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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 (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.

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).

Fusarium wilt, caused by Fusarium oxysporum f. sp. lactucae (FOLac), is amongst the main diseases affecting lettuce in subtropical regions. Although nationwide surveys indicated the exclusive presence of FOLac race 1 in Brazil, no detailed studies are available providing molecular evidences if these isolates were introduced into the country via contaminated seeds or if they are endemic populations. The translation elongation factor 1α (EF-1α) gene and rDNA intergenic spacer (IGS-rDNA) region represent the most comprehensive databases for comparative analyses of Fusarium isolates. Our aim was to assess the genetic relationships of 23 Brazilian FOLac race 1 isolates with a collection of FOLac isolates of worldwide origin, using the information from these genomic regions. A consistent single-cluster pattern was observed for FOLac race 1 isolates from Brazil, California-USA, Arizona-USA, Japan, Italy, as well as the novel FOLac race 4 isolates from the Netherlands based upon the EF-1α (604 nucleotides) and the IGS-rDNA (1859 nucleotides) sequences. Our analysis (based upon six single nucleotide polymorphisms identified only in the IGS-rDNA sequence) allowed the identification of intra-race 1 variation with the discrimination of four haplotypes. Isolates from Brazil, Italy, and a subset from the USA were classified into a single haplotype. The low diversity levels and the presence of only a single haplotype across the entire country are strong indications that Brazilian FOLac race 1 isolates are result of recent introduction event(s). This fast and widespread distribution of FOLac race 1 in Brazil has occurred more likely via importation and planting of contaminated seeds.

Fusarium oxysporum f. sp. lactucae (FOLac) is responsible for significant economic losses across major lettuce-producing regions around the world. Thus far, only FOLac race 1 isolates have been reported associated with Fusarium wilt outbreaks in Brazil. The most sustainable strategy for disease control is the pyramidization of race-specific resistance factors in lettuce cultivars. The loose-leafy cultivar ‘Vanda’ was found as one of the most promising sources of resistance to FOLac race 1. The genetic basis of this resistance was determined by analyzing the reaction to this pathogen of segregating populations derived from the cross ‘Gisele’ (susceptible) × ‘Vanda’ (pollen donor). A single molecular marker-genotyped F1 hybrid plant was selfed and individual plants of a segregating F2 population as well as 63 families F2:F3 were inoculated with a FOLac race 1 isolate by using the root-dipping method (3 × 106 conidia/ml). Our results confirmed the high levels of resistance of ‘Vanda’ even under very harsh experimental conditions. Overall, the reaction of the F1 plants and the segregating patterns of the F2 population (n = 82) and of the F2:F3 families (n = 838 plants) fit a single dominant gene/locus model. However, the phenotypic expression of resistance might suffer effects of additional genetic factor(s) (e.g., locus dosage, minor modifying genes, and incomplete penetrance). Notwithstanding, the high levels of FOLac race 1 resistance and its relatively simple genetic control makes ‘Vanda’ a major germplasm source for lettuce-breeding programs aiming to incorporate this trait in a wide array of elite lines from distinct varietal groups.

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.

Verticillium dahliae is a soil-borne fungal pathogen responsible for vascular wilt diseases in more than 300 dicotyledonous species, including solanaceous vegetable crops. In this study, a collection of 89 Brazilian V. dahliae isolates was characterized by combing molecular information for mating type and physiological race determination, as well as via virulence bioassays employing a set of differentials. Based on the virulence assays, three isolates were classified as race 1, 76 were classified as race 2, whereas ten isolates did not cause any symptom on the tested cultivars.” In race-specific detection, a total of six isolates were identified as race 1, 70 as race 2, and 13 isolates displayed no amplicon with any primer set employed. Therefore, V. dahliae race 2 isolates are currently ubiquitous across major Solanaceae-producing areas in Brazil. Both MAT idiomorphs were detected, but a larger number of the V. dahliae isolates displayed the MAT1–1 (82%) pattern in our molecular analyses. The simultaneous presence of both MAT idiomorphs opens the possibility of sporadic events of sexual reproduction among V. dahliae populations from Brazil, enabling the potential emergence of either new recombinant isolates with broader host ranges or novel physiological races. Our results clearly indicated the need to intensify the search for effective sources of resistance to V. dahliae race 2 in tomato breeding programs under Brazilian conditions.

The best levels of broad-spectrum Tospovirus resistance reported in tomatoes thus far are conferred by the Sw-5 locus. This locus contains at least five paralogues (denoted Sw-5a through Sw-5e), of which Sw-5b represents the actual resistance gene. Here we evaluated a panel of seven PCR primer pairs matching different sequences within a genomic region spanning the Sw-5a and Sw-5b gene cluster. Primer efficiency evaluation was done employing tomato isolines with and without the Sw-5 locus. One primer pair produced a single and co-dominant polymorphism between susceptible and resistant isolines. Sequence analysis of these amplicons indicated that they were specific for the Sw-5 locus and their differences were due to insertions/deletions. The polymorphic SCAR amplicon encompass a conserved sequence of the promoter region of the functional Sw-5b gene, being located in the position −31 from its open reading frame. This primer pair was also evaluated in field assays and with a collection of accessions known to be either susceptible or resistant to tospoviruses. An almost complete correlation was found between resistance under greenhouse/field conditions and the presence of the marker. Therefore, this primer pair is a very useful tool in marker-assisted selection systems in a large range of tomato accessions.

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.

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.