Occurrence of Cotyledon Spot on Soybean Caused by Fusarium oxysporum in China

Soybean (Glycine max [L.] Merr.) is an important food and oil crop in northeast China. During June 2018, cotyledon spot was observed on soybean in Acheng district (45°32′N, 126°57′E), Heilongjiang province, China. The disease occurred on approximately 6% of soybean cotyledon in three 5-ha commercial fields with random distribution in the field. Initial symptoms were irregular brown sunken spots, cracks, and a shriveled surface on soybean cotyledons, which enlarged gradually from 1 to 15 mm in diameter. The seedlings became stunted and yellowed. Ten infected soybean cotyledons were collected from the field. Infected tissues were surface disinfested by submersing in 0.5% NaOCl for 3 min, rinsed three times in sterile distilled water, and cultured on potato dextrose agar (PDA) for 3 to 4 days at 26°C. Six fungal cultures were isolated and subcultured by transferring hyphal tips. Single-microconidium isolates were generated from the fungal cultures using methods reported previously (Leslie and Summerell 2006), and morphological characteristics of the isolates were observed. Colonies on PDA consisted of whitish, aerial mycelia with pink to purple pigments and abundant microconidia, macroconidia, and chlamydospores. Microconidia were hyaline and oval-ellipsoid to cylindrical, measured 1.8 to 3.2 μm in width and 5.6 to 10.5 μm in length (30 spores were measured), and had zero to one septa. Macroconidia were slightly curved with a prominent foot-shaped basal cell, measured 2.8 to 5.2 μm in width and 15.6 to 20.5 μm in length by tangent distance, and had three to five septa. Chlamydospores were abundant and were ellipsoidal or subglobose. Based on these characteristics, the isolates were identified as Fusarium oxysporum (Leslie and Summerell 2006). Genomic DNA of a representative isolate Ziye4 was extracted, and the internal transcribed spacer regions of rDNA (ITS1-5.8S-ITS4) and translation elongation factor 1-alpha gene were amplified and sequenced with primers ITS1 and ITS4 (Yin et al. 2012) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The DNA sequences of Ziye4 were deposited in GenBank (accession no. MK212364 and MK370016). MegaBLAST analysis of the ITS sequences showed that it had 99% similarity to a F. oxysporum strain isolate FLS75 (accession no. KU671046) and S3B1 (accession no. KT032971). Pathogenicity of isolate Ziye4 was assessed on soybean seedling cotyledons (cv. Suinong 25) using a modified cut-seedling inoculation method (Li 2018). At 7 days after planting in 10-cm-diameter pots (two plants per pot), 10 soybean cotyledons were inoculated by cut-cotyledon using sterile scissors with a 5-day-old Ziye4 culture agar plug as wound area inoculum (Li 2018). In addition, 10 cotyledons treated with sterile distilled water were used as a control. The plants were stored at 26°C with >95% relative humidity for 24 h after inoculation and then in the greenhouse at 23 ± 5°C with a light/dark (12 h/12 h) cycle for 7 days. All inoculated cotyledons showed symptoms identical to those observed in the field. No disease occurred on the control cotyledons. The fungus was reisolated from diseased cotyledons and confirmed to be F. oxysporum based on morphological characteristics. To our knowledge, this is the first report confirming F. oxysporum causing cotyledon spot on soybean in China. Soybean is the main oil crop grown in northeast China, and this disease seriously affects the seedling growth and needs to be properly managed in soybean production.