First Report of Leaf Spot Caused by Ramularia sphaeroidea on Vicia villosa var. glabrescens in China.

Abstract

Hairy vetch Vicia villosa Roth is widely grown in southwestern China for green manure and forage. In December 2019, a leaf disease occurred on 80% plants of V. villosa var. glabrescens in an eight-hectare field in Qujing(N 25°28'12″, E 103°36'22″), Yunnan Province, China. The disease leaves had irregular, brown to dark brown leaf spots with white mold. Twenty diseased leaves from five plants were randomly collected from the field. The leaf samples were sterilized with 75% ethanol for 30 s and 1% NaClO for 75 s, rinsed three times with sterile distilled water, surface water removed with sterile filter paper, and placed onto potato dextrose agar (PDA) for culture at 20oC. The obtained fungal isolates were purified by transferring 1 to 2 mm hyphal tips onto fresh PDA plates and cultured under the same temperature condition. The isolates grew slowly, at a rate of 0.7 mm/d at 20℃ for 4 weeks. A diseased plant specimen (accession MHLZU19326) and three isolates (accessions YN1931401, YN1931402, and YN1931403) were deposited in the Mycological Herbarium of Lanzhou University (MHLZU). Conidia from the PDA cultures were hyaline, spherical, smooth, aseptate, and measured 2.13 to 3.67 × 4.56 to 5.77 μm (n = 50). Conidiophores were hyaline, smooth, and straight. DNA of purified isolates was extracted and the nuclear ribosomal internal transcribed spacer (ITS), tef1-α, his3 and gapdh genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF2 (Carbone and Kohn 1999;O'Donnell et al. 1998), CylH3F/CylH3R (Crous et al. 2004), and gpd1/gpd2 (Berbee et al. 1999), respectively. DNA sequences of isolates YN1931401, YN1931402, and YN1931403 were deposited in GenBank for the ITS (accessions MW092181, MW332205, and MW332206), tef1-α (MW448172 to MW448174), his3 (MW448175 to MW448177), and gapdh (MW448178 to MW448180). These sequences had the highest similarities with sequences of Ramularia sphaeroidea Sacc. in GenBank, 99%(514∕516, 515∕517, and 514∕517 bp) for ITS, 99% (402∕403, 403∕405, and 405∕405bp) for tef1-α, 99% (377∕378, 378∕378, and 376∕378bp) for his3, and 100% (558∕557, 557∕559 and 561∕565 bp) for gapdh . A phylogenetic tree generated with the sequences clustered the fungus closely with R. sphaeroidea. Infection experiments were carried out with 50 plants of V. villosa var. glabrescens in 10 pots. A conidial suspension of 1. 0 × 106 conidia/ml with 0.01% Tween 80 was prepared by adding sterile distilled water to the YN1931401 culture and scraping with a sterile scalpel. The leaves of 25 healthy plants were sprayed with the conidial suspension, and those of the 25 control plants were sprayed with sterile water. All plants were covered with clear polyethylene bags for 3 days to maintain high humidity and then grown in a greenhouse at diurnal cycles of 18℃ for 18h with light and 22℃ for 6 h in dark. Ten days post-inoculation, the inoculated plants exhibited brown lesions similar to the symptoms observed in the field (Fig. 1-F), whereas no symptoms appeared on the control plants. The same fungus was re-isolated and identified as described above. R. sphaeroidea has been reported on V. fabae and V. sativa in Ethiopia and Israel (Braun 1998), on various Vicia species including V. villosa in California, the United States (Koike et al. 2004) and on V. craccain China (Zhang et al. 2006), but to our knowledge, this is the first report of this fungus causing leaf spot on V. villosa in China.