First report of Alternaria alternata causing leaf blight on Trollius chinensis in China.

Trollius chinensis is widely distributed in east Asian countries that include China, Siberia, and Japan, with antibacterial, antiviral, anti-inflammatory and analgesic activity for medical applications. In August 2021, leaf blight was observed on nearly 80~95% of T. chinensis plants growing in Daxinganling (51.43°N, 126.39°E) from Heilongjiang Province, China. Initial symptoms were gray-black necrosis, wilting progressing from the leaf margin, and eventual defoliation. Six T. chinensis plants with typical symptoms were randomly collected, and three fresh leaf samples were collected from each plant. Diseased leaf pieces that measured 5 mm square were disinfected in 75% ethyl alcohol for 30 s and 7% NaClO for 60 s, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA). Twelve fungal isolates, obtained by single-spore isolations, were selected for further. These isolates produced colonies that measured 63 to 73 mm in diameter after 7 days growth on PDA. Colonies were black to brown in color with gray-white aerial hyphae on their surfaces, neat edges, olive green on the back. The isolates produced conidia that were ovate to pear-shaped, brown to black in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, smooth surfaced, parietal cells extending into the beak, and measured 12.5 to 37.5 × 5.0 to 12.5 μm(n=150). Conidiophores were dark, erect or curved, branched, with pronounced spore marks, and measured 35.0 to 50.0 × 4.0 to 5.0 μm(n=150). All twelve fungal isolates were morphologically similar to Alternaria alternata (Simmons 2007). Two representative isolates jlh01 and jlh02 were used for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS4/ITS5, RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nakashima 2020) and Alt-for/Alt-rev (Woudenberg et al.2015). The resulting sequences were deposited in GenBank (ITS, OM095427, OM108099; RPB2, OM131213, OM131214; GAPDH, OM201165, OM201166; TEF1, OM131211, OM131212; Alta1, OM201167, OM201168). Phylogenetic tree results showed 100% similarity between jlh01, jlh02 and the type strain CBS 118812. Morphological and molecular analysis results confirmed the identity of the fungus as A. alternata. Pathogenicity tests were done by spraying water-spore suspensions containing 106 spores per ml of A. alternata isolates jlh01 and jlh02 on leaves of six healthy T. chinensis plants, separately. Six control plants were sprayed with distilled water and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25⁰ C. Plastic bags were removed from all plants after 48 h. Black brown lesions and concentric rings developed on spore-inoculated plants after 15 days and control plants remained symptomless. The pathogenicity tests were conducted three times. A. alternata was reisolated and identified based on morphological and molecular traits, thus fulfilling Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf blight on T. chinensis in China. Based on the plant's medicinal value, this report provides the basis for further research and control of T. chinensis leaf blight.