First Report of Fusarium incarnatum-equiseti Species Complex Causing Fruit Rot on Muskmelon (Cucumis melo) in China

Abstract

Muskmelon (Cucumis melo L.) is an economically important fruit crop in China. In September 2018, fruit rot was observed on approximately 20% of muskmelon fruits in Harbin, Heilongjiang Province, China. Brown water-soaked lesions were observed on the fruit side in contact with soil initially, which gradually extended to most of or the entire fruit. Internal decay was observed with white to dark brown mycelium on the fruit surface. Diseased muskmelon tissues were surface disinfested with 1% NaOCl for 3 min, 70% ethanol for 10 s, and then washed three times with sterile water. The disinfested tissues were cut into 1-cm pieces and placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/liter) and incubated at 25°C for 1 week. Isolations were done on 10 fruit fragments, and the same fungus was obtained. The cultures were purified using the hyphal-tip technique and used for morphological and molecular analyses. Morphological characteristics were observed on 1-week-old PDA cultures grown at 28°C. The aerial mycelium changed from white to light yellow, and the back of the plate turned pale brown with time. Microconidia were single celled, hyaline, nonseptate, ovoid, and 8.5 to 10.6 × 3.2 to 4.4 μm. Hyaline macroconidia (mostly three-septate) were slightly curved at the apex and ranged from 19.4 to 35.2 × 3.8 to 7.7 μm. Chlamydospores with thick, roughened walls were abundant in clumps or chains, ellipsoidal or subglobose. Primers ITS1/ITS4 (White et al. 1990) and EF-1/EF-2 (O’Donnell et al. 2000) were used to amplify and sequence the internal transcribed spacer (ITS1-5.8S-ITS2) and translation elongation factor 1-α (TEF1) region. ITS and TEF1 gene sequences were deposited in NCBI GenBank nucleotide database with accession numbers MH910492 and MH920853, respectively. BLASTn analysis showed 99% nucleotide sequence identity with Fusarium incarnatum-equiseti species complex (FIESC). The TEF1 gene sequence of isolate NEAU-TG1 was compared with sequences in the FUSARIUM-ID database (Geiser et al. 2004), which indicated that the pathogen was most closely (99% identity) related to phylogenetic species within the FIESC. Pathogenicity of these isolates was confirmed by following Koch’s postulates. Ten muskmelon fruits of cultivar Xiangfei were surface disinfested with 2% NaOCl for 2 min and rinsed with sterile distilled water three times. Spores were produced on PDA for 7 days at 28°C and washed with sterile distilled water; the concentrations were adjusted to 1 × 10⁶ spores/ml using a hemocytometer. Ten muskmelon fruits were inoculated by injecting the suspension (2 ml, 1 × 10⁶ spores/ml) and covered with plastic bags for 24 h, and another 10 surface-disinfested fruits treated with sterile distilled water were used as a control. The fruits were placed in a humidified chamber (>95% relative humidity) at 25°C for 48 h after inoculation and kept in a growth chamber at 25°C with 12-h day/night cycle for 8 days. All inoculated fruits showed symptoms identical to those observed in the field. No disease occurred on the controls. The pathogen was reisolated from diseased fruits, and species identification was confirmed by the morphological and molecular method described above. Members of the FIESC can cause disease on plants, such as Allium cepa, Festuca arundinacea, Morchella importuna, and Cucumis trigonus. Furthermore, the members of the FIESC have been reported to produce type A and B trichothecene mycotoxins that cause toxicosis in humans and animals (Leslie and Summerell 2006; O’Donnell et al. 2009). To our knowledge, this is the first report of fruit rot caused by a member of the FIESC on muskmelon in China. This pathogen presents a threat to muskmelon production, and its accurate identification is necessary to develop effective management strategies.