Tomato golden net virus and tomato yellow net virus: two novel New World begomoviruses with monopartite genomes.

Two novel tomato-infecting begomoviruses were discovered via high-throughput sequencing in Brazil. Both viruses were also Sanger-sequenced and displayed DNA-A components phylogenetically related to New World bipartite begomoviruses. The names tomato golden net virus (ToGNV) and tomato yellow net virus (ToYNV) were proposed. The majority of the New World begomoviruses has bipartite genomes. However, extensive analyses revealed that ToGNV and ToYNV have monopartite genomes, because no cognate DNA-B components were detected. Hence, they may comprise a unique group of monopartite New World begomoviruses, which have enormous biological, molecular, and plant breeding interest.

Confirmation of Groundnut ringspot orthotospovirus (GRSV) Infection in Eggplant Cultivars in Brazil.

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.

Complete genome sequence of a novel bipartite begomovirus infecting the legume weed Macroptilium erythroloma.

The natural occurrence of mixed infections and large populations of the polyphagous vector (Bemisia tabaci) are the main factors associated with the intensification of the genetic flow among begomoviruses in Neotropical areas, contributing to the emergence of novel recombinants. Here, high-throughput sequencing and metagenomic analyses were employed to discover and characterize a novel recombinant bipartite begomovirus, tentatively named "macroptilium bright yellow interveinal virus" (MaBYIV) in the weed Macroptilium erythroloma (Fabaceae). Recombination signals were detected in MaBYIV, involving bean golden mosaic virus (BGMV) and tomato mottle leaf curl virus (ToMoLCV) genome components. All of the original MaBYIV-infected M. erythroloma plants were found to have mixed infections with BGMV. MaBYIV was transmitted to bean and soybean cultivars via B. tabaci MEAM 1, indicating that M. erythroloma may play a role as a year-round reservoir of a potential new viral pathogen of economically important legume crops.

Stability analysis of reference genes for RT-qPCR assays involving compatible and incompatible Ralstonia solanacearum-tomato 'Hawaii 7996' interactions.

Reverse transcription-quantitative PCR (RT-qPCR) is an analytical tool for gene expression quantification. Reference genes are not yet available for gene expression analysis during interactions of Ralstonia solanacearum with 'Hawaii 7996' (the most stable source of resistance in tomato). Here, we carried out a multi-algorithm stability analysis of eight candidate reference genes during interactions of 'Hawaii 7996' with one incompatible/avirulent and two compatible/virulent (= resistance-breaking) bacterial isolates. Samples were taken at 24- and 96-h post-inoculation (HPI). Analyses were performed using the ∆∆Ct method and expression stability was estimated using BestKeeper, NormFinder, and geNorm algorithms. TIP41 and EF1α (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and TIP41 (with BestKeeper), were the best combinations for mRNA normalization in incompatible interactions at 24 HPI and 96 HPI. The most stable genes in global compatible and incompatible interactions at 24 HPI and 96 HPI were PDS and TIP41 (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and PDS/EXP (with BestKeeper). Global analyses on the basis of the three algorithms across 20 R. solanacearum-tomato experimental conditions identified UBI3, TIP41 and ACT as the best choices as reference tomato genes in this important pathosystem.

Tomato yellow vein streak virus and Tomato golden vein virus: a reappraisal of the classification status of two South American Begomovirus species based upon genome-wide pairwise identity of multiple isolates.

Tomato yellow vein streak virus (ToYVSV) and tomato golden vein virus (TGVV) are begomoviruses reported infecting tomatoes and other hosts across South America. However, their close phylogenetic relationship has generated uncertainties about their taxonomic status and nomenclature. In fact, genomic DNA-A identity levels of isolates reported with an identical virus name may range from 89-100%. In view of the potential inaccuracy regarding the classification status of these viruses (strains vs. distinct species), we carried out a comprehensive set of analyses employing all 45 available isolates with complete DNA-A sequences with either ToYVSV or TGVV designation. Two clear-cut clusters were identified and they were consistent with the current criteria for Begomovirus species demarcation. Moreover, our reappraisal confirmed a large array of misnamed isolates and recognized a distinctive set of virus species-specific genomic, biological, and ecological features. Hence, the present work gives support to the notion that these viruses are closely-related, but they are distinct and valid Begomovirus species. From the breeding standpoint, this information will be useful in guiding germplasm screening strategies searching for sources of large-spectrum resistance to isolates of both viruses.

First Report of Cichorium endivia (Asteraceae) as a Natural Host of Groundnut ringspot orthotospovirus in Brazil.

Endive (Cichorium endivia L.) is a very important cash crop for small farmers in Brazil. During inspections conducted in the summer season of 2019-2020, leaf samples of C. endivia 'La Spezia' seedlings exhibiting typical symptoms of orthotospoviruses infection (viz. concentric chlorotic spots and apical leaf deformation; ≈ 10%) were collected in commercial greenhouses in Brasília-DF, Central Brazil. Leaves of one healthy and three symptomatic plants were initially evaluated via double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antibodies (produced at CNPH) raised against the nucleoprotein 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 exclusively in extracts from symptomatic samples. In order to confirm the causal agent of those symptoms, total RNA was extracted (Trizol®; Sigma) from infected leaf samples and 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., 2001). This primer set amplifies a fragment of 453 bp including the untranslated region at the 3' terminus of the small RNA and the protein N-coding gene of at least five orthotospoviruses: TSWV, GRSV, TCSV, chrysanthemum stem necrosis orthotospovirus (CSNV) and zucchini lethal chlorosis orthotospovirus (ZLCV) (Eiras et al., 2001). The obtained amplicons (≈ 432 bp) were subsequently subjected to Sanger dideoxy nucleotide sequencing at CNPH. BLASTn analysis showed >99% identity with a wide array of GRSV isolates available in the GenBank. The nucleotide sequence of Tospo #1 (MT215222) and Tospo #3 (MT215224) isolates displayed 100% identity between them, whereas the Tospo #2 (MT215223) isolate displayed one non-synonymous point mutation in the 3' untranslated region in comparison with the former two isolates. Three plants of C. endivia, Capsicum annuum L. cv. Ikeda, tomato (Solanum lycopersicum L.) cv. Santa Clara and its isoline 'LAM-147' (with the Sw-5 resistance gene), Nicotiana rustica L., Lactuca sativa L. ('Vanda' and 'PI-342444') and Gomphrena globosa L. were mechanically inoculated individually with each GRSV isolate in order to confirm their pathogenicity. Chlorotic lesions and mosaic were observed seven days after inoculation of all plant materials, except the tomato inbred line 'LAM-147', which has the Sw-5 gene that confers broad-spectrum resistance to all Brazilian orthotospoviruses (Boiteux and Giordano, 1993). The GRSV infection was confirmed via DAS-ELISA and RT-PCR 15 days after inoculation, using the same set of antibodies and the primer pair BR60 / BR65. Transmission electron microscopy of ultrathin sections from symptomatic leaf tissues, both from field-infected and experimentally inoculated endive revealed the presence of typical orthotospovirus particles, within endoplasmic reticulum cisternae. Natural infection of endive by TSWV has been reported in Greece (Chatzivassiliou et al., 2000) and by TCSV in São Paulo State, Brazil and in Florida, USA (Subramanya Sastry et al., 2019). To our knowledge, it is the first report of GRSV naturally infecting this Asteraceae species in Brazil. Confirmation of GRSV infection of C. endivia plants is a relevant piece of information aiming to design effective disease management strategies. References: Boiteux, L.S. and Giordano, L. B. 1993. Euphytica 71: 151. Eiras, M. et al. 2001. Fitopatol. Bras. 26: 170. Chatzivassiliou, E.K. et al. 2000 Ann. Appl. Biol. 137: 127. Cortez, I., et al. 2001. Arch. Virol. 146: 265. Subramanya Sastry, K., et al. 2019. Encyclopedia of plant viruses and viroids. Springer, New Delhi. https://doi.org/10.1007/978-81-322-3912-3.

Natural Infection of Tomatoes (Solanum lycopersicum) by Euphorbia yellow mosaic virus Isolates across Four Brazilian States.

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.

Current Status of Soybean Anthracnose Associated with Colletotrichum truncatum in Brazil and Argentina.

Brazil and Argentina have a combined soybean area of 53.6 million hectares, which accounts for over half of the total global production. The soybean crop in South America extends from latitude 8-10° S to 32-36° S. Such a vast, almost contiguous area imposes a serious sanitary risk to the crop. Currently, the prevalence of anthracnose is increasing, with recurring reports of severe epidemics and expressive yield losses. Soybean anthracnose is mainly associated with Colletotrichum truncatum, although other Colletotrichum species have also been reported as causal agents of this disease. Knowledge about the morphological, cultural, and molecular variability of C. truncatum in South America is crucial for disease management. Here, we present data on the molecular, morphological, biological, cultural, and pathogenicity of C. truncatum isolates collected in Brazil and Argentina. Light microscopy and randomly-amplified polymorphic DNA (RAPD) analysis were used for estimating the variability of isolates. Colletotrichum truncatum displayed three types of conidiogenesis, viz. conidial formation from conidiogenous cells on hyphal extremities, in conidiomas in acervuli, and directly from fertile setae (a mechanism yet-unreported for C. truncatum). RAPD profiling was effective in revealing the genetic diversity among C. truncatum isolates. The intra-group similarity was greater among the Argentinian isolates when compared to the Brazilian group. Furthermore, the results indicated a strong correlation between geographical origin and molecular grouping, with the exclusive or semi-exclusive assembling of Brazilian and Argentinian isolates in distinct clades. Finally, a preliminary account of the reaction of soybean accessions to C. truncatum is also included.

Nanopore sequencing of a novel bipartite New World begomovirus infecting cowpea.

A new bipartite begomovirus (family Geminiviridae) was detected on cowpea (Vigna unguiculata) plants exhibiting bright golden mosaic symptoms on leaves under field conditions in Brazil. Complete consensus sequences of DNA-A and DNA-B components of an isolate of the virus (PE-088) were obtained by nanopore sequencing and confirmed by Sanger sequencing. The genome components presented the typical genomic organization of New World (NW) begomoviruses. Pairwise sequence comparisons revealed low levels of identity with other begomovirus species previously reported infecting cowpea around the world. Phylogenetic analysis using complete sequences of DNA-A components revealed that the closest relatives of PE-088 (85-87% nucleotide sequence identities) were three legume-infecting begomoviruses from Brazil: bean golden mosaic virus, macroptilium common mosaic virus and macroptilium yellow vein virus. According to the current classification criteria, PE-088 represents a new species in the genus Begomovirus, tentatively named as cowpea bright yellow mosaic virus (CoBYMV).

Recessive Resistance Derived from Tomato cv. Tyking-Limits Drastically the Spread of Tomato Yellow Leaf Curl Virus.

The tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato (Solanum lycopersicum L.) crops throughout tropical and subtropical regions of the world. TYLCD is associated with a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) transmitted by the whitefy Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). The tomato inbred line TX 468-RG is a source of monogenic recessive resistance to begomoviruses derived from the hybrid cv. Tyking F1. A detailed analysis of this germplasm source against tomato yellow leaf curl virus-Israel (TYLCV-IL), a widespread TYLCD-associated virus, showed a significant restriction to systemic virus accumulation even under continuous virus supply. The resistance was effective in limiting the onset of TYLCV-IL in tomato, as significantly lower primary spread of the virus occurred in resistant plants. Also, even if a limited number of resistant plants could result infected, they were less efficient virus sources for secondary spread owing to the impaired TYLCV-IL accumulation. Therefore, the incorporation of this resistance into breeding programs might help TYLCD management by drastically limiting TYLCV-IL spread.