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.

New genus of trichomatous coelomycete on Myrcia fenzliana from the Brazilian Cerrado.

Megacoelomyces (type species: Megacoelomyces sanchezii), an ascomycete asexual morph infecting Myrcia fenzliana (Myrtaceae) from the Brazilian Cerrado, is described as a new genus in the Phaeosphaeriaceae (Pleosporales, Dothideomycetes, Ascomycota), based on multilocus phylogeny (three nuclear ribosomal DNA and two protein-coding genes) in addition to morphological (light and scanning electron microscopy) and ecological data.

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.

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.

Metagenomics of Neotropical Single-Stranded DNA Viruses in Tomato Cultivars with and without the Ty-1 Gene.

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.

New Stenella and Parastenella species from the Brazilian cerrado.

Five new Stenella species were found on native cerrado plants. Stenella erythroxyli-campestris, S. erythroxyli-suberosi and S. erythroxylicola were associated with plant species belonging in the family Erythroxylaceae; S. cyrtopodii was found infecting the rare Cyrtopodium eugenii (Orchidaceae), and S. ocoteae occurred on Ocotea sp. (Lauraceae). Finally Parastenella callisthenis-fasciculatae was collected on a Vochysiaceae (viz. Callisthene fasciculate) endemic to the cerrado.