First Report on Fusarium oxysporum f. sp. niveum Causing Watermelon Fusarium Wilt in Bangladesh

Abstract

Watermelon (Citrullus lanatus) is a popular summer fruit in Bangladesh. Production of this fruit was 293,956 and 253,661 metric tons in 2014–2015 and 2016–2017, respectively (Bangladesh Bureau of Statistics 2017). Watermelon fields were affected by pest and diseases before harvest in 2018 (Star Country Desk 2018). During February to March 2018, 30 to 70% of watermelon plants wilted were observed in commercial fields of Subornachar (22.662586° N, 91.094281° E), Noakhali, Bangladesh. The infected stems and roots (2- to 3-cm pieces) were surface sterilized with 1% NaOCl for 5 min, rinsed three times with sterile distilled water, and placed on half-strength potato dextrose agar (PDA). The culture plates were incubated at 25 ± 2°C for 7 days and purified to obtain single-spore isolates. The single-spored isolates produced flat, round, light purple mycelial growth on the surface and reverse of PDA plates. Hyphae were smooth, hyaline, branched, and septate. Macroconidia were mainly fusiform with three septa. Single-celled oval microconidia were produced in monophialidic conidiophores that looked like false heads. Fungal inoculum of two isolates, NSWF001 and NSWF004, were prepared with a mixture of sterilized broken maize, wheat bran, and grass pea pod seed coat (1:10:5 w/w) for soil inoculation. Thirty-five pieces (5 mm in diameter) of PDA grown with 7-day-old mycelium of each isolate were used to inoculate into 1 kg of mixture and incubated for 10 days at room temperature with a 12-h photoperiod. To confirm their pathogenicity, 15-day-old seedlings (10 seedlings/isolate, repeated three times) of watermelon cultivar Sugar Emperor were transplanted into 9-cm-diameter pots filled with soil (treated with 10 g of inoculum/kg of soil). After 2 weeks, the transplanted seedlings in the inoculated soil showed stunted growth and wilt symptoms similar to those observed in the farmer’s fields. The control seedlings remained healthy. Light orange discoloration was observed on crown tissues in longitudinal cut of stems and roots of inoculated seedlings. Control seedlings remained symptomless. The fungus was successfully reisolated from stems and roots of infected seedlings on half-strength PDA with the same morphological characters as before, which fulfilled Koch’s postulates. The internal transcribed spacers (ITS) of the two isolates were amplified by using primers ITS-1/ITS-4 (White et al. 1990). The ITS sequences of the two isolates NSWF001 (481 bp) and NSWF004 (479 bp) (GenBank accession nos. MN148629 and MN148630) showed 100% similarity with e-value of 0 to the reference sequences of Fusarium oxysporum (MN633363) and F. oxysporum f. sp. niveum (KX275296). In Fusarium MLST database, both NSWF001 and NSWF004 isolates revealed 100% similarity with isolate CBS 159.57 of F. oxysporum f. sp. cyclaminis. O’Donnell et al. (2009) found F. oxysporum f. sp. forming a cluster with different groups of the F. oxysporum species complex because of its polyphyletic nature. Based on the aligned sequences of ITS, molecular phylogenetic analysis by the maximum likelihood method, the isolates NSWF001 and NSWF004 were identified as F. oxysporum f. sp. niveum. The isolates are stored on dried filter paper at –20°C and archived at the Plant Pathology Division, Bangladesh Agricultural Research Institute. To the best of our knowledge, this is the first report of watermelon Fusarium wilt caused F. oxysporum f. sp. niveum in Bangladesh.