Watermelon (Citrullus lanatus (Thumb.) Matsum. & Nakai) is an important crop in Brazil both for export and domestic consumption. In October 2019, watermelon plants showing decline and root rot symptoms were surveyed in 16 commercial fields in Baraúna's municipality (Rio Grande do Norte, Brazil). The disease prevalence was 12.5%, and the average incidence was 5%. Affected root segments were cut into small pieces and surface-disinfected with 70% ethyl alcohol and 1.5 % NaOCl for 1 and 2 min, respectively. Tissues were plated onto potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fungal colonies developed from the infected tissues were dark or greyish, and pure cultures were obtained by hyphal tip isolation technique. Six fungal isolates with the same morphology were obtained. Two of them were selected for morphological and molecular characterization (CFC-1123 and CFC-1124). Isolates grew rapidly in PDA, covering the entire surface of the Petri dishes within 3 days. The aerial mycelium was initially white, turning dark greenish-gray after 4 to 5 days of incubation at 25°C in the dark. Isolates produced pycnidia and conidia in water-agar medium with sterilized pine needles after 30 days of incubation at 25°C under near-UV light. The conidia were initially hyaline and brown with central transverse septum and longitudinal streaks when mature. Conidia were ellipsoid to oval (22.83 ± 3.1 µm long and 11.58 ± 1.5 µm wide). Based on morphological features, the isolates were initially identified as Lasiodiplodia sp. (Phillips et al. 2013). To confirm the identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region as well as fragments of the translation elongation factor 1-α (TEF) and β-tubulin 2 (TUB) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank under accession numbers OL841380, OL865376 and OL890691 for CFC-1123, and OL841381, OL865377 and OL890692 for CFC-1124. Maximum likelihood phylogenetic analysis of the concatenated sequences of ITS, TEF and TUB gene regions of some reference sequences and ex-types of Lasiodiplodia spp. was performed. Phylogenetic analysis revealed that the isolates grouped in the L. brasiliensis clade (Netto et al. 2014) with 80/79% of bootstrap. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi from Cariri (CFC) at the Universidade Federal do Cariri (Crato, Brazil). Pathogenicity of the two isolates was determined using colonized wheat grains as inoculum source. One watermelon seed (cv. Crimson Sweet) was placed in a sterile plastic pot (500-mL) filled with 6 cm layer of a substrate composed of soil and Tropstrato® (5:1 w/w). Three wheat grains (50 mg) colonized with each isolate were placed 10 mm above the seed and covered with the substrate. Control pots were inoculated only with sterile wheat grains. There were five replicates for each isolate. The pots with seedlings were maintained in a greenhouse at 28 ± 2°C under natural light conditions. The inoculated seedlings showed poor growth, withering and drying leaves 45 days after inoculation (DAI), and subsequently root rot symptoms and death at 60 DAI. Control seedlings remained asymptomatic. The pathogen was re-isolated from all inoculated seedlings and identified by conidia morphology to fulfill Koch's postulates. Lasiodiplodia brasiliensis has been reported to cause postharvest rot and gummosis of watermelon (Farr and Rossman 2022). However, to our knowledge, this is the first report of watermelon decline caused by this fungus in Brazil and worldwide. This finding must be considered for developing efficient control strategies for the disease.
Keywords: phylogeny; vine decline; waternelon.