LAUSR

Convallaria majalis is native to temperate zones in the northern hemisphere, Europe, Asia, North America and China, and has excellent ornamental properties and medicinal value. In July 2021, leaf blight was observed on nearly 70~90% of C. majalis plants growing in Heilongjiang University of Chinese Medicine campus (45.72°N, 126.68°E) from Harbin City, China. The typical symptom on the leaves is irregular dark brown lesions. As the brown lesions expanded and eventually coalesce, they form large necrotic areas, with chlorosis, curling, and wilting at the apical edge of the diseased leaf. Ten symptomatic leaves were randomly collected from twenty different plants at the site. Several fragments of diseased tissues (5×5mm) were disinfected in 75% ethyl alcohol for 30 s and 7% NaOCl for 60 s, rinsed three times in sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 25 °C in the dark for 7 days. Twenty purified fungal isolates were obtained by single spore isolation. The morphology of all the twenty isolates was similar, and two isolates were randomly selected (LL, LL01) for further study. Colonies of these isolates on PDA were off-white to black with abundant cotton-like aerial hyphae, and the diameter of the colony is 72 to 85 mm. On potato carrot agar (PCA) medium, these isolates produced light brown and solitary conidiophore with septum. Conidia were ovate to pear-shaped, brown to black in color, with 1-4 transverse septa and 0-2 longitudinal septa, and measured 20.5 to 38.5 × 7 to 13.5 µm (n=100). The isolates were identified as Alternaria alternata according to their morphological characteristics (Simmons 2007). Two representative isolates LL and LL01 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(White et al. 1990), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nawashima 2020) and Alt-for/Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS, OM319508, OP799847; RPB2, OM649830, OP830846; GAPDH, OM296234, OP830845; TEF1, OM393717, OP830844; Alta1, OM171258, OP830847). Phylogenetic analyses showed 100% identity between LL and LL01 and the type strain CBS 118815. Thus, the fungus was identified as A. alternata based on morphology and molecular analysis. Pathogenicity tests were done by spraying conidial suspensions containing 106 conidia/ml of A. alternata isolates LL and LL01 on leaves of six healthy C. majalis plants, separately. Another six plants were sprayed with sterile distilled water as control and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25⁰ C. Plastic bags were removed from plants after 48 h. After 15 days inoculation, the similar symptoms were observed on the inoculated plants, whereas control plants remained healthy. 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 C. majalis in China and worldwide. The result will serve as the foundation for management leaf blight of C. majalis.