% similar to the reference sequences of A. tenuissima (KT223327) in GenBank. A Maximum-likelihood tree was then reconstructed based on the ITS, CAL, and ATP sequences by MEGA7, which showed that YY-1 was most closely related to A. tenuissima. Therefore, YY-1 was identified as A. tenuissima based on its morphological and molecular characteristics. To perform Koch's postulates, 20 healthy leaves from greenhouse-grown dandelion were inoculated with 5-μL drops of a conidial suspension (1 x 105 conidia/ml) of isolate YY-1. Sterile water was used as a control. The inoculated plants were placed in a growth chamber at 28℃ and 80 to 90% relative humidity. After 10 days, similar symptoms were observed on plants inoculated with YY-1, while control plants did not produce symptoms. The pathogen was reisolated from the inoculated leaves and identified by morphological and molecular methods as A. tenuissima. To our knowledge, this is the first report of A. tenuissima causing purple spot on T. mongolicum in China.