First Report of Leaf Spot of Maize (Zea mays) Caused by Bipolaris setariae in China

Abstract

In September 2016, leaf spots were observed on maize (Zea mays L.) cultivar Tieyan 58 at the Zhangzhuang farm near TieLing in Liaoning province in China. Symptoms of early infection on leaves were small, water-soaked, translucent circular to oval spots that later enlarged, with the centers turning gray color. Spots were elliptic to round shaped with a chlorotic halo. Fifteen percent incidence was observed in this farm. The incidence of this disease ranged from 5 to 20% in 15 counties of Liaoning province in 2017. Yield losses of maize attributed to leaf spot varied from 4 to 11%, with estimated losses as high as 30% when severe epidemics occurred. The causal agent was recovered from symptomatic leaf samples using standard phytopathological procedure. Leaf pieces were placed on potato dextrose agar (PDA) plates that were incubated at 24 to 26°C in the dark for 5 to 6 days. Single spores of six colonies were transferred to fresh PDA and incubated in the dark for 7 to 8 days for morphological identification. Colony margins were irregular to circular and were gray to black in color on PDA. Conidiophores were single or in clusters, geniculate, slightly flexuous or straight, and two- or three-septate. Conidia were slightly curved, truncated, spindle-shaped to fusiform, dark olive, four- to 10-septate, and 8 to 16 × 37 to 106 µm in size, with a slightly protuberant hilum. Conidia with a distinct hilum are the characteristic of Bipolaris maydis. Based on these cultural and morphological characteristics, the fungus was identified as Bipolaris setariae (Saw.) Shoem. (da Cunha et al. 2012). To confirm the morphological identification, mycelia were scraped from PDA plates, and genomic DNA was isolated by kit (Aidlab Biotech, Beijing, China). The internal transcribed spacer regions (ITS1-5.8S-ITS2) and partial translation elongation factor 1-alpha (TEF1-alpha) gene of six isolates were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The PCR products were cloned into pMD-19T Cloning Vector (Sangon Biotech, Shanghai, China). The clones were purified with the TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China) to get the full-length ITS sequence and partial TEF1-alpha sequence. BLAST analysis indicated 100% nucleotide sequence identity with B. setariae for six isolates (GenBank accession nos. CBS141.31 and EF452444). Sequences were submitted to GenBank (accession nos. MK002698 and MK439841). Pathogenicity of isolates was tested by spraying conidial suspension of six isolates (1 × 10⁶ conidia/ml). Leaves of two maize plants, hybrid Tieyan 58 (seven/eight-leaf stage) were inoculated for one isolate. Twelve plants sprayed with sterile water served as the noninoculated controls. Plants were maintained at 25 to 27°C in the greenhouse for 10 to 12 days. Small, translucent circular to oval lesions appeared in all inoculated plants. Lesions were similar to leaf spots found in the field, whereas noninoculated control plants were asymptomatic. According to Zhang’s method, disease intensity index ranged 31.5 to 33.3 for six isolates, with no significant difference (Zhang et al. 2019). Koch’s postulates were fulfilled by reisolation of B. setariae from inoculated plants. This fungus was previously described as a pathogen of coconut in China (Niu et al. 2014), but this is the first record of B. setariae on maize in China.