First Report of Fusarium proliferatum Causing Fruit Rot on Muskmelon (Cucumis melo) in China.

Abstract

Muskmelon is an economically important crop in the world, especially in China, the largest producer of muskmelon with an annual output up to 12.7 million tonnes (Gómez-García et al. 2020). Since 2018, fruit rot was observed on muskmelon in Malianzhuang Base, the main muskmelon producing area in Shandong Province, whose disease incidence was about 25-30%. Water-soaked dark brown spots were initially appeared on the side of the fruit near the ground, then gradually expanded and covered with white mold with time. To isolate the pathogens, ten muskmelon fruits with typical symptoms were collected from different greenhouses in the base. Small tissues taken from the edge of the diseased and healthy tissues were immersed in 1% NaClO for 2 min, then soaked in 75% ethanol for 30 s, and rinsed 3 times with sterile distilled water (SDW). The sterilized tissues were naturally dried and placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/L) for 7 days at 28℃. The emerging fungal mycelia were transferred to fresh PDA using the hyphal tip technology. Ten colonies were purified by single spore method and cultured on PDA for 7 days at 28℃ in the dark for morphological and molecular analyses. All colonies were flocculent with abundant white to light purple aerial hyphae, and the undersides of the colonies were observed to be from white to purple over time. Microconidia produced on PDA were hyaline, fusiform, ovoid, single cell without septum, and 4.5 to 12.7 × 2.0 to 3.6 μm in size (n=50). Macroconidia produced on carboxymethylcellulose agar (CMC) were slightly curved at both ends with three to five septa, and 17.6 to 35.7 × 2.8 to 4.0 μm in size (n=30). According to the morphological characteristics, these isolates were preliminarily identified as Fusarium sp. (Leslie and Summerell 2006). To further identify these isolates, genomic DNA of five isolates was extracted by CTAB method (Wu et al. 2001). The internal transcribed spacer (ITS) region of ribosomal DNA, translation elongation factor 1-α (TEF1) region, and the RNA polymerase II second largest subunit (RPB2) were amplified by PCR amplification with primers ITS1/ITS4, EF-1/EF-2, and RPB2-5F2/fRPB2-7cR, respectively (White et al. 1990; O'Donnell et al. 2008; Liu et al. 1999). Sequences of the five isolates were identical. The ITS, EF1-α, and RPB2 gene sequences of isolate NEAU-Mf-10-2 were submitted to NCBI GenBank with accession numbers of MZ950914, MZ960928, and MZ960929, respectively, having 100% similarity to those of Fusarium proliferatum (MK372368, MK952799 and MN245721). Phylogenetic trees were constructed based on the concatenated sequences of EF1-α and RPB2 genes using neighbour-joining and maximum-likelihood algorithms with MEGA 7.0. Two similar tree topologies both showed isolate NEAU-Mf-10-2 clustered with F. proliferatum NRRL 43665. Therefore, isolate NEAU-Mf-10-2 was identified as F. proliferatum based on morphological characteristics and phylogenetic analysis. To fulfill Koch's postulates, ten muskmelon fruits (var. Tianbao) were soaked in 2% NaClO for 2 min, and then washed three times with SDW. Muskmelon fruits were inoculated by injecting conidia suspension (200 μL, 1×106 spores/mL) with a sterile injector. Ten other surface sterilized muskmelon fruits inoculated with sterile water were used as control. The fruits were placed in a light incubator at 28℃ with 12h light cycles for 7 days. All inoculated fruits showed symptoms highly similar to those of infected muskmelon fruits observed in the field. No symptoms were observed on fruits used as control. The Fusarium isolates were successfully re-isolated from the symptomatic fruits, and identified based on above morphological and molecular biological methods. Previous studies have reported that F. proliferatum can infect Polygonatum cyrtonema, Salvia miltiorrhiza, Allium cepa, A. sativum, and so on. To our knowledge, this is the first report of F. proliferatum causing fruit rot on muskmelon in China, which will provide basic information for designing effective prevention and control strategies on this disease.