First Report of Epicoccum sorghinum Causing Leaf Sheath and Leaf Spot on Maize in China.

Abstract

In November 2020, leaf sheath on maize (Zea mays) was detected in southeastern Jiangsu (Nantong municipality; 120.54° E, 31.58° N) in China. Physiologically mature plants, 13 weeks of cultivation (at the harvest stage), exhibited red-brown lesions in stem and leaves, and dried-up stem (Figure 1). The symptoms were observed on approximately 95% of the maize plants in a 0.8 ha maize field surrounded by old sorghum fields and the crop yield was decreased by 70-85% with respect previous years, when no disease symptoms were detected. Small pieces, approximately 0.3 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated (one isolate was obtained) and cultured on PDA medium, containing chloramphenicol (50 µg/mL), under darkness at 26 ºC for 3 days. Amplification of internal transcribed spacer (ITS), large subunit (LSU), actin (ACT) and β-tubulin (TUB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R and T1/Bt2b primers, respectively (Ma et al. 2021). Sequences were submitted to GenBank under accession numbers MW800180 (ITS), MW800361 (LSU), MW845677 (ACT) and MW892439 (TUB2). Blast search revealed that the ITS sequence had 100% (492/492 bp) homology with E. sorghinum LY-D-1-1, MT604999, LSU had 98% (1075/1091 bp) homology with E. sorghinum GZDS2018BXT010, MK516207, ACT had 96% (214/222 bp) homology with E. sorghinum M3, MK044832, and TUB2 had 99% (498/499 bp) homology with E. sorghinum BJ-F1, MF987525. Molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest likelihood (-1774.9882) is shown in Figure 2. Bipolaris, Curvularia and Fusarium spp. found causing leaf spot on maize were included in the phylogenetic tree (Liu et al. 2021; Reyes Gaige et al. 2020; Chang et al. 2016). To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the stem and leaves of 2-week old maize plants. A solution containing 1 × 108 spores/mL (20 µL) was injected in the wound, whereas sterilized ddH2O was used in the control experiment. Inoculated plants were maintained in a growth chamber at 28 °C and 60% relative humidity for 3 days, observing fast-growing necrotic lesions in both stem and leaves. The pathogen was recovered from the infected plants and its identity was confirmed by morphological and sequence analyses. Microscope observations indicated the presence of chlamydospores, oval conidia (3 × 5 µm) and round pycnidia (60-100 µm diameter), and agree with those previously reported for the morphology of E. sorghinum (Bao et al. 2019). During last 2 years, E. sorghinum was reported to cause leaf spot on a number of relevant agricultural crops in China, including taro, Brassica parachinensis, tea, rice and wheat (Du et al. 2020; Li et al. 2020; Liu et al. 2020a, 2020b), confirming the expansion and host promiscuity of this pathogen. The pathogen was also reported to cause leaf spot on maize in Brazil in 2004 (Do Amaral et al. 2004); however, this is the first report of E. sorghinum causing leaf sheath and leaf spot on maize in China. Maize an important agricultural crop in China with more than 168 million tons produced in 2019. The observed yield loss and disease incidence of the isolated strain suggest that E. sorghinum may be a threat to maize production in China.