Evaluation of Tomato Germplasm against Tomato Brown Rugose Fruit Virus and Identification of Resistance in Solanum pimpinellifolium.

The tomato is one of the most important vegetable crops grown worldwide. Tomato brown rugose fruit virus (ToBRFV), a seed-borne tobamovirus, poses a serious threat to tomato production due to its ability to break the resistant genes (Tm-1, Tm-2, Tm-22) in tomatoes. The objective of this work was to identify new resistant source(s) of tomato germplasm against ToBRFV. To achieve this aim, a total of 476 accessions from 12 Solanum species were tested with the ToBRFV US isolate for their resistance and susceptibility. As a result, a total of 44 asymptomatic accessions were identified as resistant/tolerant, including thirty-one accessions of S. pimpinellifolium, one accession of S. corneliomulleri, four accessions of S. habrochaites, three accessions of S. peruvianum, and five accessions of S. subsection lycopersicon hybrid. Further analyses using serological tests identified four highly resistant S. pimpinellifolium lines, PI 390713, PI 390714, PI 390716, and PI 390717. The inheritance of resistance in the selected lines was verified in the next generation and confirmed using RT-qPCR. To our knowledge, this is a first report of high resistance to ToBRFV in S. pimpinellifolium. These new genetic resources will expand the genetic pool available for breeders to develop new resistant cultivars of tomato against ToBRFV.

Genomic and pangenomic analyses provide insights into the population history and genomic diversification of bottle gourd.

Bottle gourd (Lagenaria siceraria (Mol.) Strandl.) is an economically important vegetable crop and one of the earliest domesticated crops. However, the population history and genomic diversification of bottle gourd have not been extensively studied. We generated a comprehensive bottle gourd genome variation map from genome sequences of 197 world-wide representative accessions, which enables a genome-wide association study for identifying genomic loci associated with resistance to zucchini yellow mosaic virus, and constructed a bottle gourd pangenome that harbors 1534 protein-coding genes absent in the reference genome. Demographic analyses uncover that domesticated bottle gourd originated in Southern Africa c. 12 000 yr ago, and subsequently radiated to the New World via the Atlantic drift and to Eurasia through the efforts of early farmers in the initial Holocene. The identified highly differentiated genomic regions among different bottle gourd populations harbor many genes contributing to their local adaptations such as those related to disease resistance and stress tolerance. Presence/absence variation analysis of genes in the pangenome reveals numerous genes including those involved in abiotic/biotic stress responses that have been under selection during the world-wide expansion of bottle gourds. The bottle gourd variation map and pangenome provide valuable resources for future functional studies and genomics-assisted breeding.

Comparative Evaluation of Volatile Organic Compounds in Two Bottle Gourd Accessions with Distinct Fruit Shapes.

Bottle gourd (Lagenaria siceraria L.) belongs to the cucurbit family and has a long history of cultivation in tropical and subtropical regions worldwide, both for food and medicine. Popularized by its unique fruit shapes, gourds are used to make ornaments and musical instruments. However, there is limited information on volatile organic compounds (VOCs) in the bottle gourd fruit. In the present study, we conducted a comparative analysis of VOCs profiled in two accessions (USVL5 and USVL10) with distinct fruit shapes: bottle and cylinder. While USVL5 only produced long cylinder fruits, USVL10 produced two fruit types, cylinder (USVL10CYN) and bottle (USVL10A and USVL10B). VOCs in each line were analyzed using headspace solid-phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS). Aliphatic aldehydes and alcohols were the most abundant compounds found in these bottle gourd accessions. Based on the functional profile of the identified VOCs, our results reveal the suitability of our tested line (USVL10), enriched in functionally important VOCs such as hexanal (abundance = 381.07), nonanal (abundance = 9.85), 2-methoxy-2-methylpropane (abundance = 21.26) and D-limonene (abundance = 31.48). The VOCs profiling and functional analyses support the notion that the bottle gourd accession USVL10 can be a good candidate for its use in agriculture, the health care industry and domestic uses.

Comparative Analysis of Tomato Brown Rugose Fruit Virus Isolates Shows Limited Genetic Diversity.

Tomato is an important vegetable in the United States and around the world. Recently, tomato brown rugose fruit virus (ToBRFV), an emerging tobamovirus, has impacted tomato crops worldwide and can result in fruit loss. ToBRFV causes severe symptoms, such as mosaic, puckering, and necrotic lesions on leaves; other symptoms include brown rugose and marbling on fruits. More importantly, ToBRFV can overcome resistance in tomato cultivars carrying the Tm-22 locus. In this study, we recovered ToBRFV sequences from tomato seeds, leaves, and fruits from the U.S., Mexico, and Peru. Samples were pre-screened using a real-time RT-PCR assay prior to high-throughput sequencing. Virus draft genomes from 22 samples were assembled and analyzed against more than 120 publicly available genomes. Overall, most sequenced isolates were similar to each other and did not form a distinct population. Phylogenetic analysis revealed three clades within the ToBRFV population. Most of the isolates (95%) clustered in clade 3. Genetic analysis revealed differentiation between the three clades indicating minor divergence occurring. Overall, pairwise identity showed limited genetic diversity among the isolates in this study with worldwide isolates, with a pairwise identity ranging from 99.36% and 99.97%. The overall population is undergoing high gene flow and population expansion with strong negative selection pressure at all ToBRFV genes. Based on the results of this study, it is likely that the limited ToBRFV diversity is associated with the rapid movement and eradication of ToBRFV-infected material between countries.

The tomato yellow leaf curl virus C4 protein alters the expression of plant developmental genes correlating to leaf upward cupping phenotype in tomato.

Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus in the family Geminiviridae, is efficiently transmitted by the whitefly, Bemisia tabaci, and causes serious economic losses to tomato crops around the world. TYLCV-infected tomato plants develop distinctive symptoms of yellowing and leaf upward cupping. In recent years, excellent progress has been made in the characterization of TYLCV C4 protein function as a pathogenicity determinant in experimental plants, including Nicotiana benthamiana and Arabidopsis thaliana. However, the molecular mechanism leading to disease symptom development in the natural host plant, tomato, has yet to be characterized. The aim of the current study was to generate transgenic tomato plants expressing the TYLCV C4 gene and evaluate differential gene expression through comparative transcriptome analysis between the transgenic C4 plants and the transgenic green fluorescent protein (Gfp) gene control plants. Transgenic tomato plants expressing TYLCV C4 developed phenotypes, including leaf upward cupping and yellowing, that are similar to the disease symptoms expressed on tomato plants infected with TYLCV. In a total of 241 differentially expressed genes identified in the transcriptome analysis, a series of plant development-related genes, including transcription factors, glutaredoxins, protein kinases, R-genes and microRNA target genes, were significantly altered. These results provide further evidence to support the important function of the C4 protein in begomovirus pathogenicity. These transgenic tomato plants could serve as basic genetic materials for further characterization of plant receptors that are interacting with the TYLCV C4.

Complete Genome Sequences of Tomato Leaf Curl Guam Virus, a Novel Tomato-Infecting Begomovirus from Guam, USA.

Genome sequences of a novel begomovirus infecting tomato on Guam were obtained using primer-walking and sequencing. The complete genome sequences are 2,750 nucleotides long with a typical monopartite organization and display less than 91% nucleotide sequence identity to other begomoviruses. A provisional name, tomato leaf curl Guam virus (ToLCGuV), is proposed.

Comparative Analysis of Host Range, Ability to Infect Tomato Cultivars with Tm-22 Gene, and Real-Time Reverse Transcription PCR Detection of Tomato Brown Rugose Fruit Virus.

Tomato (Solanum lycopersicum L.) is one of the most important vegetables in the world. However, tomato is also susceptible to many viral diseases. Several tobamoviruses, including tomato mosaic virus (ToMV), tomato mottle mosaic virus (ToMMV), and tomato brown rugose fruit virus (ToBRFV), are highly contagious pathogens that could result in significant economic losses if not controlled effectively. Tobamoviruses have been managed relatively well with broad adaptation of tomato cultivars with resistance genes. However, emergence of ToBRFV was shown to break down resistance conferred by the common resistance genes, resulting in serious outbreaks in many countries in Asia, Europe, and North America. The objective of this study was to conduct a comparative analysis of biological properties, including host range and disease resistance of ToMV, ToMMV, and ToBRFV. Results showed that despite many similarities in the host range, there were some unique host plant responses for each of the three viruses. In a comparative evaluation of disease resistance using the same tomato cultivars with or without Tm-22 gene, there was a striking difference in responses from tomato plants with Tm-22 gene inoculated with ToBRFV, ToMV, or ToMMV. Whereas these test plants were resistant to ToMV or ToMMV infection, all test plants were susceptible to ToBRFV. Further, for ToBRFV detection, a sensitive and reliable multiplex real-time reverse transcription (RT)-PCR assay using TaqMan probe with an internal 18S rRNA control was also developed. With simple modifications to RNA extraction and seed soaking, real-time RT-PCR could consistently detect the virus in single infested seed in varied levels of contamination, suggesting its usefulness for seed health assay.

Effectiveness of disinfectants against the spread of tobamoviruses: Tomato brown rugose fruit virus and Cucumber green mottle mosaic virus.

BACKGROUND: Tobamoviruses, including tomato brown rugose fruit virus (ToBRFV) on tomato and pepper, and cucumber green mottle mosaic virus (CGMMV) on cucumber and watermelon, have caused many disease outbreaks around the world in recent years. With seed-borne, mechanical transmission and resistant breaking traits, tobamoviruses pose serious threat to vegetable production worldwide. With the absence of a commercial resistant cultivar, growers are encouraged to take preventative measures to manage those highly contagious viral diseases. However, there is no information available on which disinfectants are effective to deactivate the virus infectivity on contaminated hands, tools and equipment for these emerging tobamoviruses. The purpose of this study was to evaluate a collection of 16 chemical disinfectants for their effectiveness against mechanical transmission of two emerging tobamoviruses, ToBRFV and CGMMV. METHODS: Bioassay was used to evaluate the efficacy of each disinfectant based on virus infectivity remaining in a prepared virus inoculum after three short exposure times (10 s, 30 s and 60 s) to the disinfectant and inoculated mechanically on three respective test plants (ToBRFV on tomato and CGMMV on watermelon). Percent infection of plants was measured through symptom observation on the test plants and the presence of the virus was confirmed through an enzyme-linked immunosorbent assay with appropriate antibodies. Statistical analysis was performed using one-way ANOVA based on data collected from three independent experiments. RESULTS: Through comparative analysis of percent infection of test plants, a similar trend of efficacy among 16 disinfectants was observed between the two pathosystems. Four common disinfectants with broad spectrum activities against two different tobamoviruses were identified. Those effective disinfectants with 90-100% efficacy against both tobamoviruses were 0.5% Lactoferrin, 2% Virocid, and 10% Clorox, plus 2% Virkon against CGMMV and 3% Virkon against ToBRFV. In addition, SP2700 generated a significant effect against CGMMV, but poorly against ToBRFV. CONCLUSION: Identification of common disinfectants against ToBRFV and CGMMV, two emerging tobamoviruses in two different pathosystems suggest their potential broader effects against other tobamoviruses or even other viruses.

Large-Scale Seedling Grow-Out Experiments Do Not Support Seed Transmission of Sweet Potato Leaf Curl Virus in Sweet Potato.

Sweet potato leaf curl virus (SPLCV) threatens global sweet potato production. SPLCV is transmitted by Bemisia tabaci or via infected vegetative planting materials; however, SPLCV was suggested to be seed transmissible, which is a characteristic that is disputed for geminiviruses. The objective of this study was to revisit the validity of seed transmission of SPLCV in sweet potato. Using large-scale grow-out of sweet potato seedlings from SPLCV-contaminated seeds over 4 consecutive years, approximately 23,034 sweet potato seedlings of 118 genotype entries were evaluated. All seedlings germinating in a greenhouse under insect-proof conditions or in a growth chamber were free of SPLCV; however, a few seedlings grown in an open bench greenhouse lacking insect exclusion tested positive for SPLCV. Inspection of these seedlings revealed that B. tabaci had infiltrated the greenhouse. Therefore, transmission experiments were conducted using B. tabaci MEAM1, demonstrating successful vector transmission of SPLCV to sweet potato. Additionally, tests on contaminated seed coats and germinating cotyledons demonstrated that SPLCV contaminated a high percentage of seed coats collected from infected maternal plants, but SPLCV was never detected in emerging cotyledons. Based on the results of grow-out experiments, seed coat and cotyledon tests, and vector transmission experiments, we conclude that SPLCV is not seed transmitted in sweet potato.

Deep Sequencing of Small RNAs in the Whitefly Bemisia tabaci Reveals Novel MicroRNAs Potentially Associated with Begomovirus Acquisition and Transmission.

The whitefly Bemisia tabaci (Gennadius) is a notorious insect vector that transmits hundreds of plant viruses, affecting food and fiber crops worldwide, and results in the equivalent of billions of U.S. dollars in crop loss annually. To gain a better understanding of the mechanism in virus transmission, we conducted deep sequencing of small RNAs on the whitefly B. tabaci MEAM1 (Middle East-Asia Minor 1) that fed on tomato plants infected with tomato yellow leaf curl virus (TYLCV). Overall, 160 miRNAs were identified, 66 of which were conserved and 94 were B. tabaci-specific. Among the B. tabaci-specific miRNAs, 67 were newly described in the present study. Two miRNAs, with predicted targets encoding a nuclear receptor (Bta05482) and a very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase 2 (Bta10702), respectively, were differentially expressed in whiteflies that fed on TYLCV-infected versus uninfected plants. To better understand the regulatory effects of identified miRNAs and their target genes, we correlated expression profiles of miRNAs and their target transcripts and found that, interestingly, miRNA expression was inversely correlated with the expression of ~50% of the predicted target genes. These analyses could serve as a model to study gene regulation in other systems involving arthropod transmission of viruses to plants and animals.

Complete Genome Sequence of a Tomato Brown Rugose Fruit Virus Isolated in the United States.

The complete genome sequence of a U.S. isolate of a Tomato brown rugose fruit virus (ToBRFV) (CA18-01) was obtained through Illumina and MinION sequencing. The U.S. ToBRFV isolate shared a high nucleic acid sequence identity (>99%) with known ToBRFV isolates. Phylogenetic analysis revealed a tight cluster for ToBRFV isolates throughout the world, suggesting a short evolutionary history.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus.

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

QTL mapping of resistance to Fusarium oxysporum f. sp. niveum race 2 and Papaya ringspot virus in Citrullus amarus.

KEY MESSAGE: A Citrullus amarus mapping population segregating for resistance to Fusarium oxysporum f. sp. niveum race 2 and Papaya ringspot virus was used to identify novel QTL, important for the improvement in watermelon disease resistance. Multiple disease screens of the USDA Citrullus spp. germplasm collection have highlighted the value of Citrullus amarus (citron melon or wild watermelon) as a resource for enhancing modern watermelon cultivars (Citrullus lanatus) with resistance to a broad range of fungal, bacterial and viral diseases of watermelon. We have generated a genetic population of C. amarus segregating for resistance to two important watermelon diseases: Fusarium wilt (caused by the fungus Fusarium oxysporum f. sp. niveum; Fon race 2) and Papaya ringspot virus-watermelon strain (PRSV-W). QTL mapping of Fon race 2 resistance identified seven significant QTLs, with the major QTL representing a novel genetic source of resistance and an opportunity for gene pyramiding. A single QTL was associated with resistance to PRSV-W, which adhered to expectations of a prior study indicating a single-gene recessive inheritance in watermelon. The resistance loci identified here provide valuable genetic resources for introgression into cultivated watermelon for the improvement in disease resistance.

Genome of the African cassava whitefly Bemisia tabaci and distribution and genetic diversity of cassava-colonizing whiteflies in Africa.

The whitefy Bemisia tabaci, a species complex consisting of many morphologically indistinguishable species divided into distinct clades, is one of the most globally important agricultural pests and plant virus vectors. Cassava-colonizing B. tabaci transmits viruses that cause cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Half of all cassava plants in Africa are affected by these viral diseases, resulting in annual production losses of more than US$ 1 billion. Here we report the draft genome of the cassava whitefly B. tabaci Sub-Saharan Africa - East and Central Africa (SSA-ECA), the super-abundant population that has been associated with the rapid spread of viruses causing the pandemics of CMD and CBSD. The SSA-ECA genome assembled from Illumina short reads has a total size of 513.7 Mb and a scaffold N50 length of 497 kb, and contains 15,084 predicted protein-coding genes. Phylogenetic analysis suggests that SSA-ECA diverged from MEAM1 around 5.26 million years ago. A comprehensive genetic analysis of cassava-colonizing B. tabaci in Africa was also conducted, in which a total of 243 whitefly specimens were collected from 18 countries representing all major cassava-growing regions in the continent and genotyped using NextRAD sequencing. Population genomic analyses confirmed the existence of six major populations linked by gene flow and inferred the distribution patterns of these populations across the African continent. The genome of SSA-ECA and the genetic findings provide valuable resources and guidance to facilitate whitefly research and the development of strategies to control cassava viral diseases spread by whiteflies.

Comprehensive transcriptome analysis and functional characterization of PR-5 for its involvement in tomato Sw-7 resistance to tomato spotted wilt tospovirus.

Tomato spotted wilt tospovirus (TSWV), one of the most important plant viruses, causes yield losses to many crops including tomato. The current disease management for TSWV is based mainly on breeding tomato cultivars containing the Sw-5 locus. Unfortunately, several Sw-5 resistance-breaking strains of TSWV have been identified. Sw-7 is an alternative locus conferring resistance to a broad range of TSWV strains. In an effort to uncover gene networks that are associated with the Sw-7 resistance, we performed a comparative transcriptome profiling and gene expression analysis between a nearly-isogenic Sw-7 line and its susceptible recurrent parent (Fla. 8059) upon infection by TSWV. A total of 1,244 differentially expressed genes were identified throughout a disease progression process involving networks of host resistance genes, RNA silencing/antiviral defense genes, and crucial transcriptional and translational regulators. Notable induced genes in Sw-7 include those involved in callose accumulation, lignin deposition, proteolysis process, transcriptional activation/repression, and phosphorylation. Finally, we investigated potential involvement of PR-5 in the Sw-7 resistance. Interestingly, PR-5 overexpressed plants conferred enhanced resistance, resulting in delay in virus accumulation and symptom expression. These findings will facilitate breeding and genetic engineering efforts to incorporate this new source of resistance in tomato for protection against TSWV.

Understanding the Transmissibility of Cucumber Green Mottle Mosaic Virus in Watermelon Seeds and Seed Health Assays.

Cucumber green mottle mosaic virus (CGMMV), an emerging tobamovirus, has caused serious disease outbreaks to cucurbit crops in several countries, including the United States. Although CGMMV is seed-borne, the mechanism of its transmission from a contaminated seed to germinating seedling is still not fully understood, and the most suitable seed health assay method has not been well established. To evaluate the mechanism of seed transmissibility, using highly contaminated watermelon seeds collected from CGMMV-infected experimental plants, bioassays were conducted in a greenhouse through seedling grow-out and by mechanical inoculation. Through natural seedling grow-out, we did not observe seed transmission of CGMMV to germinating seedlings. However, efficient transmission of CGMMV was observed using bioassays on melon plants through mechanical inoculation of seed extract prepared from CGMMV-contaminated seeds. Understanding the seed-borne property and the ease of mechanical transmission of CGMMV from a contaminated seed to seedling is an important finding. In comparative evaluation of various laboratory techniques for seed health assays, we found that enzyme-linked immunosorbent assay and loop-mediated isothermal amplification were the most sensitive and reliable methods to detect CGMMV on cucurbit seeds. Because CGMMV is a seed-borne and highly contagious virus, a new infection might not result in a natural seedling grow-out; it could occur through mechanical transmission from contaminated seeds. Therefore, a sensitive seed health test is necessary to ensure CGMMV-free seed lots are used for planting.

Genome of 'Charleston Gray', the principal American watermelon cultivar, and genetic characterization of 1,365 accessions in the U.S. National Plant Germplasm System watermelon collection.

Years of selection for desirable fruit quality traits in dessert watermelon (Citrullus lanatus) has resulted in a narrow genetic base in modern cultivars. Development of novel genomic and genetic resources offers great potential to expand genetic diversity and improve important traits in watermelon. Here, we report a high-quality genome sequence of watermelon cultivar 'Charleston Gray', a principal American dessert watermelon, to complement the existing reference genome from '97103', an East Asian cultivar. Comparative analyses between genomes of 'Charleston Gray' and '97103' revealed genomic variants that may underlie phenotypic differences between the two cultivars. We then genotyped 1365 watermelon plant introduction (PI) lines maintained at the U.S. National Plant Germplasm System using genotyping-by-sequencing (GBS). These PI lines were collected throughout the world and belong to three Citrullus species, C. lanatus, C. mucosospermus and C. amarus. Approximately 25 000 high-quality single nucleotide polymorphisms (SNPs) were derived from the GBS data using the 'Charleston Gray' genome as the reference. Population genomic analyses using these SNPs discovered a close relationship between C. lanatus and C. mucosospermus and identified four major groups in these two species correlated to their geographic locations. Citrullus amarus was found to have a distinct genetic makeup compared to C. lanatus and C. mucosospermus. The SNPs also enabled identification of genomic regions associated with important fruit quality and disease resistance traits through genome-wide association studies. The high-quality 'Charleston Gray' genome and the genotyping data of this large collection of watermelon accessions provide valuable resources for facilitating watermelon research, breeding and improvement.

Genome-wide profiling of piRNAs in the whitefly Bemisia tabaci reveals cluster distribution and association with begomovirus transmission.

The whitefly Bemisia tabaci MEAM1 is a notorious vector capable of transmitting many plant viruses, resulting in serious crop loss and food shortage around the world. To investigate potential sRNA-mediated regulatory mechanisms in whiteflies that are affected by virus acquisition and transmission, we conducted small RNA (sRNA) deep sequencing and performed genome-wide profiling of piwi-interacting RNAs (piRNAs) in whiteflies that were fed on tomato yellow leaf curl virus (TYLCV)-infected or non-infected tomato plants for 24, 48, and 72 h. In the present study, piRNA reads ranging from 564,395 to 1,715,652 per library were identified and shown to distribute unevenly in clusters (57 to 96 per library) on the whitefly (B. tabaci MEAM1) genome. Among them, 53 piRNA clusters were common for all treatments. Comparative analysis between libraries generated from viruliferous and non-viruliferous whiteflies identified five TYLCV-induced and 24 TYLCV-suppressed piRNA clusters. Approximately 62% of piRNAs were derived from non-coding sequences including intergenic regions, introns, and untranslated regions (UTRs). The remaining 38% were derived from coding sequences (CDS) or repeat elements. Interestingly, six protein coding genes were targeted by the TYLCV-induced piRNAs. We identified a large number of piRNAs that were distributed in clusters across the whitefly genome, with 60% being derived from non-coding regions. Comparative analysis revealed that feeding on a virus-infected host caused induction and suppression of only a small number of piRNA clusters in whiteflies. Although piRNAs primarily regulate the activity of transposable elements, our results suggest that they may have additional functions in regulating protein coding genes and in insect-virus interactions.

Cucurbit Genomics Database (CuGenDB): a central portal for comparative and functional genomics of cucurbit crops.

The Cucurbitaceae family (cucurbit) includes several economically important crops, such as melon, cucumber, watermelon, pumpkin, squash and gourds. During the past several years, genomic and genetic data have been rapidly accumulated for cucurbits. To store, mine, analyze, integrate and disseminate these large-scale datasets and to provide a central portal for the cucurbit research and breeding community, we have developed the Cucurbit Genomics Database (CuGenDB; http://cucurbitgenomics.org) using the Tripal toolkit. The database currently contains all available genome and expressed sequence tag (EST) sequences, genetic maps, and transcriptome profiles for cucurbit species, as well as sequence annotations, biochemical pathways and comparative genomic analysis results such as synteny blocks and homologous gene pairs between different cucurbit species. A set of analysis and visualization tools and user-friendly query interfaces have been implemented in the database to facilitate the usage of these large-scale data by the community. In particular, two new tools have been developed in the database, a 'SyntenyViewer' to view genome synteny between different cucurbit species and an 'RNA-Seq' module to analyze and visualize gene expression profiles. Both tools have been packed as Tripal extension modules that can be adopted in other genomics databases developed using the Tripal system.

Reverse transcription loop-mediated isothermal amplification for species-specific detection of tomato chlorotic spot orthotospovirus.

Tomato chlorotic spot orthotospovirus (TCSV) is an emerging orthotospovirus that can cause severe disease on tomato plants. There are at least four orthotospoviruses infecting tomato, and mixed infection of two or more orthotospoviruses in a single tomato plant is quite common in the field. With similarity in the symptomatology and cross serological reactivity among tomato-infecting orthotospoviruses, especially between TCSV and groundnut ringspot orthotospovirus (GRSV), the current serological tests could not achieve definite and accurate species-specific determination in disease diagnosis. Here, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for TCSV. Under optimum conditions, the virus was detected in as little as 0.2 ng of total RNA or in 1:10,000 dilution of a simple diluted tissue extract, which was ten times more sensitive than a conventional RT-PCR assay. The RT-LAMP assay was highly specific for TCSV, with no cross reaction with the other two orthotospoviruses: GRSV and tomato spotted wilt orthotospovirus (TSWV). These results demonstrate that this simple and sensitive RT-LAMP could be used to achieve species-specific detection for TCSV under field conditions.