First Report of Leaf Blight Caused by on in China.

Leonurus japonicus is cultivated throughout China and is commonly used for medicinal, cosmetic, ornamental and culinary purposes. A leaf blight on L. japonicus was first observed in September 2021 in a field at a research and development farm in Liupu Town, Zhuji City (120.23°N, 29.72°E), Zhejiang Province, China. Disease incidence was more than 90% across the 30 ha. Symptoms included nearly round black to brown spots on the leaf margins that gradually enlarged causing leaves to wither. To isolate and identify the causal organism, 12 L. japonicus leaves from four different plants with typical symptoms were collected, and 5×5 mm tissues were excised at the junction of the diseased and healthy tissue. Samples were surface-sterilized in 75% ethanol for 30s, followed by 7% NaOCl for 1 min, and rinsed three times with sterile distilled water (Sun et al. 2022), and placed on potato dextrose agar (PDA) at 25℃. After 7 d, single-spore isolations were conducted. (Zhu et al. 1992) After 8 d, the colonies on PDA were 75 to 86 mm diam, dark brown, with an irregular shape. A total of 150 conidia on PDA were an inverted rod shape or oval, dark brown, 20 to 45 × 7.5 to 11.3 μm, with a short beak and no septa; or columnar or conical, 2.5 to 20 × 2.5 to 5 μm, with 0 to 6 transverse septa, 0 to 3 longitudinal or oblique septa. The conidiophores were dark or branched, with multiple conidial scars, 15 to 62.5 × 3.0 to 5.0 μm. According to morphological characteristics observation, the 12 isolates were most similar to A. alternata (Simmons 2007). To further identify the fungal species, internal transcribed spacer (ITS) rDNA regions, and the following genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen (Alt a 1), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF) were amplified and sequenced using the primers ITS4/ITS5, RPB2-5F/RPB2-7CR, gpd1/gpd2, EF1-728F/EF1-986R, and Alt-for/Alt-rev (Woudenberg et al. 2015). Sequences were uploaded (ITS: OM095432, OM095433; RPB2: OM275409, OM275410; GAPDH: OM275411, OM275412; TEF1: OM160771, OM160772; Alta1: OM160773, OM160774). The similarity of YMCLZL, YMCLZL01 and the type strain CBS 59593 T (KP124320, KP124175, KP125096, KP124788, JQ646399) on the phylogenetic tree was 97%. To evaluate pathogenicity, a conidial suspension (106 conidia/ml) of isolates YMCLZL or YMCLZL01 was sprayed on the leaves of six 15-day old healthy plants. The same number of plants were also sprayed with only distilled water as non-inoculated controls. Plants were covered with plastic bags at 25℃ for 48 h. After 8 d, inoculated plants had round, gray and black spots on leaves, while the control plants did not. The experiment was repeated three times. The fungus was reisolated from all diseased leaves fulfilling Koch's postulates. To our knowledge, this is the first report of L. japonicus leaf blight caused by A. alternata on L. japonicus worldwide. The occurrence of leaf blight will be challenging for the commercial production of L. japonicus.