First report of tomato fruit blotch virus infecting tomatoes in Brazil

Recently, a new blunervirus was reported in tomatoes showing fruit chlorotic lesions. This virus, named tomato fruit blotch virus (ToFBV), was found associated with the tomato fruit blotch disease in Italy and Australia, even though Koch’s postulates were not fulfilled and no viral particles were seen in leaf dips observed with an electron microscope (Ciuffo et al. 2020). In December 2019, symptoms of circular or irregular chlorotic blotches were observed in tomato fruits in an organic farm in Distrito Federal, Brazil. Five different tomato cultivars (2100 plants of cv. Sweet grape, 1700 of Giacomo, 560 of Grazianni, 160 of Tropical, and 160 of DRC 5640) were being grown in two greenhouses and all of them presented the symptoms in at least one fruit, particularly in older fruits. No virus-like symptoms were observed in young and middle leaves, but older leaves could not be examined because they were removed as a routine activity of the farm; and also due to the moderate infestation of the tomato russet mite Aculops lycopersici, associated with leaf and stem necrosis. No viral particles were observed in an electron microscope analysis of symptomatic fruit tissues, and sap inoculation and grafting of stems did not produce any symptom in indicator plants. Two young and asymptomatic plants with the first fruits still in development were removed from another greenhouse of the farm and transported to our greenhouse, but the typical blotch symptoms neither appeared in the fruits nor the necrosis symptoms in the leaves. Serological tests performed for all collected leaf and fruit samples using antibodies produced in-house against common tomato-infecting tospoviruses and potyviruses were negative, as well as a polymerase chain reaction (PCR) detection test for begomoviruses (Rojas et al. 1993). Total RNA from newly collected samples consisting of one symptomatic fruit sample and five asymptomatic leaf samples from distinct plants were individually extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and pooled for next generation sequencing (NGS). The library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina, San Diego, USA) and sequenced at Macrogen, Inc. (Seoul, South Korea) in an Illumina Novaseq6000 platform. The 4,621,977,958 reads obtained were trimmed using Trimommatic 0.35 (Bolger et al. 2014) and contigs were assembled using Velvet (Zerbino and Birney 2008). Following tblastx analysis on Geneious 9.1.8 (Biomatters Ltd.) and BLAST on the NCBI platform (Altschul et al. 1990), seven contigs matching tomato chlorosis virus (ToCV) and five contigs matching ToFBV were identified. Sequences for each of the four genome components of ToFBV (MK517477-MK517480) already present in databases were used as reference using the Map to Reference function in Geneious. A total of 338,402, 78,039, 555,302 and 461,474 reads mapped to virus genome components 1 to 4, respectively, with >99% coverage for each. Four final consensus sequences were used for BLAST analyses on NCBI and presented 97 to 99.7 % nucleotide identity with those used for mapping. These sequences were deposited in GenBank as isolate MAL under accession numbers MW546267 (RNA 1, 5770 nt), MW546268 (RNA 2, 3612 nt), MW546269 (RNA 3, 2826 nt) and MW546270 (RNA 4, 1950 nt). The primer pair Bluner1F (5’-ATTCCTGTTCCTTCGGATAAACTCGT-3’) and Bluner1R (5’-CACACGTGCAGGAAATGGAAAGA-3’) directed to RNA 1 was used to specifically detect the virus. Three leaf samples and two fruit samples, each from a different plant with typical symptoms, were tested positive for ToFBV and negative using ToCV-specific primers in RT-PCR (Dovas et al. 2002). This confirmed that although some plants pooled in the HTS library were infected with ToCV, the chlorotic blotch symptom was clearly associated with the presence of ToFBV. Furthermore, the ~0.5 kbp amplicon for ToFBV-specific primers from one randomly selected sample was sequenced with both primers and the resulting sequence shared 100% nt identity with the RNA 1 of ToFBV isolate Fondi2018 from Italy (MK517477). Then, the virus was detected in the tissue from the surface of another fruit, but not from its internal part, suggesting a superficial infection. The findings presented here are of high phytosanitary significance, given the strong symptoms associated with tomato fruit blotch disease and the identification of ToFBV in the tomato samples from Brazil.