First Report of Botryosphaeria dothidea Causing Gray Mold on Tartary Buckwheat in Southwest China.

Tartary buckwheat (Fagopyrum tataricum, Polygonaceae) is an annual plant originating in Southwest China. It has a short growth cycle, barren soil tolerance, and strong stress resistance (Zhang et al. 2021). Because of its high content of proteins, starch, trace elements, phenols, and dietary fiber, Tartary buckwheat is beneficial to the human body and hence has received widespread attention (Joshi et al. 2019; Dc ja, B, et al. 2020). In the period from September to November 2020, a diseased plant infected with gray mold was found among M2 generation plants treated using ethyl methanesulfonate (EMS) in a location with potted Tartary buckwheat plants in Huaxi District, Guiyang City, Guizhou Province, China. The diseased plant started to show symptoms during the initial flowering stage; water-soaked spots appeared at first, that the spots increased in size and turned into light brown patches, with the leaf edges scorched brown. In severe cases, the leaves turned yellow, the diseased spots became dry, and finally the leaves necrotic (Figure 1A). Among the leaves that showed disease symptoms, severely susceptible leaves were selected; a piece of tissue (2×2 mm) was removed at the junction of the diseased and healthy tissues. The tissue was then soaked in 75% ethanol for 2 to 3 s, transferred to 1% sodium hypochlorite solution and soaked for 3 min, rinsed three times with sterile water, and placed on sterilized filter paper to dry. Sterile tweezers were used to transfer the tissue blocks to Potato Dextrose Agar medium (Bio-Rad Ltd. Com, USA) containing a Streptomyces-Penicillium mixture (100 µg/mL), and they were incubated on this medium for 7 to 10 days at 25°C and 70% humidity under 16 h light and 8 h dark conditions. The colonies were white at the early stages, with developed aerial hyphae; subsequently, they gradually turned gray-green (Figure 1B). In the later stages, the back of the colony was black and piles of conidia could be seen (Figure 1C). The conidia are scattered, which were colorless and transparent, fusiform or fusiform, with a size of 8.02-11.13 µm×2.06-3.22 µm (average=9.51 µm×2.69 µm, n=50) (Figure 1D). Based on their morphological characteristics, These cultural and morphological characteristics were consistent with the descriptions of as B. dothidea (Fan et al. 2021). The ITS1/ITS4 (Mills et al. 1992), Bt-2a/Bt-2b primers (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Slippers et al. 2004) were amplified and sequenced to analyze the ITS region, ß-tubulin genes translation elongation factor 1-α (TEF1-α), and translation elongation factor 1-α (TEF1-α), respectively. According to BLAST search in GenBank, the sequences of ITS (MZ326853), TUB2 (MZ399162) and TEF1-α (MZ399163) had 99.40%, 100% and 100% similarity to sequences NR111146.1, AY236927.1, and AY236898.1 of B. dothidea ex-type strain CMW8000, respectively. The three nucleotide sequences were concatenated together, and MEGA-X (with the neighbor-joining method) with 1,000 bootstraps was used to construct a phylogenetic tree. The results showed that our isolate was closely related to B. dothidea (Figure 2). Healthy Tartary buckwheat from the M2 generation was used for the pathogenicity test. Disinfect with 75% alcohol and 1×105 mL-1 of spore suspension was sprayed on the leaves. Each treatment included three plants, and it was repeated three times with sterile water as control. The treatments were kept in a houseat25°C for 24 h, then transferred it to the natural environment of 22℃ to 28℃,and sterile water was sprayed every morning and evening to keep the leaves moist. After 10 days, the symptoms seen in the field appeared on the treated plants (Figure 1E), but the control plants did not show any symptoms (Figure 1F). The diseased parts of the leaves were isolated and cultured again, and the isolates were consistent with the original inoculum. Thus, the study conformed to Koch's postulates. B. dothidea is a fungus with no host preference in the genus Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales). It can cause canker, leaf spots, trunk diseases, fruit rot and die-back of many important wood plants all over the world (Marsberg et al.2017). Recently, it was reported that B. dothidea caused soybean canker in China (Chen et al.2021), but there have been no reports of B. dothidea causing Tartary buckwheat gray mold. To the best of our knowledge, this is the first report of B. dothidea causing gray mold on Tartary buckwheat. This finding will provide a basis for the prevention and treatment of Tartary buckwheat gray mold.