Caracterização do groundnut ringspot orthotospovirus (GRSV) em áreas produtoras de melancia do Rio Grande Norte e Ceará

Abstract

Watermelon (Citrullus lanatus) is mainly cultivated in China, Turkey, Iran, and Brazil. Diseases caused by viral species such as Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus are considered economically important for the watermelon crop. In 2015, the groundnut ringspot orthotospovirus (GRSV) was reported for the first time in watermelon fields in Brazil, affecting productive areas in the state of São Paulo. In 2020, watermelon plants showing symptoms of virus infection were found in fields in the state of Rio Grande do Norte - Mossoró and Ceará, Brazil. Therefore, this study aimed to perform the diagnosis, biological and molecular characterization of GRSV and verify the possibility of other viral infections in watermelon plants from the region of Mossoró and Ceará. Therefore, two data collections were investigated and sampled: one in January 2020 (96 samples) and another in January 2021 (33 samples), represented by a total of 129 leaf samples. The collected samples were submitted to the following serological tests: immunoenzymatic nitrocellulose assay (Dot-ELISA) and Enzyme-Linked Immunosorbent Assay (ELISA) using anti-GRSV (0781-DSMZ, Germany), anti-potyvirus (0573/1-DSMZ), and anti-TSWV (0106/3-DSMZ) antibodies. Next, Nicotiana Benthamiana plants were mechanically inoculated and symptomatic leaf samples were subsequently dried at 4° C and sent to the Leibniz Institute - DSMZ for total RNA extraction and high-performance sequencing (HTS) by the Illumina Nextera platform (1-2Gb, 2x301 bp). Host range studies were performed using the following species: N. benthamiana, Nicandra physalodes, Physalis florida, Capsicum baccatum, Capsicum annuum, Solanum lycopersicum – Solanaceae and Citrullus lanatus, Cucumis anguria, Cucumis sativus – Cucurbitaceae. Each plant species was evaluated in experiments with ten replications. For the molecular analysis, specific oligonucleotides were designed and used in RT-PCR assays. With the generated reads the sequences corresponding to the S (3039 nt), M (4857 nt) and L (8882 nt) segments of the GRSV_RN isolate were assembled. Blastn analysis using nucleotide sequences of the S, M and L segments revealed the highest identities of 98.94%, 98.45%, 98.86%, respectively, with sequences of the GRSV isolate found in Piauí state, deposited at NCBI (MK503848, MK503850, MK503849). Twenty-six watermelon leaf samples from the second collection (January 2021) were tested via ACP-ELISA against GRSV and potyvirus, of which 12 were positive for both viruses, characterizing the occurrence of mixed infection. DAS-ELISA assays (anti-TSWV) revealed that 14 of 28 watermelon plants were positive for TSWV. Moreover, watermelon (Crimson sweet) (6), N. benthamiana (1) and N. physalodes (1) samples tested positive via RT-PCR for GRSV. Regarding the results of the host range study, species of N. benthamiana; N. physalodes; C. annuum; S. lycopersicum; P. florida and C. lanatus were susceptible to GRSV_RN, confirmed by Dot - ELISA tests. Furthermore, the recombination analysis did not indicate significant recombination events within GRSV sequences. Taken together, it was found that GRSV infects watermelon fields in Mossoró and Ceará, and that probably mixed infections with other viral species may contribute to the symptoms observed in the field.