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Plant-virus–derived vectors are versatile tools with multiple applications in agricultural and medical biotechnology. In this study, we developed pepino mosaic virus (PepMV) (family Alphaflexiviridae; genus Potexvirus) into a vector for heterologous protein expression in plants. PepMV was initially cloned in a step-wise manner, fully sequenced and the full-length infectious clone was tested for infectivity in Nicotiana benthamiana. Initial infectious clones resulted in poor replication of PepMV and lack of systemic movement. Mutations in the viral sequence affected systemic infection. Two suspected mutations were altered to restore systemic infectivity. PepMV infection was apparent as early as 4 days post agroinfiltration (dpa) inoculation in N. benthamiana. A multiple cloning site was inserted into the PepMV genome for introduction and expression of foreign genes. Several modifications to the wild-type vector were made, such as a replacing the native subgenomic promoter (SGP) with a heterologous SGP, and introduction of translational enhancers and terminators, to improve heterologous expression of the foreign gene-of-interest. GFP was used as a reporter for monitoring virus infection and protein production. Strong GFP expression was observed as early as 4 dpa with a translational enhancer. The PepMV-based vector produces rapid expression of the foreign gene in comparison to two other potexvirus-based vectors. GFP production was monitored over time and optimal protein production was recorded between 5 and 7 dpa. GFP protein levels reached up to 4% and decreased to 0.5% total soluble protein at 7 and 14 dpa, respectively. Future studies will evaluate this virus-based vector for large-scale production of pharmaceutical compounds.Key points• A pepino mosaic virus isolate was developed into a plant-based expression vector.• Expression levels of the heterologous protein were comparable or exceeded previously developed viral vectors.• Protein levels in plants were highest between 5 and 7 days and decreased gradually.

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.

Viroids are the smallest pathogens of angiosperms, consisting of non-coding RNAs that cause severe diseases in agronomic crops. Symptoms associated with viroid infection are linked to developmental alterations due to genetic regulation. To understand the global mechanisms of host viroid response, we implemented network approaches to identify master transcription regulators and their differentially expressed targets in tomato infected with mild and severe variants of PSTVd. Our approach integrates root and leaf transcriptomic data, gene regulatory network analysis, and identification of affected biological processes. Our results reveal that specific bHLH, MYB, and ERF transcription factors regulate genes involved in molecular mechanisms underlying critical signaling pathways. Functional enrichment of regulons shows that bHLH-MTRs are linked to metabolism and plant defense, while MYB-MTRs are involved in signaling and hormone-related processes. Strikingly, a member of the bHLH-TF family has a specific potential role as a microprotein involved in the post-translational regulation of hormone signaling events. We found that ERF-MTRs are characteristic of severe symptoms, while ZNF-TF, tf3a-TF, BZIP-TFs, and NAC-TF act as unique MTRs. Altogether, our results lay a foundation for further research on the PSTVd and host genome interaction, providing evidence for identifying potential key genes that influence symptom development in tomato plants.

Bacterial spot of tomato (BST) is a disease that severely afflicts tomato crops, especially in geographic areas such as the Southeastern U.S., where the environmental conditions favor rapid disease development. Farmers usually use chemical treatments such as copper–mancozeb mixtures and acibenzolar-S-methyl, among other methods, to manage BST. However, these chemical treatments generally fail to improve marketable yields, thus raising the question of whether the BST treatments are economical. We evaluated the efficacy and profitability of bactericide treatments consisting of copper-mancozeb, acibenzolar-S-methyl, and streptomycin, as well as three inoculation levels of Xanthomonas euvesicatoria pv. perforans, on the management of BST in Florida. Across three separate field trials, BST severity was inversely correlated with marketable tomato yields; however, bactericide treatments provided no statistical improvement in marketable yields. By accounting for yield and the BST treatment costs, our profitability analysis showed that the BST treatments did not pay off economically; the net returns of these treatments were statistically equivalent to the untreated controls.

In the past two decades, viruses in the genera Crinivirus and Begomovirus, transmitted by whiteflies, have emerged as threatening diseases to cucurbit cultivation. The criniviruses Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) occur in mixed infection at high rates in cucumber greenhouses in Lebanon. The begomovirus Squash leaf curl virus (SLCV) is also present in the country infecting cucumber but at a lower incidence. The effect of single, dual or triple virus infections on cucumber was studied. Single infection by SLCV did not lead to any symptoms or yield reduction. CYSDV or CCYV infections led to development of characteristic yellowing symptoms. In single infections, CYSDV caused the greatest reductions in height and yield that ranged from 25 to 62 % and 52 to 63 %, respectively, depending on season and variety. CCYV induced less severe reduction in height, 10 to 33 %, and in fruit number, 10 to 12 %. Yellowing symptoms induced by dual infections in any combination were similar to those of singly-infected plants. Triple infections resulted in substantial reductions in plant height and yield as compared to single or dual infections. This is the first report that characterizes a significant increase in disease severity upon co-infection between cucurbit-infecting criniviruses and begomoviruses. The implications of these observations on integrated disease management strategies are discussed.

In the past decade, crinviruses have gained interest due to their rapid widespread and destructive nature for cucurbit cultivation. Several members of the genus Crinivirus are considered emerging viruses. Currently, four criniviruses: Beet pseudo - yellows virus , Cucurbit chlorotic yellows virus , Cucurbit yellow stunting disorder virus and Lettuce infectious yellows virus have been reported to infect field- or greenhouse- grown cucurbits. Apart from their cucurbit hosts, criniviruses infect other cash crops and weeds. Criniviruses are exclusively transmitted by whiteflies. The virion titer and the vector genus or species complex are predominant factors affecting virus transmission. These criniviruses maintain genetic stability with limited intra-species variability. They share similar core genome structure and replication strategies with some variations in the non-core proteins and downstream replication processes. Management of the diseases induced by criniviruses relies on integrated disease management strategies and on resistant varieties, when available. This review will cover their epidemiology, molecular biology, detection and management.

Disease symptoms Symptoms include water‐soaked areas surrounded by chlorosis turning into necrotic spots on all aerial parts of plants. On tomato fruits, small, water‐soaked, or slightly raised pale‐green spots with greenish‐white halos are formed, ultimately becoming dark brown and slightly sunken with a scabby or wart‐like surface. Host range Main and economically important hosts include different types of tomatoes and peppers. Alternative solanaceous and nonsolanaceous hosts include Datura spp., Hyoscyamus spp., Lycium spp., Nicotiana rustica, Physalis spp., Solanum spp., Amaranthus lividus, Emilia fosbergii, Euphorbia heterophylla, Nicandra physaloides, Physalis pubescens, Sida glomerata, and Solanum americanum. Taxonomic status of the pathogen Domain, Bacteria; phylum, Proteobacteria; class, Gammaproteobacteria; order, Xanthomonadales; family, Xanthomonadaceae; genus, Xanthomonas; species, X. euvesicatoria, X. hortorum, X. vesicatoria. Synonyms (nonpreferred scientific names) Bacterium exitiosum, Bacterium vesicatorium, Phytomonas exitiosa, Phytomonas vesicatoria, Pseudomonas exitiosa, Pseudomonas gardneri, Pseudomonas vesicatoria, Xanthomonas axonopodis pv. vesicatoria, Xanthomonas campestris pv. vesicatoria, Xanthomonas cynarae pv. gardneri, Xanthomonas gardneri, Xanthomonas perforans. Microbiological properties Colonies are gram‐negative, oxidase‐negative, and catalase‐positive and have oxidative metabolism. Pale‐yellow domed circular colonies of 1–2 mm in diameter grow on general culture media. Distribution The bacteria are widespread in Africa, Brazil, Canada and the USA, Australia, eastern Europe, and south‐east Asia. Occurrence in western Europe is restricted. Phytosanitary categorization A2 no. 157, EU Annex designation II/A2. EPPO codes XANTEU, XANTGA, XANTPF, XANTVE. In this review we provide a historical perspective as well as an updated overview on the aetiology, epidemiology, and management strategies of bacterial spot of tomato and pepper.

Tomato is an important crop grown worldwide. Various plant diseases cause massive losses in tomato plants due to diverse biotic agents. Bacterial spot of tomato (BST) is a worldwide disease that results in high losses in processed and fresh tomato. Xanthomonas perforans, an aerobic, single-flagellated, rod-shaped, Gram-negative plant pathogenic bacterium, is one of the leading causes of BST. Over the past three decades, X. perforans has increasingly been reported from tomato-growing regions and became a major bacterial disease. X. perforans thrives under high humidity and high temperature, which is commonplace in tropical and subtropical climates. Distinguishing symptoms of BST are necrotic lesions that can coalesce and cause a shot-hole appearance. X. perforans can occasionally cause fruit symptoms depending on disease pressure during fruit development. Short-distance movement in the field is mainly dependent on wind-driven rain, whereas long distance movement occurs through contaminated seed or plant material. X. perforans harbors a suite of effectors that increase pathogen virulence, fitness, and dissemination. BST management mainly relies on copper-based compounds; however, resistance is widespread. Alternative compounds, such as nanomaterials, are currently being evaluated and show high potential for BST management. Resistance breeding remains difficult to attain due to limited resistant germplasm. While the increased genetic diversity and gain and loss of effectors in X. perforans limits the success of single-gene resistance, the adoption of effector-specific transgenes and quantitative resistance may lead to durable host resistance. However, further research that aims to more effectively implement novel management tools is required to curb disease spread.Key points• Xanthomonas perforans causes bacterial spot on tomato epidemics through infected seedlings and movement of plant material.• Genetic diversity plays a major role in shaping populations which is evident in loss and gain of effectors.• Management relies on copper sprays, but nanoparticles are a promising alternative to reduce copper toxicity.

A novel betaflexivirus, tentatively named “miscanthus virus M” (MiVM), was isolated from Miscanthus sp. The complete genome of MiVM is 7,388 nt in length (excluding the poly(A) tail). It contains five open reading frames and has a genome organization similar to those of members of the families Alphaflexiviridae and Betaflexiviridae (subfamily Quinvirinae). The amino acid sequences of both the replicase and coat protein shared less than 45% identity with the corresponding sequences of members of either family. Phylogenetic analysis confirmed that MiVM belongs to the family Betaflexiviridae and subfamily Quinvirinae but it was too distantly related to be included in any currently recognized genus in this family. We therefore propose that miscanthus virus M represents a new species and a new genus in the family Betaflexiviridae.

Modern agricultural practices increase the potential for plant pathogen spread, while the advent of affordable whole genome sequencing enables in-depth studies of pathogen movement. Population genomic studies may decipher pathogen movement and population structure as a result of complex agricultural production systems. We used whole genome sequences of 281 Xanthomonas perforans strains collected within one tomato production season across Florida and southern Georgia fields to test for population genetic structure associated with tomato production system variables. We identified six clusters of X. perforans from core gene SNPs that corresponded with phylogenetic lineages. Using whole genome SNPs, we found genetic structure among farms, transplant facilities, cultivars, seed producers, grower operations, regions, and counties. Overall, grower operations that produced their own transplants were associated with genetically distinct and less diverse populations of strains compared to grower operations that received transplants from multiple sources. The degree of genetic differentiation among components of Florida’s tomato production system varied between clusters, suggesting differential dispersal of the strains, such as through seed or contaminated transplants versus local movement within farms. Overall, we showed that the genetic variation of a bacterial plant pathogen is shaped by the structure of the plant production system.