First Report of Corn Kernel Brown Spot Disease Caused by Mucor irregularis in China.

Abstract

In October 2012, a brown spot disease was found on corn kernels during a field survey in Nanyang city (33°01' N, 112°29' E), China. The incidences of affected ears and kernels were 2 to 10% (n = 600) and 0.08 to 0.4% (n = 25,000), respectively. Symptoms first appeared as circular or irregular brown spots on the endosperm. These spots subsequently enlarged or coalesced, resulting in the formation of a large light-brown or light-yellow irregular speckle commonly surrounded by a dark-brown edge. Pure fungal cultures with similar morphological characteristics were obtained from surface-disinfected symptomatic kernels using a conventional method for isolation of culturable microbes. The isolated fungal cultures were purified by single-spore isolation (3). A representative isolate F1 was randomly selected, used for pathogenicity tests, and identified using morphological and molecular methods. Colonies on PDA were circular with abundant villiform aerial mycelia. The color of colonies was white-gray at first and turned to light yellow or became ochraceous after 3 days of incubation at 28°C. Hyphae were hyaline and less septate, with rectangular branches. Sporangiophores were erect and unbranched or branched, with globose sporangia formed on their tips. Sporangiospores were elliptical to round, 3.6 to 7.3 × 1.6 to 3.7 μm (n = 100) in size. Two gene regions were amplified for multilocus sequence typing. The D1/D2 region of the nuclear large subunit ribosomal RNA gene (nucLSU) was amplified with primers NL1 and NL4 and the rDNA internal transcribed spacer (ITS) with primers ITS1 and ITS4. PCR products were purified using an Axygen nucleic acid purification kit for sequencing. Both rDNA D1/D2 and rDNA-ITS sequences were submitted to GenBank with accession numbers KM093834 and KM203872, respectively. The isolate F1 showed 98% identity with two isolates of Mucor irregularis (KC524427 and KC461926) in rDNA-ITS sequences and 99% identity with multiple isolates (JX976221, JX976203, and JX976219) of M. irregularis in rDNA D1/D2 sequences. Pathogenicity tests of isolate F1 were conducted based on Koch's postulates. Thirty kernels of fresh ears (milk stage) were pricked by sterilized toothpicks and separately inoculated with a sporangiospore suspension (1 × 106 spores/ml) and 5-day-old mycelial plugs (5 × 5 mm) of isolate F1. Kernels on ears that were inoculated with sterilized water and pure PDA plugs were separately used as controls. After 7 days of incubation, brown spot symptoms developed on the F1-inoculated kernels, which were similar to those observed on the naturally infected ears from the field samples. The control ears remained symptomless during the inoculation tests. Fungal cultures showing the same morphological characteristics as those of isolate F1 were consistently recovered from the diseased cobs inoculated by isolate F1, indicating that M. irregularis was responsible for corn kernel brown spot disease. M. irregularis was reported as a pathogen causing human skin diseases in China (5), America (1), and India (2) and as a phytopathogen causing fruit rot on durian (4). This is the first report of M. irregularis causing corn kernel brown spot disease in China.