A complex of begomoviruses (Geminiviridae) can cause severe tomato yield losses in the neotropics. Here, next-generation sequencing was employed for large-scale assessment of single-stranded (ss)DNA virus diversity in tomatoes either harboring or lacking the large-spectrum begomovirus tolerance Ty-1 gene. Individual leaf samples exhibiting begomovirus-like symptoms (n = 107) were field-collected, circular DNA-enriched, subdivided into pools (with and without Ty-1), and Illumina-sequenced. Virus-specific PCR and Sanger dideoxy sequencing validations confirmed 15 distinct ssDNA virus/subviral agents (occurring mainly in mixed infections), which highlight the potential drawbacks of employing virus-specific resistance in tomato breeding. More viruses (14 versus 6 species) were observed in tomatoes without the Ty-1 gene. A gemycircularvirus (Genomoviridae), a new alpha-satellite, and two novel Begomovirus species were identified exclusively in samples without the Ty-1 gene. A novel begomovirus was found only in the Ty-1 pool, being the only species associated with severe symptoms in Ty-1 plants in our survey. Our work is the first step towards the elucidation of the potential begomovirus adaptation to Ty-1 and its specific filtering effects on a subset of ssDNA viral/subviral agents.

An increased incidence and severity of Orthotospovirus species has been observed in the peanut crop. The typical symptoms of the virus, usually known as ringspot in peanut and spotted wilt in other crops, include mosaic, chlorotic ring-shaped spots, necrosis, and plant stunting. This study aimed to verify the occurrence of this virus in peanut growing areas in the São Paulo State, Brazil, identify the predominant virus species, and determine losses resulting from the presence of this virus. Surveys were carried out in four peanut commercial areas sown in the municipalities of Santa Adélia and Cândido Rodrigues in the 2014/2015 season using the cultivar Granoleico. The following parameters were evaluated: plant stand, number of plants with symptoms, and severity of these symptoms through a scoring scale of visual symptoms. The results showed that the predominant virus species is Groundnut ringspot tospovirus (GRSV). Also, the lower the stand is, the higher the percentage of plants with virus symptoms. The mean incidence of GRSV in commercial peanut areas is 40%, with mean losses estimated at 38% when GRSV is present and 64% when there is the presence of virus and reduction in plant density. Michelotto, M. D. et al. notas de sintomas visuais. Pelos resultados obtidos, constatou-se que a espécie de vírus predominante é Groundnut ringspot tospovirus (GRSV). Conclui-se que quanto menor o estande maior a percentagem de plantas com sintomas da virose. A incidência do GRSV nas áreas comerciais de amendoim é em média de 40% sendo as perdas médias estimadas em 38% quando há a presença do GRSV e em 64%, quando há a presença do vírus e a redução na densidade de plantas. Palavras-chave: Arachis hypogaea L. Incidência. Produtividade. Severidade. Tospovirus.

We report the first full-length genome of a Bougainvillea chlorotic vein banding virus, isolate BCVBV-UNB-01, causing leaf yellowing in a Bougainvillea spectabilis from Brasília, Brazil. The genome, recovered using high throughput sequencing (Illumina HiSeq) and Sanger, consisted of 8665 nucleotides and four typical badnavirus ORFs: ORF I with 456 nt in length, ORF II with 432 nt, ORF III with 6636 nt and ORF IV with 594 nt. Phylogenetic analysis using Bayesian inference of the 1228 bp of the RT + RNase H region along other 35 badnaviruses clustered isolate BCVBV-UNB-01 with BCVBV accession (NC011592) from Taiwan. The accurately aligned region of 1228 bp using Sequence Demarcation Tool (SDT) software, confirmed this result, evidencing a robust identification. In addition, further studies involving biological characteristics, distribution and diversity of BCVBV in Brazil will be conducted in an attempt for better understanding the role of interactions among virus, plant and vector.

Severe yield losses induced by a complex of whitefly–transmitted Begomovirus species (family Geminiviridae) have been reported in tomatoes in Brazil (Reis et al. 2020). Nine isolates were obtained from tomato plants exhibiting begomovirus–like symptoms (viz. apical and interveinal chlorosis, yellow spots, and stunting) during independent field surveys: one isolate in Sumaré, São Paulo–SP State (isolate SP–066) in 2001, two in Serra Negra, Minas Gerais–MG (MG–012 and MG–016) in 2002, five in Caxias do Sul, Rio Grande do Sul–RS (RS–039, RS–045, RS–046, RS–047 and RS–058) in 2011 and one in Domingos Martins, Espírito Santo–ES (ES–148) in 2016. Disease incidence across all sampled fields ranged from 30% (in Domingos Martins–ES) to 90% in Sumaré–SP. Total DNA extraction was done by a modified CTAB method (Boiteux et al., 1999). Begomovirus infection was confirmed in all isolates by selective amplification of viral DNA–A segments using the primer pairs ‘PAL1v1978 / PAR1c496’ (Rojas et al., 1993) and ‘BegomoAFor1’ / ‘BegomoARev1’ (Ha et al., 2006), which produce two large and non–overlapping segments (≈1120 bp and ≈1205 bp, respectively). These PCR amplicons were initially characterized via direct Sanger dideoxy sequencing at CNPH. BLASTn analysis of the partial DNA–A genomes of these nine isolates indicated identity levels of 95–97% to three euphorbia yellow mosaic virus (EuYMV) reference isolates (= KY559532, JF756674, and KY559583) found infecting the weed Euphorbia heterophylla L. The entire DNA–A (2,609 nts = MN746971) and DNA–B (2,579 nts = MN746970) components of the MG–016 isolate were obtained via high–performance sequencing using Illumina HiSeq 2500 system (Macrogen Inc., South Korea). Sequences were assembled with the CLC Genomics Workbench program 10. Contigs were validated by BLASTx and BLASTn and compared to the ssDNA virus database at NCBI (www.ncbi.nlm.nih.gov). The fully–characterized MG–016 isolate displayed identity levels ranging from 97 to 99% to the EuYMV reference isolates as well as similar genomic features such as the conserved TATA box, nonanucleotide, and iterons (that were in agreement with a cognate nature of the DNA–A and DNA–B components). A partial sequence of the DNA–B genome was also obtained for the MG–012 isolate (MT7831942). The isolates MG–012 and MG–016 were found in mixed infections with tomato severe rugose virus (ToSRV) and tomato golden vein virus (TGVV), respectively. In addition, the complete DNA–A genomes of ES–148 (MN746972) and SP–066 (MN782438) were also obtained via a combination of primer walking and Sanger dideoxy sequencing, displaying 96–98% identity to EuYMV isolates. To our knowledge, this is the first report of multiple and independent events of natural infection of tomatoes by EuYMV isolates. Our results confirm the natural host status of tomatoes to EuYMV isolates as indicated in previous infectivity assays using biolistic inoculation (Barreto et al., 2013). The weed E. heterophylla is widely disseminated and very often present within tomato fields due to its higher levels of tolerance to the major herbicide (metribuzin) employed in this crop. Therefore, this weed may act as a persistent reservoir of tomato–infecting EuYMV isolates, which may allow the selection of viral populations potentially more adapted to this vegetable crop.

Eggplant (Solanum melongena L.) is an economically important vegetable crop in Brazil, especially in family-based farming. Eggplant hybrids ‘Ciça’ and ‘Napoli’ (≈ 400 plants) were detected exhibiting virus-like symptoms (5-20% incidence) in field surveys (2015–2018) in Brasília–DF (Figure 1). Symptoms included chlorosis, mosaic and apical leaf deformation. Six symptomatic leaf samples were collected from fruit-bearing plants (around 100 days after planting) aiming at verifying the potential orthotospovirus infection. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out with polyclonal antibodies (produced at Centro Nacional de Pesquisa de Hortaliças – CNPH) against the N gene coat protein of the three major orthotospoviruses: tomato spotted wilt orthotospovirus (TSWV), groundnut ringspot orthotospovirus (GRSV) and, tomato chlorotic spot orthotospovirus (TCSV). Strong serological reactions were observed only against GRSV antibodies in the extracts from symptomatic samples, but not in the controls. To confirm the causal agent of those symptoms, total RNA was extracted from infected leaf samples via the standard Trizol® (Sigma) protocol and subsequently used in a two-step reverse transcriptase polymerase chain reaction (RT–PCR) approach. Synthesis of the cDNA was carried out with the J13 primer (5’–CCC GGA TCC AGA GCA AT–3’) (Cortez et al., 2001) followed by PCR assays with the primer pair BR60 (5’–AGA GCA ATC GTG TCA–3`) and BR65 (5`–ATC AAG CCT TCT GAA AGT CAT–3’) (Eiras et al., 2002). This primer set amplifies a fragment of 453 bp including the 3’ untranslated region at the 3’ terminus of the S RNA and the protein N-coding gene of at least five species: TSWV, GRSV, TCSV, chrysanthemum stem necrosis orthotospovirus (CSNV) and zucchini lethal chlorosis orthotospovirus (ZLCV). In addition, GRSV-specific primers (LNA Reis, unpublished) were used for amplification of all three segments: L segment: LF/LR (5’–AAC AGG ATT CAG CAA TAT GG–3’/ 5’–AAT TCC TTG AAG ACA ATT GTG T –3’); M segment: MF/MR (5’–TTT GTC CAA CCA TAC CAG ACC C– 3’ / 5’–GGC TTC AAT AAA GGC TTG GG–3’) and, S segment: SF/SR (5’–TTC AAA CTC AGT TGT ACT CTG A–3’/5’–TTA CTT TCG ATC TGG TTG AA– 3’). Amplicons with 509 bp (MT043204), 289 bp (MT043205), and 901 bp (MT043203) were obtained for L, M and S segments of the eggplant isolate DF-687. PCR amplicons corresponding to a segment of the N-coding gene (396 bp) of a second eggplant isolate (BJL02; MK176337) were obtained with the primer pair BR60/BR65 and subjected to Sanger dideoxy sequencing at CNPH. Alignments of nucleotide sequences of both isolates revealed identity levels varying around 99% to the corresponding genomic regions of a large set of GRSV isolates from GenBank database. PCR assays using total RNA as template yielded 494 bp amplicons solely with GRSV-specific primers (Webster et al., 2011), but no products were obtained with TSWV-specific primers (Adkins and Rosskopf, 2002), confirming the former as the sole causal agent of the field symptoms. Leaves of eggplant cv. ‘Ciça’ and indicator hosts, including Nicotiana rustica, Capsicum chinense ‘PI 159236’ (with the Tsw gene), and S. lycopersicum cv. Santa Clara were rub inoculated with extracts prepared from eggplant samples naturally infected with GRSV. Mosaic, necrotic ringspots and systemic leaf deformation symptoms were observed around ten days after inoculation on newly emerged leaves of all inoculated plants. GRSV infection was confirmed by DAS-ELISA and RT-PCR ten days after inoculation. Eggplant was erroneously listed as a host of GRSV in Brazil (Kitajima, 2020). Hence, this is the first report of eggplant infection by this virus in South America. No significant yield losses have been observed in eggplant due to GRSV infection since the overall symptoms are often mild. However, this natural host of GRSV might impact disease management strategies since eggplant is quite often cultivated under family-based farming conditions as a companion crop of highly susceptible tomato, sweet-pepper, and lettuce cultivars. References: Adkins, S., and Rosskopf, E. N. 2002. Plant Dis. 86: 1310. Cortez, I., et al. 2001. Arch. Virol. 146:265. Eiras, M. et al., 2002. Fitopatol. Bras. 27:285. Kitajima, E.W. 2020. Biota Neotrop. 20: e2019932. Webster, C. G., et al. 2011. Virology 413: 216.

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