First Report of Alternaria alternata Causing Leaf Spot on Mungbean (Vigna radiata) in the Republic of Korea

Abstract

Mungbean, Vigna radiata (L.) R. Wilczek, is an important legume crop in many countries, providing protein and other essential nutrients. In October 2017, at the Seoul National University Suwon Experimental Farm (Suwon, Republic of Korea), we observed several individual plants across various mungbean accessions with dark-brown leaf spots. Spots were most prominent on the underside of the leaf and were circular to irregular in shape. On average, the diameter of spots was approximately 2 mm. A standard tissue isolation method was used, and a total of seven fungal isolates were made. The produced conidia were beaked or beakless, had transverse and sometimes latitudinal septa, ellipsoid or obclavate, with an average width of 11 µm and an average length of 22 µm, similar to the description of Alternaria alternata by Woudenberg et al. (2015). Genomic DNA of the fungi from two out of seven isolates was extracted using GeneAll Exgene Plant SV mini (Seoul, Republic of Korea). Using each DNA as a template, we amplified the internal transcribed ribosomal spacer region (ITS) and the 28s large subunit ribosomal RNA D1/D2 region using ITS1/ITS4 primers (White et al. 1990) and NL1/NL4 primers (Reynolds and Taylor 1993), respectively. BLAST analysis of these amplicon sequences showed 100% identity match to those of A. alternata, and the sequence was deposited in GenBank (no. MH633697-700). Furthermore, we compared ITS and D1/D2 sequences to the A. alternata reference sequence from Woudenberg et al. (2015) by aligning the sequences using ClustalW (BioEdit, version 7.0.5.3). Both of the isolates had >99.99% sequence identity with the reference. Based on both sequencing and morphological information, we concluded that the isolates were A. alternata. To fulfill Koch’s postulates, we carried out both in vivo and in vitro inoculation tests. For in vitro test, we harvested the conidia by flooding the fungal plate with 0.1% Tween 20. The concentration of spores was adjusted to 1 × 10⁵ conidia/ml by further diluting with 0.1% Tween 20. Detached leaves from 3-week-old mungbean plants were sprayed with the conidia suspension until there was surface run-off, and the leaves were placed in a humidity chamber. The control leaves were sprayed with 0.1% Tween 20. After 5 days, we observed brown spots on the surface of the leaves, but no symptoms appeared on the control plants. For the in vivo test, 1-month-old potted mungbean plants were inoculated with conidia suspended in sterile distilled water adjusted to 1 × 10⁴ conidia/ml. Each plant was sprayed with 5 ml of solution. The in vivo test showed a similar symptom to the in vitro test, producing brown leaf spots. From the brown leaf spots, we again isolated the fungi and sequenced the 28s large subunit ribosomal RNA D1/D2 region. The sequencing result showed 100% similarity of the reisolate with the original inoculum, fulfilling Koch’s postulates. To our knowledge, this is the first report that A. alternata causes leaf spot disease in mungbean in Korea. This report helps to develop mungbean cultivars resistant to biotic stresses affecting mungbean yield.