Sweet corn (Zea mays L.) is widely consumed as fresh or frozen vegetable worldwide, and Zhengtian68 is a popular commercial variety cultivated extensively in southeast China. In May 2021, 40% of the inbred line YK063 (the female parent of Zhengtian68) showed early yellowing of the leaves at flowering time in a commercial seed production field with a total area of 0.5 ha in Guangzhou, Guangdong Province after a heavy rain. Black and rotten roots were observed in the diseased plants after digging the whole plant out of the soil. Grain filling was also severely affected, adversely impacting seed production. Diseased plants were more easily found in the lower section of the field, where water accumulated after rainfall. Three plants with rotten roots were collected randomly from the field to identify the causal pathogen. The diseased roots were cut into 2-3 mm sections, washed in 75% ethanol for 2 minutes and rinsed three times in sterile distilled water. Four to five sections per plant were placed on potato dextrose agar (PDA) and incubated at 28℃ in the dark for three days. Three isolates GF1, GF2, and GF3 from different plants were purified by hyphal tip isolation and transferred to new PDA and 10% V8 juice agar (16 g agar, 3 g CaCO3, 100 ml V8 juice, and 900 ml distilled water) and incubated at 28℃ for 10 days in darkness for further investigation. Translucent, glassy mycelial growth was observed on the PDA media. Morphological characteristics of the 3 isolates were observed under a microscope from the 10%V8 media. The hyphae were aseptate and 2.7 to 4.5 μm wide (mean±SD,3.3±0.44μm, n=44). Sporangia were inflated, or lobulate, terminal, or intercalary. Oogonia were globose, smooth-walled, terminal, or occasionally intercalary, with a diameter of 17.2-24.1 μm (mean±SD, 21.3±2.14μm, n=29). Oospores were globose, plerotic, smooth, and 14.5-21.2 μm (mean±SD, 18.7±2.07μm, n=35) in diameter. The antheridia were diclinous or monoclinous, not intercalary, and one to six antheridia were attached to each oogonium. Based on these morphological characteristics, 3 isolates were identified as Pythium spp. including Pythium graminicola (Van der Plaats-Niterink 1981). Genomic DNA was extracted from the mycelia grown on PDA using a Fungal Genomic DNA kit (Scintol, Beijing, China) according to the manufacturer's instructions. The cytochrome oxidase II (Cox II) gene and internal transcribed spacer (ITS) region of the rDNA were amplified using the primers FM58/FM66 (Martin 2000) and ITS4/ITS5 (White et al. 1990) respectively. Amplification was performed in a 50μl reaction volume using 25 μl PCR Mix (Trans Gene, Beijing, China), 3 μl genomic DNA (50 ng/μl), 1 μl each forward and reverse primer (10 μM), and 20 μl ddH2O. The PCR program was as follows: initial denaturation at 95°C for 30 s, 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 60 s, extension at 72°C for 60 s, and a final extension at 72°C for 10 min. PCR products were sequenced and submitted to GenBank (accession no. OQ504322, OQ933130, and OQ933212 for ITS; OQ512002, OQ942203, and OQ942204 for Cox II). BLASTn analysis revealed that the ITS and Cox II sequences showed more than 98.62% similarity (721/724bp, 722/724bp,723/724bp for ITS; 514/514bp, 506/507bp, 500/507bp for Cox II) to P. graminicola ATCC96234 (accession no. AB095045 for ITS, and AB160849 for Cox II), respectively, supporting the morphological analysis. A neighbor-joining phylogenetic analysis of the ITS and Cox II concatenated sequence further confirmed that the isolates were P. graminicola. To test the pathogenicity of GF1, GF2, and GF3 a wheat seed inoculum was prepared as previously described (Qu et al. 2016). Sweet corn YK063 plants were planted in sterilized nutrient soil in plastic pots (one plant per pot) and grown in a greenhouse at 28℃ with 60% humidity and a 12-h/12-h light-dark cycle. For each isolate,10 plants were inoculated with 20 infected wheat seeds around the roots at the V5 stage, while 10 other YK063 plants were inoculated with the non-infected wheat seeds as a control. The experiment was repeated once. Three weeks later, the non-inoculated plants were asymptomatic. In contrast, inoculated plants showed stunning, yellowing of the leaves, root rot, and decreased production of lateral roots, exhibiting symptoms similar to those originally described for the disease. P. graminicola was successfully reisolated from the diseased roots and identified by morphological characteristics and sequencing of the ITS and Cox II as the causal agent for this root rot disease, fulfilling Koch's postulate for defining a causal agent. P. graminicola was reported as a causal agent of damping-off on dent corn in Georgia (Li et al. 2018). To our knowledge, this is the first report of P. graminicola causing root rot in sweet corn in southeast China. Identification of this pathogen will facilitate further research on this disease and the development of effective strategies to control the disease.
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Nov 28, 2024
Ui Wook Hwang + 1
Open Access