Southern Blight on Macleaya microcarpa Caused by Sclerotium rolfsii in China

Abstract

Macleaya microcarpa (Maxim.) Fedde is a perennial herb belonging to the family Papaveraceae. This plant and the better-known species M. cordata (Willd.) R. Br. are native to central China and now planted worldwide for medicinal purposes (Qing et al. 2015). In August 2018, M. microcarpa in the Chinese herbal medicine planting base (approximately 4.3 ha) of Hunan Agriculture University in Changsha City, Hunan Province, showed what seemed to be a soilborne disease, in which approximately 25% of the plants were affected. The characteristic symptoms consisted of rotting, wilting, and blighting. Infected plants gradually withered, and white mycelial mats and sclerotia appeared on the surface of roots and stems at the soil line. Five sclerotia collected from three infected plants were surface disinfested in 0.5% NaOCl for 30 s, followed by 75% alcohol for 10 s, rinsed in sterile water three times, air dried, plated onto potato dextrose agar (PDA) with one sclerotium per dish, and incubated at 30°C in the dark. After 5 days, the aerial white mycelium usually formed many narrow hyphal strands, 4 to 9 μm wide. The white mycelium formed typical clamp connections. Numerous globoid sclerotia formed on PDA after 15 days of growth. The sclerotia (1 to 3 mm in diameter) were white at first and then gradually turned dark brown. Isolates were identified as Sclerotium rolfsii on the basis of mycelial characteristics, color, and sclerotia. Sequence analyses were conducted on sclerotia by extracting fungal DNA with the DNAsecure Plant Kit (Tiangen Biotech, China). To confirm the isolate identification, part of the elongation factor 1-alpha (EF1-α) gene and the complete internal transcribed spacer (ITS) region of the fungus were amplified using the primers EF595F/EF1160R (Wendland and Kohe 1997) and ITS1/ITS4 (White et al. 1990), respectively. BLAST searches revealed that the EF1-α sequence (MK275299) had 99% sequence identity to S. rolfsii (GenBank accession nos. JN790645.1, KM521757.1, and JF267794.1). The ITS sequence (MK275300) had 96% identity to S. rolfsii (GenBank accession nos. KU885934.1, KT428156.1, and MH858139.1). To fulfill Koch’s postulates, five healthy 60-day-old M. microcarpa seedlings were inoculated with sclerotia obtained from a 15-day-old culture. The seedlings were grown in greenhouse pots with a sterilized mixture of peat moss and vermiculite (3:1). Dry sclerotia were placed directly in the wounds made with a sterile blade on the root (three sclerotia per plant). Five control plants were inoculated with clean PDA plugs. All plants were packed in plastic bags that had been sprayed with water to maintain high humidity and kept at approximately 30°C in the laboratory. Five days postinoculation, the inoculated M. microcarpa seedlings presented wilt and collar rot symptoms, whereas those not inoculated were symptomless. S. rolfsii could be reisolated from the infected plants. The experiment was repeated with similar results. To our knowledge, this is the first report of southern blight caused by S. rolfsii on M. microcarpa in China. Southern blight caused by S. rolfsii is a serious soilborne disease and able to cause plant death. Understanding its etiology may help to the control of this plant fungal pathogen, thus reducing economic losses.