| Abstract |
Centranthera cochinchinensis (Lour.) Merr., or swamp foxglove, is a perennial Chinese medicinal plant in the Yunnan Province of China and is of significant medical importance and economic value. It is medically effective in hemoptysis, hematemesis, blood stasis, pain relief, and so on. The current local price is about 3,000 CNY/kg for its roots and 60,000 CNY/kg for its seeds. In June 2018, a leaf spot disease was observed on swamp foxglove in Pingbian (23°03′10′′N, 103°45′47.0′′E), Yunnan Province, China. To determine incidence of symptoms, five different plots, each of which was about 600 m2, were investigated. Disease incidence within each plot was recorded between 30 and 70%, causing serious damage. Leaf spot symptoms began as round, oval, reddish-brown speckles at leaf margins and tips. Spots gradually grew into subcircular, elliptical or irregular, black or black brown lesions, 20 to 44 × 23 to 56 mm (30 representative lesions measured). The spots coalesced into large lesions as infection progressed, ranged from 62 to 101 mm (average 80.5 mm), and caused the leaves to dry, wilt, and defoliate. Stems were also symptomatic with pale brown, circular or subcircular sunken lesions, up to 31.5 mm when confluent. Twenty-five diseased plants were collected and taken to the lab. Diseased tissues (0.5 × 0.5 cm) cut from the margin of typical leaf spots were surface disinfected by immersion in 70% ethanol for 30 s and then in 1% NaOCl for 1 min, rinsed three times in sterile distilled water, placed on potato dextrose agar (PDA) plates, and incubated at 25°C in the dark. A total of 23 single-spore isolates were obtained from different samples and examined morphologically. Colonies were woolly, floccose, with a ripple edge, changing from white to gray to black with age, reaching 8 cm diameter in 5 days at 25°C. Conidiophores were flexuous or straight, pale brown, smooth, 4 to 10 μm in diameter, occasionally reduced to conidiogenous cells. Conidiogenous cells were monoblastic, diterminate, ampulliform or subspherical, subhyaline to hyaline, and pale brown when mature. Conidia were solitary, globose to subglobose, black, smooth, aseptate, ranging from 20.1 to 31.9 μm diameter (average = 22.6 μm, n = 50). According to colony and conidia morphology, the isolates were identified as Nigrospora oryzae (Berk. & Broome) Petch (Wang et al. 2017). The internal transcribed spacer (ITS1, 5.8s, and ITS2) region of isolate YY1 was amplified using ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) primers. Sequence comparison revealed 100% sequence identity (GenBank accession no. MH880188) to N. oryzae (MH619723) and confirmed the morphological identification. To confirm pathogenicity, 45 1-year-old, potted, healthy seedlings of C. cochinchinensis were inoculated with a conidial suspension (107 conidia/ml) by wound-inoculating method using sterilized needles and non-wound-inoculating methods, respectively (90 plants total). Forty-five control plants were wound inoculated with sterile distilled water. Inoculated and control plants were covered with plastic bags for the beginning 48 h and then kept in separate growth chambers at 25°C and 90% relative humidity. No disease symptoms developed on any control seedlings 30 days after inoculation. The wound- and non-wound-inoculated seedlings developed identical disease symptoms as observed on the naturally infected plants by the fourth observation day and died within 30 days of inoculation. The fungus was reisolated from these inoculated plants 10 days postinoculation, by using the abovementioned morphological and microscopic analyses. Fungal morphology was confirmed to be similar with the inoculation isolates, thus completing Koch’s postulates. To our knowledge, this is the first report of C. cochinchinensis leaf spot disease caused by N. oryzae in China.
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