First report of Bipolaris sorokiniana leaf spot disease on watermelon (Citrullus lanatus) in Florida

Watermelon is an important crop in Florida, representing $88.2 million in cash receipts in 2015 (USDA/NASS 2017). In April and May 2021, the UF/IFAS Plant Diagnostic Center in Gainesville, Florida received eight diseased watermelon leaf samples from Alachua, Gilcrest, Levy, and Suwannee counties in Florida. Lesions were round to oblong, light gray to tan with reddish brown margins and white to light gray center, and some were coalescing resulting in about 15% disease severity. Symptomatic leaf tissue (0.5 cm2) was surface sterilized in 0.6% sodium hypochlorite for one minute, rinsed with sterile tap water, plated onto water agar media plates, and incubated at 27°C under 12-h light/dark cycle for 7 days. Characteristic Bipolaris conidia with gray to black brownish cottony mycelial growth were consistently found growing from plated lesions. The pathogen was isolated from two of the eight samples using a 0.5 mm diameter sterile metal needle to transfer a single conidium onto DifcoTM Potato Dextrose Agar (PDA) plates. Three isolates were designated G21-562 from Levy and G21-599a and G21-599b from Alachua County. All three isolates produced curved or straight, cylindrical, obclavate, distoseptate brownish gray conidia with 3 to 8 septa, mostly tapering towards ends with dark brownish to black hilum, that ranged from averaged 62um x 25um (n=30, SD=8 for length and 3 for width). Conidiophores were brownish, septate, smooth, and straight, single or in small groups, simple or branched, and swollen at the upper tip. Internal transcribed spacer region (ITS) and partial glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene sequences were amplified using primers ITS1/ITS4 and GPD-1/GPD-2 (Berbee et al. 1999). Reference sequences (Adhikari et al. 2020 and Manamgoda et al. 2014) were aligned using MUSCLE and trimmed to consistent length. Using concatenated sequence alignments of both loci, a maximum likelihood phylogenetic tree was constructed based on K2+G substitution model selected by BIC using Mega X (Kumar et al. 2018) with 1,000 bootstrap. The ITS and GPDG sequences of G21_599b, G21_599a and G21_562 (GenBank accessions OK614094 to 96, OP297398 to 400) showed 100% identity across 888 nucleotides across both loci to B. sorokiniana isolates CBS_110.14 and CBS_ 120.24 and were distinct from other reference isolates. To fulfill Koch’s postulates, all three isolates were grown on PDA at 27°C and 12-h light/dark cycle. After a week, conidia were harvested in sterile water, and the conidial suspensions were adjusted to 105 conidia/ml using a hemocytometer. Each conidial suspension and Tween 20 water control was sprayed onto three seedlings of ‘Sugar Baby’ watermelon until runoff, and inoculated seedlings were sealed in a plastic bag for 24 hrs. The experiment was done in a greenhouse (20- 25°C) and repeated once. After a week of incubation, the same leaf lesion symptoms described above were observed on seedlings inoculated with conidia, whereas seedlings sprayed with the control were asymptomatic. Isolations from symptomatic tissue produced gray to black mycelia with conidia that were the same as described from field samples. To our knowledge, this is the first report of leaf spot on watermelon caused by B. sorokiniana. B. sorokiniana is a common pathogen of grasses and agronomic crops (Farr and Rossman 2020). The extent to which this emerging disease of Florida watermelon may negatively impact production is unknown and should be the subject of future observation and research.