First report of Pythium aristosporum causing corn stalk rot in China.

Corn (Zea mays L.) stalk rot, caused by various pathogens, is one of the most prevalent corn diseases worldwide. In October 2019, a survey was carried out to determine pathogenic fungi causing corn stalk rot in 3 fields (~120 ha) in Harbin city (44.04°N 125.42°E), Heilongjiang Province, China. In each field, 100 plants at 5 sampling points were assessed at the milk stage (R3) of development. Disease incidence was 12%. Symptomatic plants showed rapid death of the upper leaves, drooping ears and stalks were soft, hollow, watersoaked with white hyphae present on teh outside of the stalk. Pieces of tissue (0.25 cm2) from 15 individual diseased stalks (5 plants/field) were surface disinfested in 0.5% NaOCl for 5 min, rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) containing streptomycin (50 μg/mL). After three days of incubation, a total of twelve fungal cultures with uniform characteristics were isolated and subcultured by transferring hyphal tips onto V8. Colonies on V8 selective medium were creamy white and floccus, with a growth rate of 20 mm/day at 26°C in darkness. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick (n = 50); globose oogonia, 23.9 to 30.5 μm in diameter (n = 50), and had 1 to 8 antheridia. Based on these characteristics, the isolates were identified as Pythium sp. (van der Plaats-Niterink 1981). Genomic DNA was extracted from single conidial cultures of representative isolates (MZYJF1, MZYJF3 and MZYJF7), and the internal transcribed spacer (ITS) region and cytochrome coxidase subunit II (CoxII) gene were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012) and COX2f/COX2r (Hudspeth et al. 2000), respectively. Partial nucleotide sequences of 796 bp and 573 bp for the ITS and COX11 amplicons, respectively, were obtained and deposited in GenBank (accession no. MW447501 for ITS, and MW471006 for COXII). MegaBLAST analysis of the ITS and CoxII sequences of MZYJF1 isolate showed 100% similarity with sequences from P. aristosporum strain ATCC 11101. The isolates were identified as P. aristosporum based on the fact that P. aristosporum has aplerotic oospores and less antheridia per oogonium than P. arrhenomanes (van der Plaats-Niterink 1981). A pathogenicity test was performed on corn cv. Xianyu 335 at tasseling stage (VT) in the field. An oospore suspension, obtained from isolate MZYJF1 grown on V8 agar media for 4 weeks (Green and Jensen, 2000) and diluted to 1×104 oospores/mL using blood cell counting method, was injected into the base of the maize stems of 6 healthy plants (1.5 ml/plant ) using a syringe. Control plants were injected with distilled sterile water. All inoculated plants showed symptoms 25 days after inoculation that were similar to those observed in the field. The oomycete of P. aristosporum was reisolated from symptomatic plants on V8 agar media and identified according to morphological and molecular characteristics. No symptoms were observed on the control plants. P. aristosporum has previously been reported on causing damping-off of pea in the Columbia basin of Central Washington (Alcala et al. 2016) and on soybean in North Dakota (Zitnick-Anderson and Nelson 2015). To our knowledge, this is the first report of P. aristosporum causing corn stalk rot in China. Corn stalk rot caused by P. aristosporum poses a threat to significantly reduce the quality of corn. Thus, its distribution needs to be investigated and effective disease management strategies developed.