First Report of Lasiodiplodia theobromae Causing Maize Ear Rot in Hainan Province in Southern China

Maize (Zea mays L.) is an important food and feed crop worldwide. In February 2015, about 4% of maize ears with black gray mold was observed in seed fields at Ledong, Hainan Province, China (18°29′N, 108°54′E). Samples of the symptomatic black gray-molded maize ears were collected for analysis. Molded kernels were surface-disinfested with 75% ethanol and 10% sodium hypochlorite, rinsed three times with sterile distilled water, and then plated onto potato dextrose agar (PDA). After 1 day of incubation at 25°C in the dark, mycelia growing from kernels were transferred to fresh PDA plates. Species were identified based on morphological characteristics (Alves et al. 2008; Phillips et al. 2013), and sequence analysis of the internal transcribed spacer (ITS) region and the translation elongation factor 1-alpha (TEF-1α) gene regions. On PDA, a 9 cm petri dish was covered with fast-growing mycelium after 2 days. Young colonies were gray white, rotund or subcircular, and mycelium showed sprawled growth; gradually colonies turned gray brown to gray black, villiform; cultures showed a dark underside as the colonies changed from greenish to black. Primary hyphae were hyaline and aseptate, whereas mature hyphae were septate with irregular branches. After 32 days, colonies produced black brown globose pycnidia; paraphyses among the conidia were hyaline, cylindrical, and septate. Conidia were hyaline, single-spore, elliptical, apex rounded, truncated at the base, and thin walled when immature, averaged 18.41 × 8.20 μm, becoming dark brown to black, didynoporae, thick walled, and longitudinally striate when mature, averaged 24.73 × 15.19 μm. The morphology of the fungus was similar to Lasiodiplodia theobromae (Pat.) Griff. & Maubl. To confirm the identity of the isolates, the ITS region of ribosomal DNA was amplified with the universal primers ITS1/ITS4 (White et al. 1990) and the TEF-1α gene regions was amplified with the universal primers EF1-728F/EF1-986R (Carbone and Kohn 1999). BLASTn searches at GenBank showed a high identity with reference sequences of ex-type culture (Zhu et al. 2014) for L. theobromae (isolate ML: ITS, 100%; TEF-1α, 100%). Sequences of two gene regions were deposited as GenBank accessions KT445902 (ITS) and KT985635 (TEF-1α). Koch’s postulates were completed by using ears of maize cultivar Prestige growing in the experimental field of Hebei Province (median temperature 30.94°C/22.09°C, July). Each of the six maize ears was injected with 2 ml conidial suspension of the isolate through the silk channel. Control groups were inoculated with sterile distilled water. About 10 days later, all maize kernels on the top of the inoculated corn ears showed punctiform discoloration. After 35 days, the seed coat of all inoculated kernels was white or showed white stripes radiating out from a central point when the infection was mild. The kernel became black and mildewed, the seed dehisced, and the endosperm was black. The cob was also infected, becoming gray black when the infection was severe. When air humidity was high, the surface of the focus was covered with gray mold layer. The pathogen could also infect the bract directly. Meanwhile, the water control ears did not exhibit any disease symptoms. The same fungus was successfully reisolated from the inoculated kernels. L. theobromae has been reported to cause cankers in a wide range of hosts, such as fruit and vegetable crops (Alves et al. 2008), and it is also reported to attack maize in India and Cuba. To our knowledge, this is the first report of L. theobromae causing ear rot on maize in China. Therefore, it is necessary to strengthen quarantine measures against spread of the pathogen from Hainan to the mainland through seed-breeding. Also, significant research efforts are needed to develop disease-resistant varieties, as this is the best option to combat the pathogen. © 2016, American Phytopathological Society. All rights reserved.