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Identification of Colletotrichum liriopes as the Causative Agent of Anthracnose in Buckwheat (Fagopyrum esculentum) in China.

Buckwheat (Fagopyrum esculentum), belonging to the Polygonaceae family, is one of the most important "functional food" crops in China. In fall 2020, buckwheat plants grown in field exhibiting stem canker symptoms were found in Tongxin county, Ningxia province, China. Symptoms included stem canker, dieback and extensive vascular discoloration. Cankers were bleached, silvery-white to dark gray, slightly sunken, oval to linear with slightly tapered tips, pycnidia formation was also observed within the cankers. Disease incidence was approximately 30% and moderate to high severity across the field. Symptomatic tissues were cut into 1-2 cm pieces, surface sterilized (75% ethanol for 30 s and 0.1% NaClO for 2 min) and washed four times with sterile distilled water, dried in sterile filter paper for 3 times, and placed on potato dextrose agar (PDA) at 25 ℃. Fluffy mycelium was visible for all isolates after 48 h of incubation. Twenty-five single isolates were hyphal-tip purified on PDA. Six representative isolates were used for further study. The fungal colonies on PDA were flat with an entire margin, gray aerial hyphae, light brown pigmentation, appressed slimy mycelium within which numerous brown-black perithecia formed. Colonies on oatmeal agar (OA) were flat, with flocculent mycelium, conidiomata and conidia and the reverse side was black to smoke-grey. Sparse brown-black perithecia were observed within the mycelium. Conidia were hyaline, one-celled, smooth-walled, rarely finely verruculose, aseptate, slightly curved, both sides gradually tapering towards the round to slightly acute apex and truncate base, measured (15.7-23.7) µm (length) × (2.8-5.7) µm (width), (avg. 20.2 µm×4.2 µm, n=100). Genomic DNA was extracted from the same six isolates, the internal transcribed spacer (ITS) region and the genes encoding beta-tubulin (TUB), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 (HIS3) were independently amplified with primers V9G/ITS4, T1/Bt-2b, CHS-354R/CHS-79F, GDF1/GDR1 and CYLH3F/CYLH3R, respectively (Damm et al., 2019). Sequences for all six isolates were identical. The sequences of the representative isolate 9J1 were deposited in GenBank (accession nos. MW819604, MW836580, MW836577, MW836578 and MW836579). The results of BLASTn showed that the ITS, TUB, CHS, GAPDH and HIS3 sequences of 9J1 were greater than 98% (555/557bp, 477/486bp, 258/259bp, 242/248bp and 339/345bp, respectively) identical to those of Colletotrichum liriopes (GenBank: MT645674 (ITS), GU228098 (TUB), MT663546 (CHS), MH291255 (GAPDH) and MH292811 (HIS3)). MrBayes phylogenetic analysis using concatenated sequences of ITS, TUB, CHS, GAPDH and HIS3 showed that the isolate clustered monophyletically with strains of C. liriopes. Based on morphological and molecular characteristics, the isolate was identified as C. liriopes. To fulfill Koch's postulates, spores of the isolate 9J1 grown on OA for 14 days were harvested in 0.01% Tween 20, and the suspension were adjusted to 104 spores/ml. Six one-month-old potted plants of buckwheat were inoculated by spraying the spore suspension until run-off. Plants were kept inside a plastic bag for 3 d to maintain high relative humidity and maintained in the greenhouse. Six control plants were sprayed with sterile deionized water and kept under the same conditions. Two weeks after inoculation, all inoculated plants showed stem canker symptoms as described above, whereas control plants remained healthy. The pathogen was successfully reisolated from leaf and stem symptomatic tissue, and identified as C. liriopes based on morphological features and DNA sequence analysis, thereby fulfilling Koch's postulates. C. liriopes has been reported causing anthracnose on Eria coronaria (Yang et al., 2011), Liriope spicata (Chen et al., 2019) in China, Liriope muscari in Mexico (Damm et al., 2009), Rohdea japonica in Korea (Kwon and Kim, 2013) and in the United States (Trigiano et al., 2018). To our knowledge, this is the first report of C. liriopes causing anthracnose on buckwheat worldwide. Occurrence of this disease may represent a significant impact for buckwheat production because this crop is the major agricultural commodity in some parts of China. More studies are needed to understand the epidemiology of this disease and foster disease management programs in China.