First Report of Sclerotium rolfsii Causing Southern Blight of Bletilla Orchid in China

Abstract

Bletilla striata, known as Chinese ground orchid, is economically important because of its medicinal and ornamental properties around the world, especially in China. The wild population of B. striata is decreasing, it was listed in appendix II (species that may be threatened or extinct) of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (CITES 2013). Therefore, farm-based cultivation of B. striata is emerging to meet the demands of the industry. In June 2016, disease symptoms similar to orchid southern blight (Punja 1985) were observed in a B. striata cultivation field (N30°21′27.37″, E112°19′58.37″) in Jingzhou, Hubei Province, China. In a follow-up survey, the disease was also found in nearby counties and provinces. Disease incidence reached 25% in the most severely affected field. Symptoms first appeared at the junction of the stem base and the soil, as necrotic dark brown stem lesions. Additional symptoms included leaf yellowing and wilting, root rot, and death during warm and humid weather. White mycelium and tan to brown sclerotia were found on the stems and roots of plants in the field and plants incubated in the lab. To isolate the pathogen, tissue from plant samples collected from a nursery (Orchid Biotechnology, Jingzhou, China) was surface sterilized with 2% sodium hypochlorite (NaOCl) for 3 min and then rinsed and incubated on potato dextrose agar medium with 50 μg/ml of ampicillin and rifampin. After 7 days of incubation at 28°C, sclerotial primordia developed followed by sclerotia approximately 2 mm in diameter after 10 days. No spores were observed. Total genomic DNA of isolate BH-2 was extracted with the cetyltrimethylammonium bromide method and then amplified and sequenced with fungal ribosomal primers ITS1/ITS4 (White et al. 1990). The length of the amplicon was 683 bp (GenBank accession no. KY606616), 99% similarity to Athelia rolfsii (teleomorph) isolate ATCC201126 (accession no. AF499018). Phylogenetic analysis of the internal transcribed spacer sequences with the neighbor-joining method in MEGA6 (Pennsylvania State University, University Park, PA) showed BH-2 was clustered with six Sclerotium rolfsii isolates selected from NCBI database (accession nos. AF499018, JN081867, JN241565, GQ3585118, JF966208, and KJ002764). Therefore, isolate BH-2 was identified as S. rolfsii. To produce inoculum for Koch’s postulates, autoclaved oat grain was inoculated with BH-2 then incubated at 30°C for 10 days. Infected oat grains were placed near the roots of 90-day-old healthy seedlings (n = 3), and sterilized oat grain was used as a control (n = 3). All seedlings were placed in sealed plastic bags with air valves and grown in a greenhouse (25 ± 2°C, relative humidity 65%). Water-soaked streaks and white hyphae were observed after 2 to 3 days. Symptoms typical of southern blight, necrotic stems, and leaf lesions were observed after 9 days. Reisolation was performed with surface-sterilized tissue from symptomatic seedlings, and isolates were confirmed as S. rolfsii based on morphological and molecular characters. To the best of our knowledge, this is the first report of S. rolfsii causing southern blight of Bletilla spp. in China.