First Report of Leaf Spot Disease on Fagopyrum esculentum Caused by Bipolaris zeae in China.

Abstract

Common buckwheat (Fagopyrum esculentum Moench), a dicotyledonous plant in family Polygonaceae, is recognized as a valuable nutritional source of fatty acids, phytosterols, phenolic compounds and tocopherols. It has received increased attention as a so-called "functional food" in China. During scouting of common buckwheat in August and September 2018, unfamiliar symptoms were observed on leaves in 20 fields in Yanchi County, Ningxia, China, with 35% incidence and moderate to high severity across the field. Brown spots most commonly occurred on lower leaves of buckwheat beginning in late July. The spots were initially light brown with an irregular border and pale brown center. Older spots were almost dark brown, and often coalesced although spots were restricted by veins. Symptomatic leaf samples were collected in late-August, and washed with flowing water for 2 min. Tissue samples were excised from the margins of the lesions and sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, before being rinsed with sterilized water four times, dried on sterile paper towels, and cultured on Potato Dextrose Agar medium at 20 °C. After 48 to 72 h, hyphae grew from tissue pieces. The obtained isolates were purified using the single-spore isolation technique. A total of thirteen isolates were recovered and characterized. Colonies of all isolates on PDA were identical, and were pale brown to brown, velvety, with irregular borders. Hyphae were hyaline to brown, branched, septate, smooth, sometimes verruculose, 3-7 μm wide. Conidiophores were (83-297) µm×(5.5-9) µm (avg. 205 µm×8 µm, n=30), dark brown, cylindrical, straight to slightly flexuous, geniculate at the upper end, septate, single, smooth. Conidia were rostrate, obclavate, straight to slightly curved, hyaline to pale brown when immature and brown to reddish brown when mature, and measured (38-104) µm×(11-19) µm (avg. 67 µm×16 µm, n=50). End cells were often paler than middle cells and with a thick dark hilum. Based on the cultural and morphological characteristic, these isolates are preliminary identified as Bipolaris zeae (Manamgoda et al., 2014; Sivanesan, 1985). Genomic DNA of the representative isolate qyj-5A was extracted and amplified using GAPDH primers (gpd 1/2) and ITS primers (ITS 1/4) respectively. The sequence of the amplicons was compared with reference sequences. The ITS sequence (GenBank: MT645704) showed 100% (571/571bp, 564/564bp) identity with B. zeae (GenBank: KU356179 and KU571464, respectively). The GAPDH sequence (GenBank: MW426530) also showed 100% (594/594bp) identity values with B. zeae (GenBank: MF415650 and KU571468). Pathogenicity tests were performed on three healthy and asymptomic buckwheat plants, which were surface sterilized with 75% ethanol and rinsed with sterilized distilled water. The leaves were sprayed with 1×105 conidia/ml of the suspensions which contain 0.01% Tween 20 and three control plants sprayed with same volume of sterile distilled water. A strip of parafilm was wrapped around the inoculated leaves for 48 h to maintain high relative humidity. At 6 days postinoculation, all the inoculated leaves showed symptoms identical to those described above. While no symptoms were observed on the control plants. The fungus was reisolated and identified as B. zeae based on morphological features and DNA sequence analysis, it was identical to the original isolate to satisfy Koch's postulates. B. zeae has been reported to be pathogenic on Acer truncatum (Sun et al., 2011), Helianthus tuberosus (Zhao et al., 2017) and Hemarthria altissima (Xue et al., 2016) in China. To our knowledge this is the first report of B. zeae causing leaf spot on F. esculentum in China. This fungal pathogen represents a severe threat and has the potential to cause yield losses of F. esculentum, so further research is required to define effective management strategies.