Cucurbitaceae crops are widely cultivated in the Northeast region of Brazil, which is the biggest producer of melon and watermelon in the country (Oliveira, 2020). Between November and December 2020 leaves of pumpkins (Cucurbita maxima L.) and watermelon (Citrullus lanatus L.), and leaves and fruits of melon plants (Cucumis melo L.) were collected with moderate to severe necrotic, irregular, and brown lesions from farms in the state of Rio Grande do Norte, Brazil. Fragments of diseased tissues were cut into small pieces and surface disinfested in 70% ethanol for 30 seconds, then in 2% sodium hypochlorite for 1 minute, and washed in sterile distilled water. Disinfested pieces of tissue were plated on potato dextrose agar (PDA) and incubated for seven days in the dark at 28 ± 2 °C. A total of 12 fungal isolates (four from pumpkins, one from watermelon, and seven from melons) were isolated from leaves and symptomatic fruits. All isolates in this study shared similar morphological characteristics. The colonies were dark gray to olive green in color with a velvety texture and surrounded by gray-white hyphae. The conidiophores were erect, tall, dark, and irregularly branched at the apex containing dark conidia, with 0 to 3 septa, variable in shape and size, forming chains that were often branched, globose, or subglobose with 3 to 4.5 μm in diameter. DNA from each isolate was extracted using the SDS method (Smith et al., 2001) and submitted to PCR amplification of the ITS and TEF1α regions with the primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The amplicons were sequenced and deposited in GenBank: ITS (OP493545-OP493556) and TEF1α (OP536836-OP536847). Blastn analysis of the ITS and TEF1α partial sequences revealed that all 12 isolates belong to the species Cladosporium tenuissimum, with 100% nucleotide similarity with sequences of many C. tenuissimum isolates deposited in GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis, with the concatenated sequences (ITS-TEF1α) on MEGAX software (version 11.0.8) (Tamura et al, 2018). All 12 isolates clustered in the same clade and were closely related to isolates A2PP5, A3I1, and XCHK2 with the respective accession numbers KU605789.1, KU605790.1, and MG873071.1 from GenBank, with 99% bootstrap support. The pathogenicity of the 12 isolates was evaluated in pumpkin and melon plants in a greenhouse. Spore suspensions (10 6 conidia/ml -1) were sprayed on the leaves of healthy seedlings until runoff, only water was sprayed on control plants as the mock, and five seedlings of each crop (melon and pumpkin) were inoculated in each treatment. All plants were covered with plastic bags for two days. Spots, similar to those observed on diseased plants in the field, developed on the inoculated leaves (after seven days from the inoculation day, no symptoms were observed on plants from the mock treatment) and the fungal morphology was identical to that observed on the originally diseased leaves, fulfilling Koch's postulate. The pathogenicity test was repeated and yielded the same results. The fact that all 12 isolates were pathogenic on pumpkin and melon leaves, indicates that many Cucurbits are susceptible to C. tenuissimum infection. Many growers in the region are reporting similar symptoms in their melon plantations and it appears that the disease incidence is getting more severe year after year, based on growers's reports. Therefore, more research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to Cucurbits to develop strategies for disease control. To the best of our knowledge, this is the first report of C. tenuissimum causing disease in Cucurbits in Brazil.
Keywords: Cladosporium tenuissimum; Cucurbits; melon; spot diseases.