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First Report of Gray Leaf Spot of Maize Caused by Cercospora zeina in China

Gray leaf spot of maize (Zea mays L.) is an important foliar disease in many parts of China. The causal organism of gray leaf spot in China is generally regarded as Cercospora zeae-maydis (3). In October 2011, symptoms similar to gray leaf spot were observed on 77% of maize plants in 25 locations (about 3,000 ha.) of Yunnan Province, China, and the disease could cause yield losses of 35 to 50%. The symptoms of leaf spot were different from those caused by C. zeae-maydis. The lesions on leaves were oblong, pale gray to pale brown, 2 to 3 × 5 to 40 mm, and confined by leaf veins that eventually coalesced. To identify the pathogen, 75 leaf samples were collected from 25 fields (three leaf samples for each field) at the kernel maturity stage. Single, well-separated lesions were excised and surface-sterilized by placing them in 75% ethanol for 5 s, then disinfested in 2% sodium hypochlorite for 5 min and rinsed with sterilized water. The lesions were incubated on water agar (WA) at 24°C for 48 to 72 h to allow sporulation. Seventy-five single-conidial isolates were obtained and cultured as described in Crous (1). Morphology of the isolates was determined on plates containing maize leaf powder agar (MLPA). After 5 days, isolates produced pale brown mycelia that consisted of 3- to 4-μm-wide, septate, branched hyphae. Conidiophores were 5 to 7 × 55 to 100 μm, straight to slightly flexuous, and usually 1- to 5-septate. Conidia were average 7.5 × 68 μm, fusiform, apex subobtuse, base subtruncate, and 3- to 6-septate. These characteristics are similar to C. zeina (2). The internal transcribed spacer (ITS) region of rDNA was amplified from each of the 75 isolates using primers ITS1/ITS4 and sequenced. The same sequences were obtained and the sequence of isolate YNGLS was submitted to GenBank (Accession No. KC878692). BLAST analysis of the sequence showed 100% confirmation to C. zeina (DQ185081). Additionally, a PCR-based diagnostic test using species-specific primers (2) confirmed the identification of the 75 isolates as C. zeina. The pathogenicity of the isolates was tested on greenhouse grown maize variety Huidan 4. The test was performed on 40 plants and replicated three times. The plants were inoculated at the 10 leaf stage by injecting 2 ml of conidial suspensions (2,500 conidia ml^–1) into leaf whorl using a hypodermic syringe, and control plants were injected with sterile water. Conidia were collected from 5-day-old cultures grown on MLPA and suspended in sterile water. Forty days after inoculation, all inoculated plants showed characteristic lesions on leaves, but control plants remained asymptomatic. C. zeina was reisolated from the lesions, and the identity of the reisolates was confirmed by the morphological and molecular characteristics as stated above. C. zeina was previously reported as the causal agent of maize gray leaf spot (2). To our knowledge, this is the first report of C. zeina causing gray leaf spot of maize in China.

References: (1) P. W. Crous. Mycologia Memoir. 21:1, 1998. (2) P. W. Crous et al. Stud. Mycol. 55:189, 2006. (3) C. H. Lu et al. J. Southwest China Normal Univ. 37:51, 2012.