Novel plant-associated genomoviruses from the Brazilian Cerrado biome.

The discovery rate of new plant viruses has increased due to studies involving high-throughput sequencing (HTS), particularly for single-stranded DNA viruses of the family Genomoviridae. We carried out an HTS-based survey of genomoviruses in a wide range of native and exotic trees grown in the Brazilian Cerrado biome, and the complete genome sequences of two novel members of the family Genomoviridae from two distinct genera were determined. Specific primers were designed to detect these genomoviruses in individual samples. A new gemykolovirus (Tecoma stans associated gemykolovirus) was detected in Tecoma stans, and a new gemykibivirus (Ouratea duparquetiana associated gemykibivirus) was detected in Ouratea duparquetiana. A gemykrogvirus related to Gila monster associated gemykrogvirus (80% pairwise identity) was also detected in foliar samples of Trembleya parviflora. Our pilot study paves the way for a better characterization of this diverse collection of genomoviruses as well as their interactions with the associated tree species.

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.

The Sw-5b NLR Immune Receptor Induces Early Transcriptional Changes in Response to Thrips and Mechanical Modes of Inoculation of Tomato spotted wilt orthotospovirus.

The NLR (nucleotide-binding leucine-rich repeat) class immune receptor Sw-5b confers resistance to Tomato spotted wilt orthotospovirus (TSWV). Although Sw-5b is known to activate immunity upon recognition of the TSWV movement protein NSm, we know very little about the downstream events that lead to resistance. Here, we investigated the Sw-5b-mediated early transcriptomic changes that occur in response to mechanical and thrips-mediated inoculation of TSWV, using near-isogenic tomato lines CNPH-LAM 147 (Sw5b+/+) and Santa Clara (Sw-5b-/-). We observed earlier Sw-5b-mediated transcriptional changes in response to thrips-mediated inoculation compared with that in response to mechanical inoculation of TSWV. With thrips-mediated inoculation, differentially expressed genes (DEGs) were observed at 12, 24, and 72 h postinoculation (hpi). Whereas with mechanical inoculation, DEGs were observed only at 72 hpi. Although some DEGs were shared between the two methods of inoculation, many DEGs were specific to either thrips-mediated or mechanical inoculation of TSWV. In response to thrips-mediated inoculation, an NLR immune receptor, cysteine-rich receptor-like kinase, G-type lectin S-receptor-like kinases, the ethylene response factor 1, and the calmodulin-binding protein 60 were induced. Fatty acid desaturase 2-9, cell death genes, DCL2b, RIPK/PBL14-like, ERF017, and WRKY75 were differentially expressed in response to mechanical inoculation. Our findings reveal Sw-5b responses specific to the method of TSWV inoculation. Although TSWV is transmitted in nature primarily by the thrips, Sw-5b responses to thrips inoculation have not been previously studied. Therefore, the DEGs we have identified in response to thrips-mediated inoculation provide a new foundation for understanding the mechanistic roles of these genes in the Sw-5b-mediated resistance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Diversity of tomato-infecting begomoviruses and spatiotemporal dynamics of an endemic viral species of the Brazilian Atlantic rain forest biome.

Yield losses induced by a complex of begomoviruses are observed across all major tomato-producing areas in Brazil. Tomato severe rugose virus (ToSRV) is the most widespread begomovirus in the country. Conversely, tomato common mosaic virus (ToCmMV) displays a more restricted geographical distribution to areas associated with the Atlantic Rain Forest (ARF) biome, encompassing the States of Espírito Santo-ES, Minas Gerais-MG, and Rio de Janeiro-RJ. Here, we characterized 277 tomato-infecting isolates collected in fields located within the ARF biome from 2006 to 2018. ToSRV displayed the highest prevalence (n = 157), followed by ToCmMV (n = 95) and tomato interveinal chlorosis virus (n = 14). Four other begomoviruses were also detected, but with very low incidences. ToCmMV was the predominant begomovirus in the ARF biome up to 2014-2015 with very low ToSRV incidence. Subsequently, ToSRV became the most prevalent species in ES and RJ, but ToCmMV was still predominating in the "Zona da Mata" meso-region in MG. Due to the remarkable endemic distribution of ToCmMV, we carried out phylogeographical studies of this virus using information from all 28 available isolates with complete DNA-A sequences. The closest common ancestor of ToCmMV was more likely originated around Coimbra-MG area ≈ 25 years before the formal report of this viral species. So far, all surveys indicated tomatoes as the only natural hosts of ToCmMV with outbreaks occurring mainly (but not exclusively) in highland areas. ToSRV shows a more widespread incidence across both highland and lowland areas of the ARF biome.

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.

Molecular Characterization of Hovenia Dulcis-Associated Virus 1 (HDaV1) and 2 (HDaV2): New Tentative Species within the Order Picornavirales.

In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5' ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.

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).

The Sw-5 Gene Cluster: Tomato Breeding and Research Toward Orthotospovirus Disease Control.

The Sw-5 gene cluster encodes protein receptors that are potentially able to recognize microbial products and activate signaling pathways that lead to plant cell immunity. Although there are several Sw-5 homologs in the tomato genome, only one of them, named Sw-5b, has been extensively studied due to its functionality against a wide range of (thrips-transmitted) orthotospoviruses. The Sw-5b gene is a dominant resistance gene originally from a wild Peruvian tomato that has been used in tomato breeding programs aiming to develop cultivars with resistance to these viruses. Here, we provide an overview starting from the first reports of Sw-5 resistance, positional cloning and the sequencing of the Sw-5 gene cluster from resistant tomatoes and the validation of Sw-5b as the functional protein that triggers resistance against orthotospoviruses. Moreover, molecular details of this plant-virus interaction are also described, especially concerning the roles of Sw-5b domains in the sensing of orthotospoviruses and in the signaling cascade leading to resistance and hypersensitive response.

BIFURCATE FLOWER TRUSS: a novel locus controlling inflorescence branching in tomato contains a defective MAP kinase gene.

A mutant line, bifurcate flower truss (bif), was recovered from a tomato genetics programme. Plants from the control line produced a mean of 0.16 branches per truss, whereas the value for bif plants was 4.1. This increase in branching was accompanied by a 3.3-fold increase in flower number and showed a significant interaction with exposure to low temperature during truss development. The control line and bif genomes were resequenced and the bif gene was mapped to a 2.01 Mbp interval on chromosome 12; all coding region polymorphisms in the interval were surveyed, and five candidate genes displaying altered protein sequences were detected. One of these genes, SlMAPK1, encoding a mitogen-activated protein (MAP) kinase, contained a leucine to stop codon mutation predicted to disrupt kinase function. SlMAPK1 is an excellent candidate for bif because knock-out mutations of an Arabidopsis orthologue MPK6 were reported to have increased flower number. An introgression browser was used to demonstrate that the origin of the bif genomic DNA at the BIF locus was Solanum galapagense and that the SlMAPK1 null mutant is a naturally occurring allele widespread only on the Galápagos Islands. This work strongly implicates SlMAPK1 as part of the network of genes controlling inflorescence branching in tomato.

Genome Sequence of Ralstonia pseudosolanacearum Strains with Compatible and Incompatible Interactions with the Major Tomato Resistance Source Hawaii 7996.

We report here the complete genome sequences of two Ralstonia pseudosolanacearum strains, isolated from the warm northeast region of Brazil. They display divergent (compatible versus incompatible) interactions with the resistant tomato line Hawaii 7996. Polymorphisms were detected in a subset of effector genes that might be associated with these contrasting phenotypes.

Plant responses to tomato chlorotic mottle virus: Proteomic view of the resistance mechanisms to a bipartite begomovirus in tomato.

Tomato chlorotic mottle virus (ToCMoV) is a widespread bipartite Begomovirus species found in tomato fields in Brazil. In this study, plant responses and putative mechanisms associated with the 'Tyking'-derived recessive resistance to ToCMoV were investigated. Changes in the protein profile in the inoculated plants of two near isogenic tomato lines resistant ('LAM 157') and susceptible ('Santa Clara') to ToCMoV were analyzed. Seedlings were biolistically inoculated with an infectious ToCMoV clone. Leaves from infected plants (confirmed by PCR) were sampled at 15days after inoculation. Proteins were extracted using phenol and analyzed by shotgun MS (2D-nanoUPLC/HDMSE). Out of the 534 identified proteins, 82 presented statistically significant differences in abundance, including 35 unique proteins displayed in the resistant tomato inoculated with ToCMoV. Proteins associated to chromatin structure, cytoskeleton structure, cuticle biosynthesis, and ubiquitin pathway were identified and their putative roles during virus infection process were discussed. The protein profile analysis allowed for the development of a hypothetical model showing how the resistant host cell responds to ToCMoV infection. The data obtained provide a better understanding of resistant mechanisms used by the host plant to contain viral infection and could be the basis for further investigation in other plant-begomovirus pathosystems. BIOLOGICAL SIGNIFICANCE: In this study we propose a model of resistance to begomovirus in tomato and highlight host proteins, which could be targets for future investigations in plant-begomovirus pathosystems.

Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems.

Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L.) genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes ('Santa Clara', susceptible, and its near-isogenic line 'LAM 157', resistant); subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV) and its mock-inoculated control) and in two distinct times after inoculation (early and late). Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper). To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.

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.

Meloidogyne polycephannulata n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitizing carrot in Brazil.

Meloidogyne polycephannulata n. sp. is described from specimens collected from an area cultivated with carrot cv. Brasilia, in the city of Rio Paranaíba, in the region of Alto Paranaíba, Minas Gerais State, Brazil. The perineal pattern of the female is circular to ovoid with a high dorsal arch that has widely spaced, coarse annulations. The lateral field may have a deep furrow separating the dorsal and ventral arches. The medial lips are short and wide, whereas the lateral lips are large and triangular. The female stylet is 15-16 µm long with wide knobs, distinctly divided by an indentation in the center. Its tip is slightly curved dorsally. The excretory pore opens 34-65 µm from the anterior end. Females retain eggs and second-stage juveniles in their body cavity, similar to that of the cyst-forming nematodes. Males are 1.3-1.7 mm long and have a high head cap that is rounded and slopes posteriorly. The labial disc is fused to the medial lips. The head region has several irregular annulations that are similar in appearance to the first or second body annules that are likewise irregular, making the head region appear to be extremely large. The stylet of the male is 21-24 µm long; it is slender, and has small, rounded knobs, that are distinctly indented medially and appear heart-shaped. The shaft has several tiny projections throughout its length. Mean second-stage juvenile length is 411.7 µm. The juvenile head cap is elevated, the medial lips are small, and the lateral lips are elongate to triangular-shaped. The head region has several short, incomplete and irregular transverse annulations. The juvenile stylet is 14-23 µm long with small, rounded, and sloping knobs. The thin tail ends with a short hyaline portion that is variable in size (16-26 µm) and with a small, rounded tip. Isozyme profiles of esterases from Meloidogyne javanica show 3 strong bands (SB) at Rm 46, 59, and 66; profiles of M. polycephannulata n. sp. show a SB at Rm 47 and a weak band (WB) at Rm 52; M. petuniae has two SB at Rm 44 and 53; M. phaseoli has a SB at 53, 58, and 64 Rm; M. brasilensis has three SB at Rm 40, 58, and 66 and a WB at Rm 71; M. pisi has a SB at Rm 40, 60, and 64 and two WB at 46 and 50 Rm. Data from sequencing the 18S rDNA region of M. polycephannulata n. sp. confirms that it is different from M. arabicida, M. arenaria, M. ethiopica, M. incognita, M. javanica, M. paranaensis, and M. thailandica. Sequence identity among these eight species ranged between 85 to 93.4%. Meloidogyne polycephannulata n. sp. reproduces very well on carrot and tomato; poorly on pepper; and not at all on cotton, peanut, tobacco, watermelon, and sweet corn.

A chromosome-specific estimate of transmission of heterozygosity by 2n gametes in potato.

Polyploid plants are formed when numerically unreduced (2n) gametes participate in fertilization. Based on cytological and genetic analyses, modes of 2n gamete formation have been determined for a number of plant species. Gametes formed by a first-division restitution (FDR) mechanism contain nonsister chromatids near the centromere, whereas those formed by second-division restitution (SDR) contain sister chromatids. These mechanisms differ in the proportion of heterozygous loci they transmit intact to offspring. This paper estimates the transmission of heterozygosity on an individual chromosome basis through pachytene analysis of chromosomes of haploids (2n = 2x = 24) of Solanum tuberosum Andigena Group (2n = 4x = 48), a South American cultivated potato. Transmission of heterozygosity by FDR and SDR 2n gametes was calculated for 6 different cytogenetic assumptions. FDR was more than twice as effective as SDR in transmission of heterozygosity under all 6 scenarios. Rates of transmission of heterozygosity were similar in each situation. Transmission of heterozygosity by FDR was also compared with transmission of heterozygosity by tetrasomic inheritance and found to be approximately 50% more effective.