First Report of Phytopythium helicoides Causing Stalk Rot on Corn in China

Corn (Zea mays L.) is one of the most important grain crops in the world, especially in China. Besides, corn stalks are often used in production of bio-fuels (Xue et al., 2017). Recently, the production and quality of corn have been severely influenced by corn stalk rot in China caused by Fusarium spp. (Yu et al., 2017). At the end of June of 2019, a field survey of corn was carried out in Tai'an City, western Shandong Province, China. During the survey, the average day time temperature ranged between 22-28°C with intermittent rainfall, the relative humidity was 50-70%. In this survey, the symptomatic corn plants showed signs of necrosis and rotting on stalks and root collars. Five fields were surveyed and symptomatic corn plants were observed in three fields. The incidence rate of disease was about 5%, and the disease was more of a problem in low-lying areas. A total of twenty-eight symptomatic corn plants (7-12 per field), hybrid Denghai-618, at the 3-4 leaf stage were collected and tested for the presence of pathogens. The diseased tissues were excised, surface-sterilized with 75% ethanol for 30 seconds, rinsed for 3 to 5 times with sterile distilled water, and plated on potato dextrose agar (PDA). All plates were incubated at 28°C for 48 hours, emerging colonies were sub-cultured onto PDA plates. Forty-two isolates were obtained, and twenty-seven isolates were identified as Fusarium spp. The remaining fifteen isolates had similar morphology, with colonies that were white and cottony in texture after incubation at 28°C for three days on PDA. The suitable temperature range for growth of hyphae was between 15°C to 40°C, and sporangia were ellipsoidal, papillate, and 23 - 34×21 - 31 µm in diameter. Oogonia (smooth, 22 - 30 μm in diameter) were present in the cultures after 28 days at 28°C. The isolates were identified using both morphological characteristics and DNA sequencing. Identity of the oomycete was confirmed using the BLAST algorithm available through the GenBank with the DNA sequences of rDNA internal transcribed spacer region (ITS), cytochrome c oxidase Ⅰ (coxⅠ) gene and cytochrome c oxidase Ⅱ (coxⅡ) gene, which were amplified using the primers ITS1/ITS4 (White et al. 1990), FM35/FM59 and FM66/FM58 (Martin 2000), respectively. The fifteen isolates selected for sequence analysis had identical gene sequences, and hence, only sequences for isolate RMSD1 were submitted to GenBank (ITS - MW440691, coxI - MW450815 and cox II - MW450816). The ITS, coxI and coxII sequences of the isolate RMSD1 showed 97% identity (751/774 bp), 99% identity (1087/1098 bp) and 99% identity (548/554 bp) with Phytopythium helicoides Accession nos: HQ643382, FR774199, and AB108014, respectively. The pathogenicity of RMSD1 was tested on the corn hybrid Denghai-618. Three-leaf-stage corn plants (N = 15) were inoculated with mycelial agar disks (3 to 4 mm in diameter) colonized with RMSD1 placed on their root-collars. Sterile PDA disks (3 to 4 mm in diameter) served as the negative control (N = 9). Inoculated plants were placed in the growth chamber at 28°C, 60% relative humidity, 16 h / 8 h light regime cycle. Ten days post-inoculation, the inoculated plants showed necrosis, with symptoms of stem rot similar to those observed in the field. The inoculation experiments were repeated twice with the same results, fulfilling Koch's postulates. The root-collars and stems of negative control remained asymptomatic, and P. helicoides was not isolated. Previously, P. helicoides has been reported as a pathogen of strawberry (Zhan et al. 2020) and kiwi fruits (Wang et al. 2015) from China, but not from corn. To our knowledge, it is the first report of P. helicoides causing corn stalk rot in China. In the future, P. helicoides can be considered as a potential candidate causing stem and collar-rot of corn in China, but not the only one. There are other microbes that can produce similar symptoms on corn, and control methods for pathogenic oomycetes differ from those for fungi.