First Report of Didymella americana causing corn stalk rot in China.

Corn (Zea mays L.) is an important food crop and feedstuff worldwide. However, Corn stalk rot, caused by multiple pathogens, is globally an economic soil-borne disease worldwide. In September 2019, a survey was carried out to characterize pathogenic fungi in corn stalks in Nehe city (48.48°N 124.88°E), Heilongjiang Province, China. Stalk rot incidence was approximately 5% in three of the fields sampled (5 ha/per field). Symptoms included wilting of whole plants, drooping ears or rapid death of the upper leaves or whole plant from blister stage to physiological maturity (growth stages R2- R6) stage with drooping ears or rapid death of the upper leaves or whole plant. A brown to black dry rot or necrosis was observed throughout the central pith and internal tissues of the stalk and crown were observed, which resulted in hollow and soft stalks. Fifteen tissue samples (0.25 cm2) from 15 individual diseased plants were surface disinfested with 75% ethanol for 2 s, followed by 0.5% NaOCl for 5 min, rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) with 50 µg/mL streptomycin at 26°C in darkness. After 3 days, a total of eight fungal isolates with consistent characteristics were obtained from three sampling points and subcultured by transferring hyphal tips onto a new PDA plate. Single-conidium isolates were generated with methods reported previously (Leslie and Summerell 2006). Cultures on PDA were honey to olivaceous buff in the center with dense aerial mycelia and wide buff colored margins. The dimensions of conidia from 30-day-old PDA cultures were 4.5 to 15.3 µm × 1.5 to 4.3 µm (n = 50). Often, one to two oil bodies were present within the conidia. Based on these morphological features, the isolates were identified as Didymella americana (Aveskamp et al. 2010; Gorny et al. 2016). Genomic DNA was extracted from a representative isolate YJDA8 and the internal transcribed spacer regions (ITS) and translation elongation factor 1-alpha gene (TEF-1ɑ) were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The sequences of YJDA8 (accession nos. MT995077 for ITS and MW003707 for TEF-1a ) showed 99.6% (529/531 bp) and 97.6% (283/290 bp), identity to the sequences of D. americana isolate YSGYE6 (accession no. MK945663.1) and isolate K_INSO2_6_10 (MN554764.1) respectively. Pathogenicity tests were conducted by root injection of corn plants at the blister stage in the field. Conidia were obtained from 30-day-old PDA cultures grown at 20°C with a 12 h photoperiod. A conidial suspension (1.5 ml of 1×105 conidia/mL) was injected into the base of the maize stems using a 5 ml syringe. For each treatment, 5 plants were inoculated. Plants injected with 1.5 ml distilled sterile water served as the control. After inoculation, the plants were managed using conventional methods. All inoculated plants showed symptoms 25 days after inoculation that were similar to those observed in the field, while no symptoms were observed on the control plants. The fungus was re-isolated and confirmed to be D. americana. D. americana has previously been reported on corn roots and soybean pods in the USA (Aveskamp et al. 2009 as Peyronellaea americana), on lima bean in Delaware and Maryland (Everts et al. 2020). To our knowledge, this is the first report of D. americana causing stalk rot on corn in China. Therefore, its distribution needs to be investigated, monitored and managed with effective disease management strategies to protect corn.