First Report of Stemphylium Leaf Blight Caused by Stemphylium eturmiunum on Welsh Onion in China

Welsh onion (Allium fistulosum L. ) is native to Southwest China (Tomoo and Shinji 2012), with a cultivated area of about 550,000 ha and a production of 20 million tons in 2020, and widely cultivated throughout the world as a condiment. During April 2020, a Stemphylium-like disease occurred on a three-hectare field in Nanchang County (28°33'N, 115°57'E) was first observed and then monitored in Ningdu (26°23'N, 115°59'E) and Leping (28°50'N, 117°18'E), Jiangxi Province, China. The disease incidence was estimated to be nearly 30% in a survey of 300 plants. Disease symptoms were oval or irregular lesions at the center of outer leaves. The lesions were light brown at the edges and black-violet in the middle. Previous studies reported that the disease was mainly caused by S. vesicarium (Misawa et al. 2012). Besides, S. botryosum (Misawa et al. 2011) and S. solani (Dumin et al. 2021) can also cause this disease. To investigate the causal agent of the disease in Jiangxi, pieces of tissue from the margins of the symptomatic leaves were surface sterilized with 75% ethanol for 15 s, rinsed in sterile water three times, and placed on Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Colonies were initially yellowish-brown and gradually turned into reddish-brown after 2 weeks. Conidia were solitary, oblong or ellipsoid, mid to deep brown (22-45×14-25 μm) with transverse and longitudinal septa (Fig. S1). Morphological characteristics were consistent with S. eturmiunum reported by Simmons (Simmons 2001). For molecular identification, genomic DNA was extracted from two representative isolates. ITS and gapdh genes of the isolates were amplified with primers ITS1/ITS4 (White et al. 1990) and gdp1/gdp2 (Berbee et al. 1999). BLASTn analysis showed that ITS sequences (OM846570, OM846571) of the two isolates both > 99% identity with S. eturmiunum strain CBS 109845 (MH862841) (Vu et al. 2018); and also had 100% identity for gapdh gene sequences (OM867337, OM867338) with S. eturmiunum strain CBS 109845 (KU850689) (Woudenberg et al. 2017). Phylogenetic tree based on ITS and gapdh gene sequences were constructed using a maximum-likelihood method with 1000 bootstraps indicated that the two isolates were clustered with S. eturmiunum (Fig. S2). Based on morphological characteristics and molecular analysis, the fungus was identified as S. eturmiunum. A pathogenicity test was performed on healthy welsh onion leaves to fulfill Koch's postulates. Twenty leaves were inoculated with a spore suspension of 1×104 conidia/mL followed by incubation at 25℃ (relative humidity > 90%) in the greenhouse. Negative controls were inoculated with sterile water. The experiment was repeated three times. Purple brown lesions were observed on all inoculated leaves at 7 days post-inoculation that similar to those observed in the field, and control remained symptomless. S. eturmiunum was re-isolated from the diseased leaves. It has reported that S. eturmiunum can infect tomato, garlic, sweet cherry and many other crops and cause different diseases. To the best of our knowledge, this is the first report of S. eturmiunum can cause Stemphylium leaf blight on welsh onion in China. Appropriate strategies should be developed to manage this disease.