First Report of Stem Spots of Ephedra intermedia Caused by Neodidymelliopsis longicolla

Ephedra intermedia Schrenk (Gnetaceae, Gymnospermae) is a traditional Chinese medicinal plant used to treat asthma and cough and as a diaphoretic (Committee of China Pharmacopoeia 2005). It grows in rocky or sandy dry areas. Gansu Province is a main production area. Wild E. intermedia plants occupy ∼600 ha in Yumen City (continental temperate arid climate, ∼1,400 to 1,700 m altitude). However, wild Ephedra has been excessively harvested. Since 1990, ∼100 ha of cultivated Ephedra has been planted in Yumen City. Cultivation of Ephedra is controlled by the government of China, and its cultivation has not changed much since our 2007 disease survey. Stem spots were first found on wild E. intermedia in Huanghua Hacienda in Donghu Village, Yumen City. Early lesions on the stems were small, circular, pale brown spots; they became elliptical, fusiform lesions and finally faded to grayish brown in the center, with numerous black pycnidia distributed on each stem spot. Our surveys in 2007, 2012, and 2016 revealed disease incidence of 40 to 60%. When the disease was serious, lesions became connected, 5 to 10% of stem tissues were damaged, and the plants were stunted. Conidiomata on the stem lesions were pycnidial, immersed or semi-immersed, light brown to olive brown, 192.6 to 327.6 × 179.2 to 277.7 μm. Ostioles papillate or developing into elongated neck, 20 to 35 μm wide and 20 to 100 μm tall. Conidia were hyaline, smooth, cylindrical to clavate, straight to slightly curved, mainly one-septate, slightly constricted at the septa, 10.6 to 16.5 × 5.9 to 8.2 (avg. 13.9 × 6.3) μm, and occasionally aseptate and 7.5 to 15 × 1.25 to 6.25 (avg. 9.95 × 3.42) μm. Symptomatic stems were surface sterilized in 1% NaOCl (diluted domestic bleach) for 1 min and rinsed three times in sterilized distilled water, and the tissues from the lesion periphery were plated onto PDA to obtain pure cultures (the causal agent was designated AM2H). The isolation frequency of AM2H was 100%. Colonies on PDA were white at first and then turned vinaceous with black confluent pycnidia in ∼2 weeks. Conidial dimensions were within the range of those from the field samples. Representative samples were deposited in the Herbarium of Gansu University of Chinese Medicine. Pathogenicity tests were performed by spraying ten 6-month-old Ephedra plants with suspension of conidia harvested from 14-day-old colonies of AM2H growing on PDA; another ten plants sprayed with water served as controls. All plants were placed in a growth chamber and covered with a polyethylene bag for 48 h to maintain >95% humidity. Five days after inoculation, red-brown spots were observed on inoculated stems, but no symptoms appeared on control plants. After another 2 weeks similar spot symptoms on the inoculated plants were observed, and Koch’s postulates were verified by reisolating AM2H from stems with typical spots. DNA of AM2H was extracted, and ITS, LSU, BTUB, and RPB2 regions were sequenced with primers ITS1/ITS4, LROR/LR5, fRPB2-5F/fRPB2-7Cr, and T1/β-Sandy-R, respectively (Verkley et al. 2013); segments (535, 912, 1,102, and 401 bp) were submitted to GenBank (KF263920, MF476109, KM074044, and MF476108, respectively). The phylogenetic tree was constructed with Mega 6.0 using the neighbor-joining method from the combined multilocus database. Strain AM2H clusters with all accepted species in Neodidymelliopsis and forms a single clade with the maximum support rate (100%) with sequences of Neodidymelliopsis longicolla (CBS 382.96); Chen et al. (2017) proposed it as a new species that can cause lesion blight on Papaver dubium in Iran (Razaghi and Zafari 2018). Morphologically, the conidia size of AM2H (10.6 to 16.5 × 5.9 to 8.2 μm) is almost the same as N. longicolla (CBS 382.96) (12 to 15 to 16.5 × 4 to 7 μm). This is the first report of stem spots on Ephedra caused by N. longicolla in the world. Identification of the pathogen will help to work out methods to control the disease.