Beet Soil-Borne Virus Is a Helper Virus for the Novel Beta vulgaris Satellite Virus 1A.

Sugar beet (Beta vulgaris) is grown in temperate regions around the world as a source of sucrose used for natural sweetening. Sugar beet is susceptible to a number of viral diseases, but identification of the causal agent(s) under field conditions is often difficult due to mixtures of viruses that may be responsible for disease symptoms. In this study, the application of RNAseq to RNA extracted from diseased sugar beet roots obtained from the field and from greenhouse-reared plants grown in soil infested with the virus disease rhizomania (causal agent beet necrotic yellow vein virus; BNYVV) yielded genome-length sequences from BNYVV, as well as beet soil-borne virus (BSBV). The nucleotide identities of the derived consensus sequence of BSBV RNAs ranged from 99.4 to 96.7% (RNA1), 99.3 to 95.3% (RNA2), and 98.3 to 95.9% (RNA3) compared with published BSBV sequences. Based on the BSBV genome consensus sequence, clones of the genomic RNAs 1, 2, and 3 were obtained to produce RNA copies of the genome through in vitro transcription. Capped RNA produced from the clones was infectious when inoculated into leaves of Chenopodium quinoa and B. vulgaris, and extracts from transcript-infected C. quinoa leaves could infect sugar beet seedling roots through a vortex inoculation method. Subsequent exposure of these infected sugar beet seedling roots to aviruliferous Polymyxa betae, the protist vector of both BNYVV and BSBV, confirmed that BSBV derived from the infectious clones could be transmitted by the vector. Co-inoculation of BSBV synthetic transcripts with transcripts of a cloned putative satellite virus designated Beta vulgaris satellite virus 1A (BvSat1A) resulted in the production of lesions on leaves of C. quinoa similar to those produced by inoculation with BSBV alone. Nevertheless, accumulation of genomic RNA and the encoded protein of the satellite virus in co-inoculated leaves was readily detected on Northern and Western blots, respectively, whereas no accumulation of satellite virus products occurred when satellite virus RNA was inoculated alone. The predicted sequence of the detected protein encoded by BvSat1A bears hallmarks of coat proteins of other satellite viruses, and virions of a size consistent with a satellite virus were observed in samples testing positive for the virus. The results demonstrate that BSBV is a helper virus for the novel satellite virus BvSat1A.

Wheat Ym2 originated from Aegilops sharonensis and confers resistance to soil-borne Wheat yellow mosaic virus infection to the roots.

Wheat yellow mosaic virus (WYMV) is a pathogen transmitted into its host's roots by the soil-borne vector Polymyxa graminis. Ym1 and Ym2 genes protect the host from the significant yield losses caused by the virus, but the mechanistic basis of these resistance genes remains poorly understood. Here, it has been shown that Ym1 and Ym2 act within the root either by hindering the initial movement of WYMV from the vector into the root and/or by suppressing viral multiplication. A mechanical inoculation experiment on the leaf revealed that the presence of Ym1 reduced viral infection incidence, rather than viral titer, while that of Ym2 was ineffective in the leaf. To understand the basis of the root specificity of the Ym2 product, the gene was isolated from bread wheat using a positional cloning approach. The candidate gene encodes a CC-NBS-LRR protein and it correlated allelic variation with respect to its sequence with the host's disease response. Ym2 (B37500) and its paralog (B35800) are found in the near-relatives, respectively, Aegilops sharonensis and Aegilops speltoides (a close relative of the donor of bread wheat's B genome), while both sequences, in a concatenated state, are present in several accessions of the latter species. Structural diversity in Ym2 has been generated via translocation and recombination between the two genes and enhanced by the formation of a chimeric gene resulting from an intralocus recombination event. The analysis has revealed how the Ym2 region has evolved during the polyploidization events leading to the creation of cultivated wheat.

Differential Tropism in Roots and Shoots of Resistant and Susceptible Cassava (Manihot esculenta Crantz) Infected by Cassava Brown Streak Viruses.

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope® in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.

Active Components from Cassia abbreviata Prevent HIV-1 Entry by Distinct Mechanisms of Action.

Cassia abbreviata is widely used in Sub-Saharan Africa for treating many diseases, including HIV-1 infection. We have recently described the chemical structures of 28 compounds isolated from an alcoholic crude extract of barks and roots of C. abbreviata, and showed that six bioactive compounds inhibit HIV-1 infection. In the present study, we demonstrate that the six compounds block HIV-1 entry into cells: oleanolic acid, palmitic acid, taxifolin, piceatannol, guibourtinidol-(4α→8)-epiafzelechin, and a novel compound named as cassiabrevone. We report, for the first time, that guibourtinidol-(4α→8)-epiafzelechin and cassiabrevone inhibit HIV-1 entry (IC50 of 42.47 µM and 30.96 µM, respectively), as well as that piceatannol interacts with cellular membranes. Piceatannol inhibits HIV-1 infection in a dual-chamber assay mimicking the female genital tract, as well as HSV infection, emphasizing its potential as a microbicide. Structure-activity relationships (SAR) showed that pharmacophoric groups of piceatannol are strictly required to inhibit HIV-1 entry. By a ligand-based in silico study, we speculated that piceatannol and norartocarpetin may have a very similar mechanism of action and efficacy because of the highly comparable pharmacophoric and 3D space, while guibourtinidol-(4α→8)-epiafzelechin and cassiabrevone may display a different mechanism. We finally show that cassiabrevone plays a major role of the crude extract of CA by blocking the binding activity of HIV-1 gp120 and CD4.

Armillaria root rot fungi host single-stranded RNA viruses.

Species of Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named "ambiviruses" with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector.

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.

Blueberry red ringspot virus genomes from Florida inferred through analysis of blueberry root transcriptomes.

A growing number of metagenomics-based approaches have been used for the discovery of viruses in insects, cultivated plants, and water in agricultural production systems. In this study, sixteen blueberry root transcriptomes from eight clonally propagated blueberry plants of cultivar 'Emerald' (interspecific hybrid of Vaccinium corymbosum and V. darrowi) generated as part of a separate study on varietal tolerance to soil salinity were analyzed for plant viral sequences. The objective was to determine if the asymptomatic plants harbored the latent blueberry red ringspot virus (BRRV) in their roots. The only currently known mechanism of transmission of BRRV is through vegetative propagation; however, the virus can remain latent for years with some plants of 'Emerald' never developing red ringspot symptoms. Bioinformatic analyses of 'Emerald' transcriptomes using de novo assembly and reference-based mapping approaches yielded eight complete viral genomes of BRRV (genus Soymovirus, family Caulimoviridae). Validation in vitro by PCR confirmed the presence of BRRV in 100% of the 'Emerald' root samples. Sequence and phylogenetic analyses showed 94% to 97% nucleotide identity between BRRV genomes from Florida and sequences from Czech Republic, Japan, Poland, Slovenia, and the United States. Taken together, this study documented the first detection of a complete BRRV genome from roots of asymptomatic blueberry plants and in Florida through in silico analysis of plant transcriptomes.

Arbuscular Mycorrhizal Symbiosis Primes Tolerance to Cucumber Mosaic Virus in Tomato.

Tomato plants can establish symbiotic interactions with arbuscular mycorrhizal fungi (AMF) able to promote plant nutrition and prime systemic plant defenses against pathogens attack; the mechanism involved is known as mycorrhiza-induced resistance (MIR). However, studies on the effect of AMF on viral infection, still limited and not conclusive, indicate that AMF colonization may have a detrimental effect on plant defenses against viruses, so that the term "mycorrhiza-induced susceptibility" (MIS) has been proposed for these cases. To expand the case studies to a not yet tested viral family, that is, Bromoviridae, we investigated the effect of the colonization by the AMF Funneliformis mosseae on cucumber mosaic virus (CMV) infection in tomato by phenotypic, physiological, biochemical, and transcriptional analyses. Our results showed that the establishment of a functional AM symbiosis is able to limit symptoms development. Physiological and transcriptomic data highlighted that AMF mitigates the drastic downregulation of photosynthesis-related genes and the reduction of photosynthetic CO2 assimilation rate caused by CMV infection. In parallel, an increase of salicylic acid level and a modulation of reactive oxygen species (ROS)-related genes, toward a limitation of ROS accumulation, was specifically observed in CMV-infected mycorrhizal plants. Overall, our data indicate that the AM symbiosis influences the development of CMV infection in tomato plants and exerts a priming effect able to enhance tolerance to viral infection.

RNAseq Analysis of Rhizomania-Infected Sugar Beet Provides the First Genome Sequence of Beet Necrotic Yellow Vein Virus from the USA and Identifies a Novel Alphanecrovirus and Putative Satellite Viruses.

"Rhizomania" of sugar beet is a soilborne disease complex comprised of beet necrotic yellow vein virus (BNYVV) and its plasmodiophorid vector, Polymyxa betae. Although BNYVV is considered the causal agent of rhizomania, additional viruses frequently accompany BNYVV in diseased roots. In an effort to better understand the virus cohort present in sugar beet roots exhibiting rhizomania disease symptoms, five independent RNA samples prepared from diseased beet seedlings reared in a greenhouse or from field-grown adult sugar beet plants and enriched for virus particles were subjected to RNAseq. In all but a healthy control sample, the technique was successful at identifying BNYVV and provided sequence reads of sufficient quantity and overlap to assemble > 98% of the published genome of the virus. Utilizing the derived consensus sequence of BNYVV, infectious RNA was produced from cDNA clones of RNAs 1 and 2. The approach also enabled the detection of beet soilborne mosaic virus (BSBMV), beet soilborne virus (BSBV), beet black scorch virus (BBSV), and beet virus Q (BVQ), with near-complete genome assembly afforded to BSBMV and BBSV. In one field sample, a novel virus sequence of 3682 nt was assembled with significant sequence similarity and open reading frame (ORF) organization to members within the subgenus Alphanecrovirus (genus Necrovirus; family Tombusviridae). Construction of a DNA clone based on this sequence led to the production of the novel RNA genome in vitro that was capable of inducing local lesion formation on leaves of Chenopodium quinoa. Additionally, two previously unreported satellite viruses were revealed in the study; one possessing weak similarity to satellite maize white line mosaic virus and a second possessing moderate similarity to satellite tobacco necrosis virus C. Taken together, the approach provides an efficient pipeline to characterize variation in the BNYVV genome and to document the presence of other viruses potentially associated with disease severity or the ability to overcome resistance genes used for sugar beet rhizomania disease management.

Colonization of human opportunistic Fusarium oxysporum (HOFo) isolates in tomato and cucumber tissues assessed by a specific molecular marker.

Fusarium oxysporum is a large complex cosmopolitan species composed of plant pathogens, human opportunistic pathogens, and nonpathogenic isolates. Many plant pathogenic strains are known based on host plant specificity and the large number of plant species attacked. F. oxysporum is an opportunistic pathogen in humans with a compromised immune system. The objectives of this study were: (1) to develop a specific marker to detect human opportunistic F. oxysporum (HOFo) isolates; (2) to determine whether or not HOFo isolates can colonize and cause disease symptoms in plants; and (3) to assess Taiwan isolates sensitivity to two agro-fungicides. The primer pair, Primer 5/ST33-R, specifically amplifying Taiwan and international reference HOFo isolates was developed and used to detect and assess the distribution of a Taiwan isolate in inoculated tomato plants and tomato and cucumber fruit. Taiwan HOFo isolate MCC2074 was shown to colonize tomato roots, hypocotyls, and cotyledons, but did not show any visible symptoms. Four days after surface inoculation of tomato and cucumber fruit with the same isolate, MCC2074 was detected in the pericarp and locular cavities of both tomato and cucumber fruit and in columella of tomato fruit. Three Taiwan HOFo isolates were found to be moderately sensitive to azoxystrobin and highly sensitive to difenconazole.

Genome composition analysis of multipartite BNYVV reveals the occurrence of genetic re-assortment in the isolates of Asia Minor and Thrace.

Beet necrotic yellow vein virus (BNYVV) is the cause of rhizomania, an important disease of sugar beet around the world. The multipartite genome of the BNYVV contains four or five single-stranded RNA that has been used to characterize the virus. Understanding genome composition of the virus not only determines the degree of pathogenicity but also is required to development of resistant varieties of sugar beet. Resistance to rhizomania has been conferred to sugar beet varieties by conventional breeding methods or modern genome engineering tools. However, over time, viruses undergo genetic alterations and develop new variants to break crop resistance. Here, we report the occurrence of genetic reassortment and emergence of new variants of BNYVV among the isolates of Thrace and Asia Minor (modern-day Turkey). Our findings indicate that the isolates harbor European A-type RNA-2 and RNA-3, nevertheless, RNA-5 is closely related to East Asian J-type. Furthermore, RNA-1 and RNA-4 are either derived from A, B, and P-types or a mixture of them. The RNA-5 factor which enhance the pathogenicity, is rarely found in the isolates studied (20%). The creation of new variants of the virus emphasizes the necessity to develop new generation of resistant crops. We anticipate that these findings will be useful for future genetic characterization and evolutionary studies of BNYVV, as well as for developing sustainable strategies for the control of this destructive disease.

Comparative Transcriptome Analysis Provides Molecular Insights into the Interaction of Beet necrotic yellow vein virus and Beet soil-borne mosaic virus with Their Host Sugar Beet.

Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) are closely related species, but disease development induced in their host sugar beet displays striking differences. Beet necrotic yellow vein virus induces excessive lateral root (LR) formation, whereas BSBMV-infected roots appear asymptomatic. A comparative transcriptome analysis was performed to elucidate transcriptomic changes associated with disease development. Many differentially expressed genes (DEGs) were specific either to BNYVV or BSBMV, although both viruses shared a high number of DEGs. Auxin biosynthesis pathways displayed a stronger activation by BNYVV compared to BSBMV-infected plants. Several genes regulated by auxin signalling and required for LR formation were exclusively altered by BNYVV. Both viruses reprogrammed the transcriptional network, but a large number of transcription factors involved in plant defence were upregulated in BNYVV-infected plants. A strong activation of pathogenesis-related proteins by both viruses suggests a salicylic acid or jasmonic acid mediated-defence response, but the data also indicate that both viruses counteract the SA-mediated defence. The ethylene signal transduction pathway was strongly downregulated which probably increases the susceptibility of sugar beet to Benyvirus infection. Our study provides a deeper insight into the interaction of BNYVV and BSBMV with the economically important crop sugar beet.

A bushel of viruses: Identification of seventeen novel putative viruses by RNA-seq in six apple trees.

Apple decline in Washington state has been increasing in incidence, particularly on Honeycrisp trees grown on G.935 rootstock. In this disease the trees exhibit dieback with necrosis at the graft union and in the rootstock. The cause of this disease remains unknown. To identify viral candidates, RNA-seq was performed on six trees: four trees exhibiting decline and two healthy trees. Across the samples, eight known viruses and Apple hammerhead viroid were detected, however none appear to be specifically associated with the disease. A BLASTx analysis of the RNA-seq data was performed to identify novel viruses that might be associated with apple decline. Seventeen novel putative viruses were detected, including an ilarvirus, two tombus-like viruses, a barna-like virus, a picorna-like virus, three ourmia-like viruses, three partiti-like viruses, and two narna-like viruses. Four additional viruses could not be classified. Three of the viruses appeared to be missing key genes, suggesting they may be dependent upon helper viruses for their function. Others showed a specific tropism, being detected only in the roots or only in the leaves. While, like the known apple viruses, none were consistently associated with diseased trees, it is possible these viruses may have a synergistic effect when co-infecting that could contribute to disease. Or the presence of these viruses may weaken the trees for some other factor that ultimately causes decline. Additional research will be needed to determine how these novel viruses contribute to apple decline.

Response of cassava cultivars to African cassava mosaic virus infection across a range of inoculum doses and plant ages.

Cassava production in Africa is constrained by cassava mosaic disease (CMD) that is caused by the Cassava mosaic virus (CMV). The aim of this study was to evaluate the responses of a range of commonly cultivated West African cassava cultivars to varying inoculum doses of African cassava mosaic virus (ACMV). We grafted 10 cultivars of cassava plants with different inoculum doses of CMV (namely two, four, or six CMD-infected buds) when the experimental plants were 8, 10, or 12 weeks old, using non-inoculated plants as controls. Three cultivars showed disease symptoms when grafted with two buds, and four cultivars showed disease symptoms when grafted with four or six buds. Most cultivars became symptomatic six weeks after inoculation, but one ('TMS92/0326') was symptomatic two weeks after inoculation, and two ('Ntollo' and 'Excel') were symptomatic after four weeks. Root weight tended to be lower in the six-bud than in the two-bud dose, and disease severity varied with plant age at inoculation. These results indicate that the level of CMD resistance in cassava cultivars varies with inoculum dose and timing of infection. This will allow appropriate cultivars to be deployed in each production zone of Africa in accordance with the prevalence of CMD.

Root Transcriptomic Analysis Reveals Global Changes Induced by Systemic Infection of Solanum lycopersicum with Mild and Severe Variants of Potato Spindle Tuber Viroid.

Potato spindle tuber viroid (PSTVd) causes systemic infection in plant hosts. There are many studies on viroid-host plant interactions, but they have predominantly focused on the aboveground part of the plant. Here, we investigated transcriptomic profile changes in tomato roots systemically infected with mild or severe PSTVd variants using a combined microarray/RNA-seq approach. Analysis indicated differential expression of genes related to various Gene Ontology categories depending on the stage of infection and PSTVd variant. A majority of cell-wall-related genes were down-regulated at early infection stages, but at the late stage, the number of up-regulated genes increased significantly. Along with observed alterations of many lignin-related genes, performed lignin quantification indicated their disrupted level in PSTVd-infected roots. Altered expression of genes related to biosynthesis and signaling of auxin and cytokinin, which are crucial for lateral root development, was also identified. Comparison of both PSTVd infections showed that transcriptional changes induced by the severe variant were stronger than those caused by the mild variant, especially at the late infection stage. Taken together, we showed that similarly to aboveground plant parts, PSTVd infection in the underground tissues activates the plant immune response.

A portable detection assay for Apple stem pitting virus using reverse transcription-recombinase polymerase amplification.

A molecular diagnostic assay for the rapid, sensitive and specific detection of Apple stem pitting virus (ASPV) in infected samples, utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 42 °C and the designed target-specific primers, was developed. The RT-RPA assay was able to be used in ASPV-infected leaves, rootstocks and fruits. Sensitivity tests, using ASPV transcripts, showed that the RT-RPA with the ASPV-specific primers was more sensitive than the conventional RT-PCR, with a detection limit of 1 fg/µL of RNA. In addition, the reaction time for the amplification of ASPV was shortened to as little as 1 min. The assay was highly specific and did not give a positive reaction to other viruses infecting pears. Moreover, the amplified genomic fragment of ASPV produced by the assay could be determined within 4 min using a portable capillary gel electrophoresis system. The entire process, excluding the extraction of total RNA, could be completed in 5 min using portable equipment in the field. This is the first report of utilizing an RT-RPA assay to detect a pear tree virus and the assay could be used both in the laboratory and in the field for ASPV detection.

The complete genome sequence of apple rootstock virus A, a novel nucleorhabdovirus identified in apple rootstocks.

We report the complete genome sequence of a novel nucleorhabdovirus, apple rootstock virus A (ApRVA), isolated from Malus spp. in South Korea. ApRVA has a 14,043-nt single-stranded negative-sense RNA genome. In the antigenome sense, it contains seven open reading frames, encoding the putative nucleocapsid protein, phosphoprotein, cell-to-cell movement protein, matrix protein, glycoprotein, RNA-dependent RNA polymerase, and an additional hypothetical protein, the gene for which is located between the genes for the matrix protein and glycoprotein. The complete genome sequence of ApRVA showed 47.45% nucleotide sequence identity to that of black currant-associated rhabdovirus 1. The genome organization, phylogenetic relationships, and sequence similarities to other nucleorhabdoviruses suggest that ApRVA is a new member of the genus Nucleorhabdovirus.

Multi-phased internalization of murine norovirus (MNV) in Arabidopsis seedlings and its potential correlation with plant defensive responses.

Norovirus is a highly infectious human pathogen that causes acute foodborne diseases worldwide. As global diet patterns have begun to incorporate a higher consumption of fresh agricultural products, the internalization of norovirus into plants has emerged as a potential threat to human health. Here, we demonstrated that murine norovirus (MNV1) was internalized into Arabidopsis in multiple phases, and this internalization was correlated with Arabidopsis innate immunity responses. Under hydroponic conditions, continuous treatment of MNV1 retarded root growth and facilitated flower development of Arabidopsis without causing necrotic lesions. Examination of viral titers and RNA levels revealed that MNV1 was internalized into Arabidopsis in at least three different phases. In response to MNV1 treatment, the Arabidopsis defensive marker PR1 (a salicylic acid signaling marker) was transiently up-regulated at the early stage. PDF1.2, a jasmonic acid signaling marker, exhibited a gradual induction over time. Noticeably, Arabidopsis RNS1 (T2 ribonuclease) was rapidly induced by MNV1 and exhibited anti-correlation with the internalization of MNV1. Exposure to recombinant Arabidopsis RNS1 protein reduced the viral titers and degraded MNV1 RNA in vitro. In conclusion, the internalization of MNV1 into Arabidopsis was fluctuated by mutual interactions that were potentially regulated by Arabidopsis immune systems containing RNS1.

Development of a reverse transcription-loop- mediated isothermal amplification (LAMP) assay for the rapid detection of onion yellow dwarf virus.

Onion yellow dwarf virus (OYDV) is one of the most important viral pathogens of onion. In particular, on 'Rossa di Tropea' onion, granted with Protected Geographical Indication (PGI) trademarks, this pathogen represents the most limiting biotic stress in terms of spread, severity of symptoms and damage, and its detection is necessary to preserve high quality standards and avoid yield losses. A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed for detection of OYDV. The specificity, sensitivity, repeatability and reproducibility of the assay were validated according to EPPO standard PM7/98 (2). Diagnostic specificity, diagnostic sensitivity and diagnostic accuracy were determined in both leaf and bulb tissues. To enhance the feasibility of a LAMP-based method for field diagnosis, several nucleic acid extraction methods were compared to simplify sample preparation. The results showed the reliability of the method for OYDV detection, with a limit of detection (LOD) comparable to real time reverse transcription polymerase chain reaction (RT-qPCR). The ease of sample preparation, and the more than acceptable LOD, indicated that the RT-LAMP assay could be used in plant pathology laboratories with limited facilities and resources, as well as directly in the field. This work was carried out in the frame of "SI.ORTO" project.

Novel Foveavirus (the family Betaflexiviridae) species identified in ginseng (Panax ginseng).

Ginseng (Panax ginseng) is a valuable herb that is widely cultivated in Korea, China, and Japan because it contains a variety of pharmacologically active substances with a wide range of positive effects on human health. Identification and prevention of disease-causing viral pathogens of ginseng is important for improving the yield and quality of ginseng-derived bioactive molecules. In this study, the genome sequence of the virus Panax ginseng flexivirus 1 (PgFV1) was identified from a ginseng root transcriptome data set. Sequence comparison and phylogenetic analysis showed that PgFV1 is a novel plant RNA virus species of the genus Foveavirus (the family Betaflexiviridae). Foveaviruses have flexuous and filamentous virions with a single-stranded positive-sense mono-segmented RNA genome. Its infection causes diseases with mosaic and ringspot symptoms in the stems and leaves. The PgFV1 genome encodes for 5 open reading frames: a replicase polyprotein for viral genome replication, 3 triple gene block proteins for viral cell-to-cell movement, and coat protein. Phylogenetic trees inferred from replicase polyprotein or coat protein sequences showed that PgFV1 is most closely related to grapevine virus T. PgFV1 is the first foveavirus identified to be associated with ginseng. Given the potential pathogenic features of previously known foveaviruses and importance of ginseng in the health industry, the PgFV1 genome sequence may be highly useful for studying ginseng foveaviruses. Keywords: ginseng; Panax ginseng flexivirus 1; Foveavirus; Betaflexiviridae.