First Report of Black Root Rot on Cucumis melo Caused by Berkeleyomyces rouxiae in Heilongjiang Province of China

Abstract

Muskmelon (Cucumis melo L.) is a popular summer fruit, widely cultivated as an economically important crop in Heilongjiang Province, China. In April 2018, a new root rot disease on muskmelon roots was observed at the nursery stage in several plastic greenhouses in Qiqihar Meilisi district (19°25′12.00″ N, 108°54′15.13″ E) in western Heilongjiang Province, with disease incidences of 20 to 30%. Initially, the seeding roots were affected by black rots, necrosis, and shriveled stems. Subsequently, infected plants exhibited stunting, wilting, and death. To identify the causal agent, affected root pieces (5 mm²) were sterilized in 70% ethanol for 30 s, soaked in 1% sodium hypochlorite for 1 min, and then rinsed three times in sterile distilled water. The treated tissues were placed on potato dextrose agar (PDA) amended with 50 mg/liter of streptomycin sulfate and incubated at 25°C for 7 days. Pure cultures were obtained by single-spore isolation. The pure colony on PDA was olive green, with neat edges and obvious aerophytic hyphae. Colonies grew slowly, with an average growth rate of 2 mm/day. Conidiophores were dark and transparent, branched, with multiple septa, produced terminally or laterally on vegetative hyphae. Two types of spores were observed in 1-week-old isolates. Endoconidia were monosporic, colorless, short columnar, with two equal or slightly obtuse circles. Dimensions were 8.0 to 21.0 × 5.0 to 6.0 μm. Chlamydospores were produced on the top or lateral hyphal branches, clavate, dark brown, consisting of two to six spores. The size ranged from 33.0 to 96.0 × 16.0 to 22.0 μm. According to colony morphology and microscopic characteristics, the fungus was identified as Berkeleyomyces rouxiae (Berk. & Broome) W.J. Nel, Z.W. de Beer, T.A. Duong & M.J. Wingf. (Nel et al. 2017) (syn. Thielaviopsis basicola). To further confirm the identification, DNA was extracted from single-spore isolates. The internal transcribed spacer region was amplified and sequenced with primers ITS1/ITS4 (White et al. 1990). BLAST analysis of the sequence (GenBank accession no. MK164871) showed a 99% similarity to that of B. rouxiae (GenBank accession no. MF952393). Pathogenicity of isolate TG515 was performed under laboratory conditions. Thirty healthy muskmelon seedlings were equally divided into two groups. The roots of one group of seedlings were soaked in a 50-ml conidial suspension (1 × 10⁶ spores/ml) for 15 min, and the other group was treated with sterile distilled water as a control. The seedlings were transplanted in pots with sterilized soil after inoculation, and all seedlings were placed in a light incubator (25°C, light/dark = 12 h/12 h). The assay was repeated three times. Typical symptoms of black rot and necrotic lesions developed after 2 weeks in group 1, with a 100% disease incidence. However, control seedling roots did not show any disease symptoms. The pathogen was reisolated from the symptomatic roots and confirmed as B. rouxiae by morphological and molecular analysis, thus fulfilling Koch’s postulates. This fungus is globally distributed and causes a serious root rot on many important plant species and, therefore, also poses a threat to muskmelon production in China. To our knowledge, this is the first report of B. rouxiae on muskmelon in China.