First Report of Pythium selbyi Causing Corn Seed and Seedling Disease in Northeastern China

Abstract

Corn is one of the main grain crops in China. In recent years, occasional poor emergence and poor stands of corn seedlings have been reported in northeastern China. Soil samples were collected from fields with a history of poor corn seedling emergence in Heilongjiang province of China in 2017. After being planted in the collected soil for 21 days, rotted corn seeds were harvested and washed for 4 min under running water with a “Safeguard” soap. The incubation temperature was divided into four stages: 21℃/7℃ for 6 days, 10℃/3℃ for 4 days, 16℃/7℃ for 5 days, 20℃/20℃ for 6 days (16 h/ 8 h, light/dark). Seeds were then cut in half and placed on water agar amended with pimaricin (5 μg/ml), ampicillin (100 μg/ml), rifampicin (10 μg/ml), streptomycin (200 μg/ml), pentachloronitrobenzene (50 μg/ml), and benomyl (10 μg/ml) (PARP+B), which is selective for oomycetes (Jeffers and Martin 1986). Plates were incubated at 10℃ for 2-3 days in the dark. Pythium selbyi was isolated from rotted seeds. Six of isolates were molecularly identified further. Sequences were generated from each isolate using primers FM55/FM52R for the cytochrome c oxidase subunit I (COI) gene (Long et al. 2012), and using primers DC6/ITS4 for rDNA ITS (Cooke et al. 2000). For the COI gene, BLAST analyses of the 873 bp segments showed 99% identity with P. selbyi isolate (GenBank Accession No. JF895536.1). For ITS fragment, BLAST analyses of the 893 bp segments showed 99% identity with P. selbyi isolate (GenBank Accession No. KT337692.1). Both sequences were submitted to GenBank with accession numbers MK208967 and MK208681 for COI and ITS, respectively. On 10% clarified V8 juice agar (cV8A), P. selbyi produced sporangia, oogonia, antheridia and oospores. Sporangia were terminal or rarely intercalary, globose to slightly ellipsoidal, ranging from 25-41 μm diam. Oogonia were laevis, terminal, intercalary, globose, ranging from 18-28 μm diam. Oospores were plerotic, globose and laevis ranging from 15-22 μm diam. Antheridia were mostly hypogynous, rarely diclinous. Each oogonium was supplied by 1-2 antheridial cells, measuring: 10-23  7-12 μm. These morphological characters were consistent with the previous report on P. selbyi (Ellis et al. 2012). The pathogenicity tests were conducted as described by Matthiesen et al. (2016). Briefly, the autoclaved parboiled rice was inoculated with 7-day-old Pythium selbyi isolate as inoculum, incubated for 10 days at 25℃ in the dark and dried for 2 to 3 days. Then, the 10-day-old inoculum was placed in a layer approximately 2-3 cm below 12 corn seeds in a 1 L plastic case filled with coarse vermiculite. Both inoculated and non-inoculated control plants were incubated in growth chambers at 21-23℃. After 14 days, symptoms similar to those observed in the field and lab appeared in the seeds of inoculated plants, whereas control plants remained symptomless. Isolation made from diseased seeds consistently yielded P. selbyi. The extent of the geographical distribution and host range of this recently described species are still being determined. To our knowledge, this is the first report of P. selbyi in China. After identification of the pathogen, we can prevent and control the pathogen to protect the plant and to prevent yield losses.