First Report of Stemphylium lycopersici Causing Leaf spot on Sedum plumbizincicola in Hunan Province of China.

Abstract

Sedum plumbizincicola is a perennial succulent herb that can hyperaccumulate high concentrations of cadmium and zinc (Liu et al. 2017). In October 2021, a leaf spot disease occurred on S. plumbizincicola seedlings in a nursery in Changsha (28°13' N; 112°56'E), the Hunan Province of China. Almost 30% of the nearly 1 million seedlings were infected. Symptoms initially appeared as small brown spots on the leaf surface or edges, gradually enlarged, becoming oval, and bearing chlorotic lesions with dark brown borders. Eventually, the center of the lesions became sunken and then fell off. Eight symptomatic plant samples were collected by five-point sampling method (Zheng et al. 2018). Small pieces of 5×5 mm were excised from the lesion margins, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed with sterile distilled water three times, and then cultured on potato dextrose agar (PDA) at 26 °C for 5 days in the dark. Fungal colonies showing similar morphology were observed from all the isolated samples and, in total, eight fungal strains were obtained. On PDA, fungal colonies were initially white, and later become light gray. After cultured on V8 juice agar (V8A, each litre of medium contains 200 mL of V8 juice, 3 g of CaCO3 and 15 g of agarose) for 14 days (Hyowon et al. 2016), conidia of a representative isolate SY-1 were produced, which were oblong, muriform, with blunt ends and conical apex, pale to light brown, and constricted at the 1 to 3 major transverse septa, 38.34-46.68 μm×11.67-18.34 μm (n=50). These morphological characteristics were consistent with that of Stemphylium lycopersici (Nasehi et al. 2016). The internal transcribed spacer (ITS) region of rDNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of representative isolates SY-1 to SY-3 were amplified and sequenced using the primer pairs ITS4/ITS5 and gpd1/gpd2 as described previously (Woudenberg et al. 2017). BLASTn analysis showed that ITS sequences of isolates SY-1, SY-2 and SY-3 (accession nos. OP317641, OQ852042 and OQ852043) had more than 99% identity with Stemphylium sp, while GAPDH sequences (OP331223, OQ858620 and OQ858621) had 100% identity with S. lycopersici KR911813 (Sun et al. 2016). A concatenated ITS-GAPDH phylogenetic tree grouped our isolates within the S. lycopersici clade. For the pathogenicity test, one-month-old potted S. plumbizincicola seedlings were inoculated with conidia suspension (105 conidia/ml), which was induced on V8A. Four sites of each leaf of the potted S. plumbizincicola plants were dropped with a conidia suspension of strain SY-1, with 10 μL per site. Leaves treated with sterile water were served as controls. All of the inoculated seedlings were placed in a growth chamber at 26°C with a photoperiod of 12 h. The pathogenicity tests were repeated twice, with each had three replicative plants. After 7 days, all the inoculated leaves developed brown spots resembling those observed in the nursery, whereas the control plants remained symptomless. Stemphylium lycopersici was specifically re-isolated and identified by morphological and molecular methods (accession nos. OQ852045 for ITS and OQ858622 for GAPDH, respectively), thus fulfilling Koch's postulates. To our knowledge, this is the first report of S. lycopersici causing leaf spot on S. plumbizincicola in China. Since S. plumbizincicola played an important role and widely planted for heavy metal pollution treatment (Jiang et al. 2010), and this disease might seriously influence the S. plumbizincicola seedling breeding, identification of the pathogen might provide a foundation for the diagnosis and control of the disease.