First Report of Leaf Spot Disease on Rice Caused by Epicoccum sorghinum in China

Rice is being threatened by many fungal diseases, of which rice blast (RB) and brown leaf spot (BLS) are the two most important rice diseases. Recently, a leaf spot disease similar to RB and BLS was observed on leaf samples collected from Dingyuan, Anhui province. During August to September 2019, 200 plants and 800 leaves of each variety were assessed on seven varieties (Xiushui134, Nanjing46, Jiahe218, Zhongzheyou8, Yongyou12, Yongyou15, and Yongyou1540), and the disease incidence ranged from 9.5 to 69% (plant) and 8.63 to 41.38% (leaf). The symptoms appeared as small chlorotic spots with yellow halos and gray to black centers. Lesions on the older leaves enlarged and coalesced into bigger spots. The infections started from lower leaves and progressed not as rapidly as RB; therefore, lower leaves were badly damaged, whereas flag leaves mostly became slightly infected with several spots. Twenty-one samples were surface sterilized and cultured on water agar at 28°C for 24 h. Among 25 isolations, 19 showed same cultural characters. Morphological analysis was performed following published work (Zeng et al. 2018). The fungal colonies were villose and regular, gray at the center and pale at the margin, and finally turned dark gray, and the back turned scarlet. Chlamydospores, unicellular or multicellular, massively produced. Pycnidia, brown and mostly spheroid, were 67.9 to 117.5 × 44.5 to 90.1 µm, and conidia, unicellular, hyaline, oval, were 3.6 to 5.3 × 1.8 to 2.8 µm. The genes of the fungal pathogen (LSU, ITS, tub2, and rpb2) were amplified, sequenced, and multilocus phylogenetic analyzed according to described procedures (Chen et al. 2015), and the sequences were submitted to GenBank with accession numbers MT125855, MT125854, MT127630, and MN562463, respectively. The multilocus phylogenetic analysis showed the isolated fungus was the closest to Epicoccum sorghinum. In view of both morphological and molecular characteristics, the fungus strains were finally identified as E. sorghinum. The pathogenicity test was repeated three times in both mycelial and conidial inoculation ways. Rice plants at filling stage were inoculated with mycelial plugs and conidial suspension (1 × 10⁶ conidia/ml) separately. In each time, five healthy plants (three for inoculation, two for control) were used. Ten leaves of each plant were wounded and then pinned with a 0.5-cm PDA culture plug or dosed with 6 μl of conidia suspension. The inoculated areas were kept moist with soaked sterilized cotton during the study, and environmental humility was maintained at >80%. After being cultured in the dark for 36 to 48 h at 26°C, these plants were put under a 12-h light/dark cycle. Typical symptoms described in this study appeared on more than 93% of mycelium-inoculated leaves and more than 80% of conidium-inoculated leaves after 7 to 10 days postinoculation. E. sorghinum was consistently reisolated from leaves with symptoms, and hence E. sorghinum was confirmed to cause leaf spot disease on rice. To our knowledge, this is the first report of leaf spot disease on rice caused by E. sorghinum in China. The disease is not expected to cause greater yield losses based on our study. However, the symptoms could be easily mistaken for RB and incur unnecessary chemical sprays.