First Report of Southern Blight on Cynanchum stauntonii Caused by Athelia rolfsii in China

Abstract

Cynanchum stauntonii is a medicinal plant belonging to the Apocynaceae family, and its rhizome can be used as antitussive and anti-inflammatory medicine. C. stauntonii is widely cultivated in Tuanfeng, Xinzhou, Yingshan, and Qichun cities in Hubei Province. However, serious outbreaks of southern blight were observed in C. stauntonii fields in June and July in Tuanfeng County, Huanggang City (N30°48′48.943″, E115°01′15.967″) from 2018 to 2019, and the disease incidence was about 10 to 40%. The diseased C. stauntonii plants were characterized by water-soaked lesions on stems with white mycelia and white to brown sclerotia densely distributed on the infected sites and the surrounding soil. Leaf yellowing and wilting, root rot, and plant death are very common symptoms of infected C. stauntonii in warm and humid weather, especially in June and July. To isolate the fungal pathogen, mycelial fragments and sclerotia were collected from the field and cultivated directly on potato dextrose agar (PDA containing 100 µg/ml of kanamycin) medium and incubated at 27°C. The purified isolate BQ-1 showed white and fluffy aerial mycelia, which grew radially with an average growth rate of 32.3 to 37.5 mm/day (n = 10). White microsclerotia could be observed on PDA at 5 days postinoculation (dpi), and brown mature sclerotia appeared at 9 dpi. The number of mature sclerotia produced per plate ranged from 67 to 128 (92 ± 18; n = 10), and the diameter of mature sclerotia ranged from 1.00 to 4.15 mm (2.65 ± 0.83 mm; n = 50). No spores were observed under the microscope, and the fungal hyphae formed septa. For molecular identification, the 18S rDNA region of isolate BQ-1 was amplified by employing primers NS1 and NS6 (Li et al. 2014). The 18S rDNA sequence was sequenced by Sangon Biotech (Shanghai, China) and was deposited in GenBank under accession number MN243786. This sequence was 100% similar to Athelia rolfsii isolate AFTOL-ID 664 (AY665774.1). In addition, part of the elongation factor 1-alpha (TEF-1α) gene was amplified by using the primers EF595F and EF1160R (Wendland and Kothe 1997). The TEF-1α sequence was deposited in GenBank with accession number MN262526 and had 99.63% similarity to A. rolfsii strain Sr_12 (JF267794.1). The phylogenetic tree also shows that isolate BQ-1 is very close to seven A. rolfsii genes screened from GenBank. Pathogenicity tests were conducted using cleaned roots, stems, and leaves of healthy C. stauntonii. The upper, middle, and lower parts of roots (n = 3), stems (n = 3), and leaves (n = 3) were inoculated with mature sclerotia in a moist chamber for 5 days at 25 ± 2°C following the method described previously (Mahadevakumar et al. 2018). Samples inoculated with sterile water were used as a control. Sclerotia began to germinate and formed small colonies on the surface of tissues at 1 dpi. Mycelia began to infect tissues and formed black-brown spots on the tissues at 2 dpi, and the tissues were rotted at 5 dpi. In the pot experiment, stem bases of three C. stauntonii seedlings were punctured with a sterile needle and then inoculated with 5-day-old PDA-grown BQ-1 mycelia. Three seedlings treated with sterile water were used as controls. Three days later, the inoculated seedlings showed typical disease symptoms. Fungal pathogens were reisolated from the inoculated site and were reconfirmed through morphological features. This is the first report of A. rolfsii causing southern blight on C. stauntonii in China. This report will provide resources and reference for controlling of the increased incidence and economic losses of southern blight on C. stauntonii.