First Report of Alternaria alternata Causing Leaf Spot of Tartary Buckwheat (Fagopyrum tataricum) in China.

Buckwheat (Fagopyrum tataricum) is recognized as a healthy food with abundant nutrients and high levels of rutin. In April and May of 2020, an unknown tartary buckwheat leaf spot distinct from Nigrospora leaf spot (Shen et al. 2020) was observed in Xiangxiang, Hunan, China (27°49'54″N, 112°span style="font-family:'Times New Roman'; color:#0000ff">18'48″E.). Disease incidence was 60-70% within three fields (totally 7, 000 m2). The disease occurred after plants emerged. Initial symptoms began as circular, or ellipsoid, chlorotic, water-soaked spots, mostly on leaf apexes or leaf margins. The small spots gradually enlarged and often coalesced to form large circular or irregular, pale to light brown lesions, and the infected leaves eventually withered and fell off. Thirty 2 × 2 mm infected tissue pieces collected from five locations were sterilized in 70% ethanol for 10 S, in 2% NaClO for 30 S, rinsed in sterile water for three times, dried, and placed on PDA with lactic acid (3 ml/L). After 3-5 days at 28°C in the dark, 17 fungal isolates were purified using single-spore isolation method. Almost all fungal isolates had similar morphology. Colonies were initially olive green with white margin and later turned dark olive or black with profuse sporulation. Conidia were borne in long chains, tawny to brownish green, with 1-3 longitudinal and 1-7 transverse septa, pyriform, and measured 9.5-39.6 µm long, and 5.1-12.6 µm wide (n=50). Based on morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007). Partial internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α(TEF) and Alternaria major allergen (Alt a1) genes of isolate BLS-1 were amplified using ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF1-986R (Carbone and Kohn 1999), Gpd1/Gpd2 and Alt-4for/Alt-4rev (Lawrence et al. 2013), respectively. Sequences were deposited into GenBank with acc. nos MW453091 (ITS), MW480219 (GAPDH), MW480218 (TEF), and MW480220 (Alt a1). BLASTn analysis showed 99.8% (ITS, MH854758.1), 100% (GAPDH, KP124155.1), 99.8% (TEF, KP125073.1) and 100% (Alt a1, KP123847.1) identity with reference strain CBS 106.24 of A. alternata, confirming isolate BSL-1 to be A. alternata. A neighbor-joining phylogenetic tree constructed by MEGA7.0 based on concatenated sequences of the four genes indicated that BSL-1 formed a distinct clade with A. alternata CBS 106.24 with 100% bootstrap values. Pathogenicity test was triplicately performed on healthy leaves. Twenty leaves of five 20-day-old plants (cv. Pinku1) were sprayed with conidial suspension (1×106 conidia/ml) collected from PDA cultures with 0.05% Tween 20. An equal number of control leaves were sprayed with sterile water to serve as the controls. Treated plants were kept in a greenhouse at 28±3 °C with relative humidity of 80±5% for 24 h and transferred to natural conditions (22-30°C, RH 50-60%). After 4 to 6 days, all inoculated leaves developed symptoms similar to those observed in the fields, while the control leaves remained healthy. A. alternata was re-isolated from all infected leaves. Occasionally-isolated Diaporthe isolates were not pathogenic. A. alternata causes leaf spot of oat (Zhao et al. 2020) and leaf blight of F. esculentum (Lu et al. 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on F. tataricum in China and the world. Effective strategies should be developed to manage the disease.