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Grapevine fanleaf virus (GFLV) is a nepovirus that is transmitted to grapevines by the ectoparasitic nematode Xiphinema index. GFLV causes severe losses in yield and quality in viticulture worldwide. Presently, laborious and time-consuming field trials or greenhouse tests are necessary for screening putative GFLV resistance in new grape genotypes developed in breeding programmes. We developed an in vitro dual culture system for grapevines and nematode vectors that requires less time and space than inoculation experiments done in the greenhouse. Virus infection of in vitro grapevines was investigated using immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) analysis. The development of root galls induced by feeding nematodes on in vitro grapevines was also analysed. Virus infection in grapevines in the dual culture with viruliferous nematodes was detected six weeks post-inoculation. Root galls were always absent from parasitised in vitro grapevines with detectable virus infection, whereas they developed on some parasitised, but virus-negative tested grapevines. Therefore, root galls cannot be used as a reliable indicator for parasitism and virus transmission.

The tribe Longidorini within the subfamily Longidorinae (Longidorus spp. and Paralongidorus spp.) and the subfamily Xiphineminae (Xiphinema spp.) are two large nematode groups with about 260 and 230 known species, respectively. They are globally two important groups of ectoparasitic nematodes considered to be major pests because of their activity as vectors of important plant nepovirus, with some species included in the list of quarantine pathogenic organisms in many European countries. Knowledge of the biodiversity and occurrence of this nematode group is a prerequisite for the establishment of sound management strategies and control measures. According to data collected from the databases (such as EPPO, FSTA, and Web of Science) and published in specialised literature, a total of 15 Longidorus, 1 Paralongidorus and 40 Xiphinema species have been recorded as present in Portugal. However, the taxonomic status of some species is controversial, and thus needs to be clarified. A comprehensive review for unravelling the biodiversity and occurrence of nematode species of the genus Longidorus, Paralongidorus and Xiphinema in Portugal is herein provided. This review includes an updated checklist of species with information on the localities, host plants and geographical distribution. Additionally, maps on the species distributions of Longidorinae and Xiphineminae nematodes present in Continental Portugal and the Portuguese Macaronesian islands are provided, as well as unpublished data on authors and comments on the current taxonomic status. Finally, new insights and directions for future research on Longidoridae in Portugal are presented.

The complete nucleotide sequence of a new soybean-infecting member of the genus Nepovirus (provisionally named “soybean latent spherical virus” [SLSV]) was identified by high-throughput sequencing of RNAs from soybean leaf samples from North Dakota, USA. The sequences of RNAs 1 (8,190 nt) and 2 (5,788 nt) were completed by rapid amplification of cDNA ends. Each contained a single long open reading frame and a 3’ nontranslated region of greater than 1,500 nt. The predicted amino acid sequences of the two ORFs were most closely related to nepoviruses in subgroup C. Full-length cDNAs of RNAs 1 and 2 were cloned and used to inoculate soybean plants, which did not display obvious symptoms. These results suggest that SLSV represents a new species in the genus Nepovirus.

Grapevine (Vitis spp.) can be infected by numerous viruses that are often widespread and of great economic importance. Reliable detection methods are necessary for sanitary selection which is the only way to partly control grapevine virus diseases. Biological indexing and ELISA are currently the standard methods for screening propagation material, and PCR-methods are becoming increasingly popular. Due to the diversity of virus isolates, it is essential to verify that the tests allow the detection of the largest possible virus populations. We developed three quadruplex TaqMan

Background Genome-wide association study (GWAS) is a useful tool for detecting and characterizing traits of interest including those associated with disease resistance in soybean. The availability of 50,000 single nucleotide polymorphism (SNP) markers (SoySNP50K iSelect BeadChip; www.soybase.org) on 19,652 soybean and wild soybean plant introductions (PIs) in the USDA Soybean Germplasm Collection allows for fast and robust identification of loci associated with a desired phenotype. By using a genome-wide marker set to predict phenotypic values, genomic prediction for phenotype-unknown but genotype-determined PIs has become possible. The goal of this study was to describe the genetic architecture associated with sensitivity to Tobacco ringspot virus (TRSV) infection in the USDA Soybean Germplasm Collection. Results TRSV-induced disease sensitivities of the 697 soybean PIs were rated on a one to five scale with plants rated as one exhibiting mild symptoms and plants rated as five displaying terminal bud necrosis (i.e., bud blight). The GWAS identified a single locus on soybean chromosome 2 strongly associated with TRSV sensitivity. Cross-validation showed a correlation of 0.55 (P

We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR.

Arabidopsis thaliana leucine-rich repeat-containing (NLR) proteins RPS4 and RRS1, known as dual resistance proteins, confer resistance to multiple pathogen isolates, such as the bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum and the fungal pathogen Colletotrichum higginsianum. RPS4 is a typical Toll/interleukin 1 Receptor (TIR)-type NLR, whereas RRS1 is an atypical TIR-NLR that contains a leucine zipper (LZ) motif and a C-terminal WRKY domain. RPS4 and RRS1 are localised near each other in a head-to-head orientation. In this study, direct mutagenesis of the C-terminal LZ motif in RRS1 caused an autoimmune response and stunting in the mutant. Co-immunoprecipitation analysis indicated that full-length RPS4 and RRS1 are physically associated with one another. Furthermore, virus-induced gene silencing experiments showed that hypersensitive-like cell death triggered by RPS4/LZ motif-mutated RRS1 depends on EDS1. In conclusion, we suggest that the RRS1-LZ motif is crucial for the regulation of the RPS4/RRS1 complex.

A new satellite RNA (satRNA) of grapevine fanleaf virus (GFLV) was identified by high-throughput sequencing of high-definition (HD) adapter libraries from grapevine plants of the cultivar Panse precoce (PPE) affected by enation disease. The complete nucleotide sequence was obtained by automatic sequencing using primers designed based on next-generation sequencing (NGS) data. The full-length sequence, named satGFLV-PPE, consisted of 1119 nucleotides with a single open reading frame from position 15 to 1034. This satRNA showed maximum nucleotide sequence identity of 87 % to satArMV-86 and satGFLV-R6. Symptomatic grapevines were surveyed for the presence of the satRNA, and no correlation was found between detection of the satRNA and enation symptom expression.

Virus-like particles (VLPs) derived from non-enveloped viruses result from the self-assembly of capsid proteins (CPs). They generally display similar structural features to viral particles but are non-infectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self-assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N- and C-terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant-based production of nucleic-acid free VLPs. Remarkably, expression of N- or C-terminal CP fusions, resulted in the production of VLPs with recombinant proteins exposed either to the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules., Particules de type viral (comme VLP s) provenant de virus non enveloppés résultent de l'auto-assemblage des protéines de capside ( CP s). Ils présentent généralement des caractéristiques structurales similaires à des particules virales , mais sont noninfectious et leur cavité intérieure et la surface extérieure peuvent potentiellement être adaptées pour servir nanocarriers d' un grand intérêt biotechnologique. Alors qu'une VLP surface extérieure est généralement prête à des modifications chimiques ou génétiques, encagement une cargaison dans des particules peut être plus complexe et est souvent limité à de petites molécules ou des peptides. Des exemples où les deux cavité intérieure et la surface extérieure ont été utilisées pour encapsuler simultanément et exposer les protéines entières restent rares. Ici, nous décrivons la production de particules sphériques VLP exposant les protéines fluorescentes de soit à leur surface extérieure ou cavité interne à la suite de l'auto-assemblage d'une seule protéine de structure virale génétiquement modifié, le CP du virus grapevine fanleaf ( GFLV ). Nous avons trouvé que les extrémités N- et C-terminales de la GFLV CP permettent à la fusion génétique des protéines aussi grandes que 27 kD a et la production à base de plantes de l' acide nucléique libre- VLP s. De manière remarquable, l' expression de N- ou C-terminaux CP fusion a donné lieu à la production de VLP s avec des protéines recombinantes exposées soit à la cavité intérieure ou sur la surface extérieure, respectivement, tandis que la co - expression des deux protéines de fusion conduit à la formation d'un hybride VLP , mais plutôt inefficacement. De telles propriétés sont plutôt unique pour une seule protéine de structure virale et ouvrent de nouvelles avenues potentielles pour la conception de nanocarriers sûrs et polyvalents, en particulier pour l'administration ciblée de molécules bioactives.

The nucleotide sequence of RNA1 of grapevine Anatolian ringspot virus (GARSV), a nepovirus of subgroup B, was determined from cDNA clones. It is 7,288 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame (ORF), extending from nucleotides 272 to 7001, encoding a polypeptide of 2,243 amino acids with a predicted molecular mass of 250 kDa. The primary structure of the polyprotein, compared with that of other viral polyproteins, revealed the presence of all the characteristic domains of members of the order Picornavirales, i.e., the NTP-binding protein (1B(Hel)), the viral genome-linked protein (1C(VPg)), the proteinase (1D(Prot)), the RNA-dependent RNA polymerase (1E(Pol)), and of the protease cofactor (1A(Pro-cof)) shared by members of the subfamily Comovirinae within the family Secoviridae. The cleavage sites predicted within the polyprotein were found to be in agreement with those previously reported for nepoviruses of subgroup B, processing from 1A to 1E proteins of 67, 64, 3, 23 and 92 kDa, respectively. The RNA1-encoded polyprotein (p1) shared the highest amino acid sequence identity (66 %) with tomato black ring virus (TBRV) and beet ringspot virus (BRSV). The 5'- and 3'-noncoding regions (NCRs) of GARSV-RNA1 shared 89 % and 95 % nucleotide sequence identity respectively with the corresponding regions in RNA2. Phylogenetic analysis confirmed the close relationship of GARSV to members of subgroup B of the genus Nepovirus.

Population genetic analysis of grapevine fanleaf virus (GFLV) was done on the basis of the virus movement protein (MP) gene sequences from the isolates detected and identified in this study and those of all previously reported GFLV strains/isolates. These revealed that the GFLV populations of Iran and Slovenia were highly distinct, whereas those of France, Germany, Italy and the USA were composed of multiple lineages. All populations were significantly differentiated from each other. However, two GFLV isolates from Tunisia, the only recorded GFLVs from that country, were not statistically distinct from the French, German and Italian populations. The ratio of non-synonymous nucleotide diversity to synonymous nucleotide diversity (Pi(a)/Pi(s)) was less than 1, suggesting that the MP gene has been under purifying selection. The neutrality tests were indicative of a balancing selection that is operating within Iranian and USA GFLV isolates, but they show a purifying selection within the other populations. Eleven recombination events were detected in a total of 50 isolates from France, Germany, Iran, Italy, Slovenia and the USA. The results from the recombination analysis were in agreement with those of the phylogenetic analysis. This study suggests that diversity among GFLV geographical populations resulted from possible host adaptation, recombination and founder effects.

The complete sequences of RNA1 and RNA2 have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SAPCS3). The two RNAs are, respectively, 7,342 and 3,817 nucleotides in length, excluding the poly(A) tails. RNA1 has a large open reading frame (ORF) of 6,852 nucleotides and a 5'-UTR and a 3'-UTR of 243 and 244 nucleotides, respectively. RNA2 encodes for an ORF of 3,330 nucleotides and has the highest nucleotide identity (90.4 %) with GFLV-F13. The full length nucleotide sequence of GFLV-SAPCS3 RNA1 had the highest nucleotide identity (86.5 %) to the French isolate GFLV-F13. The 5'- and 3'-UTRs of GFLV-SAPCS3 RNA2 are 272 nucleotides and 212 nucleotides (nt) in length, respectively. The GFLV-SAPCS3 RNA2 5'-UTR is 32-53 nt longer compared to other GFLV isolates. The GFLV-SAPCS3 RNA2 5'-UTR is also more closely related to GFLV-GHu and Arabis mosaic virus (ArMV) isolates than to other GFLV isolates. Putative intra- and interspecies recombination events between GFLV and ArMV isolates involving GFLV-SAPCS3 RNA1 and RNA2 were investigated. Recombination analysis software has indicated that the GFLV-SAPCS3 5'-UTR might have evolved from a recombinational event between GFLV-F13-type and ArMV-Ta-type isolate.

The complete nucleotide sequence of cherry leaf roll virus (CLRV, genus Nepovirus) from a naturally infected cherry tree (Prunus avium cv. Bing) in North America was determined. RNA1 and RNA2 consist of 7,893 and 6,492 nucleotides, respectively, plus a poly-(A) tail. Each RNA encodes a single potential open reading frame. The first 657 nucleotides of RNA1 and RNA2 are 99% identical and include the 5′-UTR and the first 214 deduced amino acids of the polyproteins following the first of two in-frame start codons. Phylogenetic analysis reveals close relationships between CLRV and members of subgroup C of the genus Nepovirus. Electronic supplementary material The online version of this article (doi:10.1007/s00705-011-1208-4) contains supplementary material, which is available to authorized users.

Most Arabidopsis ecotypes display tolerance to the Tobacco ringspot virus (TRSV), but a subset of Arabidopsis ecotypes, including Estland (Est), develop lethal systemic necrosis (LSN), which differs from the localized hypersensitive responses (HRs) or systemic acquired resistance (SAR) characteristic of incompatible reactions. Neither viral replication nor the systemic movement of TRSV was restricted in tolerant or sensitive Arabidopsis ecotypes; therefore, the LSN phenotype shown in the sensitive ecotypes might not be due to viral accumulation. In the present study, we identified the Est TTR1 gene (tolerance to Tobacco ringspot virus 1) encoding a TIR-NBS-LRR protein that controls the ecotype-dependent tolerant/sensitive phenotypes by a map-based cloning method. The tolerant Col-0 ecotype Arabidopsis transformed with the sensitive Est TTR1 allele developed an LSN phenotype upon TRSV infection, suggesting that the Est TTR1 allele is dominant over the tolerant ttr1 allele of Col-0. Multiple sequence alignments of 10 tolerant ecotypes from those of eight sensitive ecotypes showed that 10 LRR amino acid polymorphisms were consistently distributed across the TTR1/ttr1 alleles. Site-directed mutagenesis of these amino acids in the LRR region revealed that two sites, L956S and K1124Q, completely abolished the LSN phenotype. VIGS study revealed that TTR1 is dependent on SGT1, rather than EDS1. The LSN phenotype by TTR1 was shown to be transferred to Nicotiana benthamiana, demonstrating functional conservation of TTR1 across plant families, which are involved in SGT-dependent defense responses, rather than EDS1-dependent signaling pathways.

The small icosahedral plant RNA nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by a nematode and causes major damage to vineyards worldwide. To elucidate the molecular mechanisms underlying the recognition between the surface of its protein capsid and cellular components of its vector, host and viral proteins synthesized upon infection, the wild type GFLV strain F13 and a natural mutant (GFLV-TD) carrying a Gly297Asp mutation were purified, characterized and crystallized. Subsequently, the geometry and volume of their crystals was optimized by establishing phase diagrams. GFLV-TD was twice as soluble as the parent virus in the crystallization solution and its crystals diffracted X-rays to a resolution of 2.7 A. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3 A by growth in agarose gel. Preliminary crystallographic analyses indicate that both types of crystals are suitable for structure determination. Keys for the successful production of GFLV crystals include the rigorous quality control of virus preparations, crystal quality improvement using phase diagrams, and crystal lattice reinforcement by growth in agarose gel. These strategies are applicable to the production of well-diffracting crystals of other viruses and macromolecular assemblies.

The 3′ untranslated regions (UTRs) of RNA1 and RNA2 of Tomato ringspot virus (ToRSV) are 1.3 kb in size and virtually identical. In this study, sequences containing most of the 3′ UTRs (1168–1265 bp) were determined from 18 ToRSV isolates collected from fruit trees, small fruits and grapevines in North America. Pairwise nucleotide sequence comparisons with sequences of two isolates available in GenBank showed identities ranging from 95.1 to 100% for the majority of ToRSV isolates. Most genetic variation occurs as point mutations and single insertions and/or deletions. Phylogenetic analysis showed that 17 of the 20 isolates grouped together into a major cluster and three isolates grouped into two minor clusters, with genetic divergence rates among the three clusters of 11.8% to 23.8%. No correlation was found between the 3′ UTR sequences and symptom expression on Nicotiana benthamiana, host plant or geographic origin of ToRSV isolates. RT-PCR using primers designed within the highly conserved 3′ U...

The complete genome sequence of grapevine Bulgarian latent virus (GBLV) has been determined. RNA-1 (7,452 nt in length) contains a single ORF of 6,285 nt, encoding a polyprotein with conserved motifs characteristic of the viral protease cofactor (Prot-cofact), the NTP-binding protein (NTP), the cysteine-like protease (Cyst-Prot) and the RNA-dependent RNA polymerase (RdRp) of members of the order Picornavirales and show high aa sequence identity with blackcurrant reversion virus (BRV, 64%). RNA-2 (5,821 nt) contains a single ORF of 4,500 nt, encoding a polyprotein in which the conserved motifs of the movement protein (MP) and coat protein (CP) have been identified. The GBLV CP aa sequence shows highest homology with that of blueberry leaf mottle virus (BLMoV, 68%). Both RNAs have a poly(A) tail and a NCR at the 3' and 5' termini, respectively. The results of this study confirm the classification of GBLV as a member of a distinct species in subgroup C of the genus Nepovirus.

EICHMEIER, A., BARANEK, M., PIDRA, M.: The demonstration of the GFLV Nepovirus isolates on naturally infected grapevine cultivars and evaluation of variability within genome region encoding movement protein. Acta univ. agric. et silvic. Mendel. Brun., 2011, LIX, No. 3, pp. 35–44 In this work, the infection of Grapevine fanleaf virus (GFLV) disease was described on the base of symptomatic diff erences within eight grapevine plants of six grape ciltivars with positive tests on GFLV. Among them, cultivars Kodrjanka, Pamjati Negrula, Kismis Lucistyj were planted in wine region of the South Moravia (Czech Republic), three infected grapevine cultivars (URS, Cinsaut, Dimrit) included in this study originating from Italy. Except symptomatic evaluation, the diff erences between isolates were emphasized at the genetic level too, exactly in the frame of RNA2 genomic region coding movement protein. The variability of the tested isolates within the eight plants was in the range from 86.59 to 97.61% at the nucleotide level. The results confi rmed very high degree of similarity between virus isolates of GFLV within studied RNA2 region. This fact was assessed by the phylogenetic analysis of obtained sequencing data too. Nepovirus, GFLV, sequence, symptomatology

There is limited information on the distribution of blueberry viruses in the U.S. or around the world other than where the viruses were first discovered and characterized. A survey for blueberry viruses was carried out in the U.S. in 2015–2017. Most blueberry viruses have been characterized to the point that sensitive diagnostic assays have been developed. These assays are based on ELISA or variations of PCR, which were employed here to determine the presence of blueberry viruses in major blueberry production and nursery areas of the U.S. The viruses included in this study were: blueberry fruit drop (BFDaV), blueberry latent (BlLV), blueberry leaf mottle (BLMoV), blueberry mosaic (BlMaV), blueberry red ringspot (BRRV), blueberry scorch (BlScV), blueberry shock (BlShV), blueberry shoestring (BlSSV), blueberry virus A (BVA), peach rosette mosaic (PRMV), tobacco ringspot (TRSV), and tomato ringspot (ToRSV). In the Pacific Northwest BlShV was the most widespread virus, with BlScV and ToRSV detected in a limited number of fields in Oregon and Washington, but BlScV was widespread in British Columbia. In the upper midwest, the nematode-borne (ToRSV, TRSV), aphid-transmitted (BlSSV and BVA) and pollen-borne (BLMoV) viruses were most widespread. In the northeast, TRSV, ToRSV, and BlScV, were detected most frequently. In the southeast, BRRV and BNRBV were the most widespread viruses. BlLV, a cryptic virus with no known symptoms or effect on plant growth or yield was present in all regions. There are other viruses present at low levels in each of the areas, but with the lower incidence they pose minimal threat to nursery systems or fruit production. These results indicate that there are hotspots for individual virus groups that normally coincide with the presence of the vectors. The information presented highlights the high risk viruses for nursery and fruit production each pose a different challenge for control.

Pollen is traded internationally as a source of germplasm and for pollination. Thirty-nine viruses and five viroids are known to be pollen transmitted. We investigated whether reverse transcription-polymerase chain reaction (RT-PCR) could be used to detect viruses reliably in pollen. Four extraction methods yielded nucleic acid in appropriate quantity and quality from Tobacco ringspot virus (TRSV)-infected pollen for RT-PCR

Grapevine fanleaf virus (GFLV) is vectored specifically from grapevine to grapevine by the ectoparasitic nematode Xiphinema index. Limited information is available on the vector competency of X. index populations from diverse geographical origins. We determined the transmissibility of two GFLV strains showing 4.6% amino acid divergence within their coat protein (e.g., strains F13 and GHu) by seven clonal lines of X. index developed from seven distinct populations from the Mediterranean basin (Cyprus, southern France, Israel, Italy, and Spain), northern France, and California. X. index lines derived from single adult females were produced on fig (Ficus carica) plants to obtain genetically homogenous aviruliferous clones. A comparative reproductive rate analysis on Vitis rupestris du Lot and V. vinifera cv. Cabernet Sauvignon showed significant differences among clones, with the single-female Cyprus line showing the highest rate (30-fold the initial population) and the Spain and California lines showing the lowest rate (10-fold increase), regardless of the grapevine genotype. However, there was no differential vector competency among the seven X. index lines for GFLV strains F13 and GHu. The implications of our findings for the dynamic of GFLV transmission in vineyards and screening of Vitis spp. for resistance to GFLV are discussed.

Sweet cherries (Prunus avium) are of economic importance in Oregon, which ranks third in sweet cherry production in the United States after Washington and California. Cherry leaf roll virus (CLRV) causes decline in sweet cherry trees. Cherry leaf roll virus is a species of the genus Nepovirus in the family Secoviridae. In May 2016, decline, rosetting (bunching of leaves due to shortened internodes), and enation symptoms in approximately 7% of trees were observed in adjacent sweet cherry orchards cvs. ‘Royal Anne’ and ‘Bing’ on Mazzard rootstock in The Dalles, Wasco County, Oregon. A symptomatic leaf sample (cv. ‘Royal Anne’) was collected and tested positive via CLRV-specific antisera in a double-antibody sandwich ELISA (DSMZ; Braunschweig, Germany). A second collection of both cultivars was made in September 2016 from the same and neighboring orchard blocks. Of the 16 samples collected, eight symptomatic leaf samples tested positive and eight asymptomatic samples tested negative with the same CLRV-specif...

Kumari S. (2009): Detection of Cherry leaf roll virus and Strawberry latent ring spot virus by one-step RTPCR. Plant Protect. Sci., 45: 140–143. A one-step reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed and used for the detection of Cherry leaf roll virus (CLRV) and Strawberry latent ring spot virus (SLRSV). The protocol was used to test infected screen house plants and also plants from orchards and vineyards where the vector (Xiphinema diversicaudatum) of SLRSV was detected from the soil. The one-step RT-PCR protocol is rapid and sensitive and has the potential to be used for the diagnosis of CLRV and SLRSV in routine diagnostic laboratories.

Mekuria, T. A., Gutha, L. R., Martin, R. R., and Naidu, R. A. 2009. Genome diversity and intra- and interspecies recombination events in Grapevine fanleaf virus. Phytopathology 99:1394-1402. Grapevine fanleaf virus (GFLV) was documented in self-rooted vines of four grapevine (Vitis vinifera) cultivars in eastern Washington. GFLV was found as mixed infection in cvs. Pinot Noir, Chardonnay, and Cabernet Franc and as single infections in cv. Merlot. Fanleaf disease symptoms were only observed in the first two cultivars. The spatial distribution of GFLV-infected grapevines was random, suggesting primary spread through planting virus-infected cuttings rather than infield transmission. RNA1 sequences of Washington isolates showed 87 to 89% nucleotide sequence identity between them and with strain F13. RNA2 of Washington isolates was variable in size, showing 85 to 99% sequence identity between them and 81 to 92% with other isolates. As in other GFLV isolates, three conserved putative stem-loop structures were present in the 5′ noncoding regions of both RNAs of Washington isolates. Phylogenetic incongruence of GFLV isolates from Washington in 2A HP and 2BMP-based trees and identification of putative recombination events suggested that their genomic RNA2 originated from inter- and intraspecies recombination events between GFLV, Grapevine deformation virus, and Arabis mosaic virus. These results confirm interspecies recombination in RNA2 of grapevine-infecting nepoviruses as an important strategy for GFLV evolution.

International audience; Recombination was assessed in a vineyard site in which grapevines cross-protected with mild strains GHu of Grapevine fanleaf virus (GFLV) or Ta of Arabis mosaic virus (ArMV) were superinfected with GFLV field isolates following transmission by the nematode vector Xiphinema index. The genetic structure and variability within RNA2 of isolates from grapevines co-infected with GFLV field isolates and either GFLVGHu or ArMV-Ta were characterized to identify intra- and interspecies recombinants. Sequence analysis and phylogenetic relationships inferred intraspecies recombination among GFLV field isolates but not between field isolates and GFLV-GHu. SISCAN analysis confirmed a mosaic structure for two GFLV field isolates for which recombination siteswere located in the movement protein and coat protein genes. One of the recombinantswas found in eight grapevines thatwere in close spatial proximity within the vineyard site, suggesting its transmission by X. index. No interspecies recombination was detected between GFLV field isolates and ArMV-Ta. Altogether, our findings suggest that mild protective strains GFLV-GHu and ArMV-Ta did not assist the emergence of viable recombinants to detectable level during a 12-year crossprotection trial. To our knowledge, this is the first extensive characterization of the genetic structure and variability of virus isolates in cross-protected plants.

We analysed the kinetics of virus distribution in Tobacco ringspot virus (TRSV)-infected tobacco. Infected plants developed asymptomatic leaves above the symptomatic leaves that exhibited ringspots. We detected virus in the shoot apical meristem (SAM). Time-course immunohistochemical microscopy showed that TRSV continuously distributed in the centres of the SAM and in leaf primordia. In contrast, TRSV infection was transient in the root meristem. In western blot and RT-PCR analyses, TRSV infection occurred only within and around the ringspot tissues of the symptomatic leaf; however, TRSV was not or hardly detected in the asymptomatic leaves of inoculated tobacco plants. These results indicate that TRSV persists in the SAM, but that TRSV infection is transient in the root meristem and asymptomatic leaves.

Grapevine fanleaf virus (GFLV) is responsible for severe fanleaf degeneration in grapevines of all major wine producing regions of the world, including South Africa. In order to successfully control the spread of the virus, specific and reliable diagnostic assays are necessary. The genetic variability of 12 GFLV isolates recovered from naturally infected grapevine plants in the Western Cape region of South Africa were characterised. These samples were subjected to RNA extraction, RT-PCR analysis and sequencing of the coat protein gene (2CCP). Sequence identities between different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the 2CCP gene sequences showed that the South African isolates form two distinct clades or sub-populations. The specificity and sensitivity of three diagnostic techniques (rapid-direct-one-tube-RT-PCR, DAS-ELISA and ImmunoStrips) for the detection of GFLV were analysed to determine the appropriate diagnostic assay for virus infection. Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for detection. This is the first report on sequence analysis of full-length 2CCP gene cDNA clones of GFLV isolates from South Africa.

International audience; The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed “Secoviridae” to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae.

The wide natural incidence of Cherry leaf roll virus (CLRV) in deciduous forest trees and nurseries in northern Europe is believed to have occurred, apart from occasional mechanical spread and transmission through grafting, mainly by seed transmission. The mode of the vertical transmission and its role in the epidemiology of the virus has not been investigated, basically due to the inconvenient host-pathogen combinations studied to date. With the aim of obtaining an appropriate system for identification of viral genes and products participating in infection processes and seed transmission of CLRV, we performed infection and seed transmissibility tests with CLRV in Arabidopsis thaliana plants. Two phylogenetically and serologically different CLRV isolates were tested. Both of them were found able to infect A. thaliana plants, exhibited clear symptoms of the infection and spread systemically in the plants. Infection of the seeds and of a remarkable number of seedlings generated from infected seeds was possible for two consecutive generations. These results, for first time, report seed transmission of CLRV in the model plant A. thaliana and allow the assumption to be made of embryo invasion during seed transmission. Furthermore, first indications are given that genetically diverse CLRV isolates exhibit different abilities for vertical transmission in A. thaliana. The CLRV-A. thaliana model system is suitable for investigating viral invasion of developing plant organs and meristematic tissue, a prerequisite for successful virus dissemination via vertical transmission through seed.

SUMMARY Grapevine fanleaf virus (GFLV) is one of the most destructive pathogens of grapevine. In this study, we generated monoclonal antibodies binding specifically to the coat protein of GFLV. Antibody FL3, which bound most strongly to GFLV and showed cross-reactivity to Arabis mosaic virus (ArMV), was used to construct the single-chain antibody fragment scFvGFLVcp-55. To evaluate the potential of this single-chain variable fragment (scFv) to confer antibody-mediated virus resistance, transgenic Nicotiana benthamiana plants were generated in which the scFv accumulated in the cytosol. Recombinant protein levels of up to 0.1% total soluble protein were achieved. The T1 and T2 progenies conferred partial or complete protection against GFLV on challenge with the viral pathogen. The resistance to GFLV in transgenic plants was strictly related to scFvGFLVcp-55 accumulation levels, confirming that the antibody fragment was functional in planta and responsible for the GFLV resistance. In addition, transgenic plants conferring complete protection to GFLV showed substantially enhanced tolerance to ArMV. We demonstrate the first step towards the control of grapevine fanleaf degeneration, as scFvGFLVcp-55 could be an ideal candidate for mediating nepovirus resistance.

Attempts to conserve and utilise autochthonous grapevine germplasm in modern breeding programmes, are sometimes faced with the challenge that virus-free plants of old grapevine varieties and clones are hard to find. From 50 year-old vineyards in Frankonia the Vitis vinifera cv. Domina was selected showing particularly interesting loose-bunch architecture with fewer berries. However this valuable germplasm was carrying an Arabis mosaic virus (ArMV) infection requiring a reliable and effective method to produce healthy mother plants for clonal selection. Somatic embryogenesis was established from anthers as the most promising technical approach. The absence of ArMV in 46 regenerated plant lines was confirmed by ELISA and IC-RT PCR, repeated after different time intervals in vitro and in vivo after acclimatisation, and after one dormancy period under glasshouse conditions. Morphologically, all grapevines appeared true-to-type, and a screening of 20 plants by flow cytometry to determine the ploidy level and to exclude the risk of undesired genetic variability confirmed that all tested plants were diploid. Field evaluations of the initially selected bunch traits are currently underway.

Two generic PCR protocols were developed to detect nepoviruses in subgroups A and B using degenerate primers designed to amplify part of the RNA-dependent RNA polymerase (RdRp) gene. It was observed that detection sensitivity and specificity could be improved by adding a 12-bp non-complementary sequence to the 5' termini of the forward, but not the reverse, primers. The optimized PCR protocols amplified a specific product ( approximately 340bp and approximately 250bp with subgroups A and B, respectively) from all 17 isolates of the 5 virus species in subgroup A and 3 species in subgroup B tested. The primers detect conserved protein motifs in the RdRp gene and it is anticipated that they have the potential to detect unreported or uncharacterised nepoviruses in subgroups A and B.

Surveys for virus diseases and nematode vectors were conducted in 95 commercial vineyards of four different Lebanese districts (Bekaa valley, Mount Lebanon, North and South Lebanon). Out of 915 randomly collected grapevine samples tested by ELISA, 511 (55.8%) were infected by one or more viruses. Grapevine virus A (30.9%) and Grapevine leafroll-associated virus 3 (23.7%) were the prevailing viruses, followed by Grapevine fleck virus (15.1%), Grapevine leafroll-associated virus 1 (10.6%) and Grapevine leafroll-associated virus 2 (8.7%). Arabis mosaic virus was not found whereas Grapevine fanleaf virus (GFLV) and Grapevine virus B were little represented. The most important Lebanese grapevine varieties, i.e. Maghdouchi, Tfeifihi and Beitamouni, had average infection rates between 70% and 87%, whereas varieties of foreign origin had a better sanitary status with the exception of cvs Cinsaut and Thompson (c. 83% infection). Grapevine rupestris stem pitting-associated virus was detected in 79 of 90 (87.8%) samples tested by RT-PCR and closteroviruses were recorded in seven of 70 (10%) vines tested. One of these viruses was identified as Grapevine leafroll-associated virus 5 by ELISA and partial genome sequencing. No nepoviruses other than GFLV were detected in any of 90 samples tested using three different sets of degenerate primers. Xiphinema index was found in 23 of 89 soil samples collected from vineyards, and in three of 15 samples collected primarily under fig trees in fields where no grapevines were grown.

Five isolates of Grapevine fanleaf virus (GFLV) collected from two viticultural regions in the north-east of Iran were characterized based on coat protein (CP) gene sequences. The CP gene of all isolates was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using its specific primers, yielding a 1500bp amplicon. The nucleotide sequences for these isolates were obtained and compared with the CPI region of seven isolates from north-west Iran and also with some other exotic GFLV-CP sequences available in the GenBank. Analysis of our data revealed that the north-east Iranian sequences have 83.7–87.5% of homology with the CPI regions of exotic GFLV isolates, and 83.0–87.1% homology with other isolates from north-west Iran. High nucleotide homology (98.6–100%) was found among the GFLV isolates sequences obtained in this study. The deduced proteins obtained for our five isolates were 92.0–96.0% identical to other sequences. The generated phylogenetic tree revealed a clustering of GFLV isolates distributed over the world in one branch, and the Iranian isolates in other branches.

We collected samples from black, red and white currants showing symptoms of blackcurrant reversion disease (BRD) and full blossom disease (FBD), cultivated in the Czech Republic. Blackcurrant reversion virus (BRV) was detected in all symptomatic plants. After amplification, a substantial part of the 3′ non-translated region (3′-NTR) of RNA2 of 15 new isolates of BRV was sequenced and compared with sequences available in the literature and GenBank. We did not find significant sequence diversity among isolates associated with either FBD or BRD. BRV was graft-transmitted from FBD infected red currant to black currant where symptoms of BRD were observed. Further sequence analysis of BRV isolates resulted in a phylogenetic tree with four branches, each consisting of six to nine isolates. No correlation with geographic origin was visible on the tree as isolates from various countries occurred in all four branches. We also found no correlation between the host and the topology of the tree: most of black currant isolates occurred in branches 3 and 4, but also occurred in branches 1 and 2. Only one white currant and one red currant isolate occurred in branches 3 and 4, respectively. The sequence identity of the Czech isolates in this region ranged from 91.9 to 99.8%. The 17 plant species growing within and in the close vicinity of the BRD-infested plantation were tested negative for BRV by RT-PCR as natural hosts of BRV. BRV was successfully transmitted by mechanical inoculation from black currant to Nicotiana occidentalis and N. tabacum cv. Xanthi, the latter being a new host for BRV. The infection was confirmed by PCR and sequencing.

Replication of Tomato ringspot virus (ToRSV) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleotide triphosphate-binding protein (NTB) of ToRSV is an ER-targeted protein and that an intermediate polyprotein containing the domains for NTB and for the genome-linked viral protein (VPg) is associated with the replication complex. We now report the detection of a 95-kDa polyprotein that contains the domains for the RNA-dependent RNA polymerase (Pol), the proteinase (Pro) and the VPg. This polyprotein appears to be a truncated version of the full-length 111-kDa VPg-Pro-Pol polyprotein and was termed VPg-Pro-Pol′. A subpopulation of VPg-Pro-Pol′ was peripherally associated with ER-derived membranes active in viral replication. However, the VPg, Pro and Pol domains did not target to membranes in the absence of viral infection. We propose a model in which VPg-Pro-Pol′ is brought to the site of replication through interaction with a viral membrane protein.

Plant viruses cause a wide array of disease symptoms and cytopathic effects. Although some of these changes are virus specific, many appear to be common even among diverse viruses. Currently, little is known about the underlying molecular determinants. To identify gene expression changes that are concomitant with virus symptoms, we performed comparative expression profiling experiments on Nicotiana benthamiana leaves infected with one of three different fruit tree viruses that produce distinct symptoms: Plum pox potyvirus (PPV; leaf distortion and mosaic), Tomato ringspot nepovirus (ToRSV; tissue necrosis and general chlorosis), and Prunus necrotic ringspot ilarvirus (PNRSV; subtle chlorotic mottling). The numbers of statistically significant genes identified were consistent with the severity of the observed symptoms: 1,082 (ToRSV), 744 (PPV), and 89 (PNRSV). In all, 56% of the gene expression changes found in PPV-infected leaves also were altered by ToRSV, 87% of which changed in the same direction. Both PPV- and ToRSV-infected leaves showed widespread repression of genes associated with plastid functions. PPV uniquely induced the expression of large numbers of cytosolic ribosomal genes whereas ToRSV repressed the expression of plastidic ribosomal genes. How these and other observed expression changes might be associated with symptom development are discussed.

Application of a simplified protocol for nucleic acid preparation for polymerase chain reaction (PCR) detection of greening bacterium (Candidatus Liberibacter asiaticus; Cla) and Citrus mosaic virus (CMBV) associated with citrus is described. Crude extracts of citrus tissues in NaOH - ethylenediaminetetraacetic acid solution, prepared without the use of liquid nitrogen, were spotted on a nitrocellulose membrane (NCM) and then eluted in water at 80 °C for 10 min. Both the greening bacterium and CMBV were detected by PCR from the eluted liquid from the spotted NCMs. The detection efficacy for the templates obtained in the NCM-eluted liquid was comparable with that of templates obtained with a multistep laboratory method or a commercial kit for nucleic acid preparation. The protocol is simple, inexpensive, rapid, and applicable to large-scale surveys of citrus trees. The spotted-membrane methodology can also be used for short-term sample storage for future testing or for long-distance sample transport to a d...

Based on the nucleotide sequence homology of RNA-1 and RNA-2 of nepoviruses isolated from grapevines, three sets of degenerate primers, one for each of the three subgroups of the genus (A, B and C), were designed and proved effective for RT-PCR detection of subgroups in infected grapevines and herbaceous hosts. Primers designed specifically for detecting subgroup A species amplified a fragment of 255 bp from samples infected by Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV) and Grapevine deformation virus (GDefV), but not from samples infected by other nepovirus species. Similarly, primers for detection of subgroup B nepoviruses amplified a 390 bp product from samples infected by Grapevine chrome mosaic virus (GCMV), Tomato black ring virus (TBRV), Grapevine Anatolian ringspot virus (GARSV) and Artichoke Italian latent virus (AILV). The third set of primers amplified a 640 bp fragment, only from samples infected by subgroup C nepoviruses, i.e Tomato ringspot virus (ToRSV) Grapevine Bulgarian latent virus (GBLV), and Grapevine Tunisian ringspot virus (GTRSV). These primers were able to detect simultaneously all viral species belonging to the same subgroup and to discriminate species of different subgroups. Multiplex-PCR detection of subgroup A and B nepoviruses was obtained using a specific primer (sense for subgroup A and antisense for subgroup B) for each of the species of the same subgroup in combination with the degenerate subgroup-specific primers. In this way it was possible to detect four different viral species in single samples containing mixtures of viruses of the same subgroup. In particular, for viruses of subgroup A (TRSV, GFLV, ArMV and GDefV) amplicons of 190, 259, 301 and 371 bp were obtained, whereas amplicons of 190, 278, 425 and 485 bp, respectively, were obtained from samples infected with viruses of subgroup B (GCMV, AILV, GARSV and TBRV).

The nucleolytic ribozymes catalyse site-specific phosphodiester cleavage and ligation transesterification reactions in RNA. The hairpin ribozyme folds to generate an intimate loop-loop interaction to create the local environment in which catalysis can proceed. We have studied the ion-induced folding using single-molecule FRET experiments, showing that the four-way helical junction accelerates the folding 500-fold by introducing a discrete intermediate that juxtaposes the loops. Using FRET we can observe individual hairpin ribozyme molecules as they undergo multiple cycles of cleavage and ligation, and measure the rates of the internal reactions, free of uncertainties in the contributions of docking and substrate dissociation processes. On average, the cleaved ribozyme undergoes several docking-undocking events before a ligation reaction occurs. On the basis of these experiments, we have explored the role of the nucleobases G8 and A38 in the catalysis. Both cleavage and ligation reactions are pH dependent, corresponding to the titration of a group with pKa=6.2. We have used a novel ribonucleoside in which these bases are replaced by imidazole to investigate the role of acid-base catalysis in this ribozyme. We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both.

The 5′ and 3′ non-translated regions (NTRs) of mRNAs of eukaryotes and their viruses often contain translational enhancers, including internal ribosomal entry sites (IRESs) comprised in the 5′ leaders of many uncapped viral mRNAs.Blackcurrant reversion virus(BRV) has a genome composed of two uncapped, polyadenylated RNAs with relatively short 5′ NTRs, almost devoid of secondary structure. In this work, a role of the RNA2 5′ NTR in translation was studied by using mono- and dicistronicPhotinus pyralisandRenilla reniformisluciferase reporter mRNAs in protoplasts ofNicotiana benthamiana. The RNA2 5′ leader was found to confer efficientin vivotranslation compared with the control 5′ NTR, and each half of the BRV leader was essential for stimulatory function. Such efficient translational enhancement was mediated, at least in part, through an IRES mechanism. Multiple RNA2 5′ NTR regions, complementary to a fragment of plant 18S rRNA demonstrated previously to be accessible for intermolecular mRNA–rRNA interactions and conserved between eukaryotes, were shown to be important for efficient translation. Similar mRNA–rRNA base-pairing potential was also predicted for the 5′ leaders of other nepoviruses.

Due to their small size, high stability and strict monomeric nature, Nanobodies (Nbs) deriving from camelids heavy chain only antibodies have proven very valuable as diagnostic and therapeutic tools. However their use in agro biotechnology remains limited.In order to apply Nbs to the study and the control of grapevine fanleaf degeneration, I produced acollection of Nbs against Grapevine fanleaf virus (GFLV), the causal agent of this devastating disease worldwide.When fused to a fluorescent protein and stably expressed in plants, one of these Nbs (calledChromobody, Cb) conferred high resistance to GFLV, whether inoculated mechanically or by vector-mediated transmission.The identification of an isolate overcoming the resistance but still bound by the Cb allowed real-time tracking of the infection showing the high potential of Cbs as biosensors.The cryoEM structure of the Nb/GFLV complex was obtained at 2,8 Å and provides a clear picture of the footprint of the Nb on the surface of the GFLV capsid.These results pave the way for the innovative use of Nbs to unravel viral life cycle and to counter viral diseases.; Par leur stabilité, leur petite taille et leur nature monomérique, les domaines variables des immunoglobulines à chaînes lourdes, ou Nanobodies (Nb), sont incontournables en diagnostic et recherche médicale. Pourtant, leur utilisation en agro-biotechnologies demeure confidentielle.Dans l'idée de les utiliser pour étudier et combattre le Grapevine fanleaf virus (GFLV), responsable de la maladie du court-noué très préjudiciable à l'économie viticole mondiale, j'ai produit une collection de Nb spécifiques du GFLV.Fusionné à une protéine fluorescente et exprimé en plante de façon stable, un de ces Nb (alors appelé Chromobody, Cb) a conféré une haute résistance au GFLV inoculé mécaniquement ou transmis par nématodes.Le potentiel du Cb comme biocapteur a été validé par le suivi in vivo d’un isolat contournant la résistance mais toujours reconnu par le Cb. La structure du complexe Nb/GFLV a été résolue à 2,8 Å et révèle la zone occupée par le Nb à la surface de la capside.Ces résultats ouvrent des perspectives innovantes pour la compréhension du cycle infectieux d'un phytovirus et l'élaboration de nouvelles stratégies de lutte antivirale.

The Peach rosette mosaic virus (PRMV) is a plant pathogen of the genus Nepovirus, and has been designated as a controlled quarantine virus in Korea. In this study, a specific reverse transcription (RT)-PCR marker set, nested PCR marker set, and modified-plasmid positive control were developed to promptly and accurately diagnose PRMV at plant-quarantine sites. The final selected PRMV-specific RT-PCR marker was PRMV-N10/C70 (967 bp), and the nested PCR product of 419 bp was finally amplified. The modified-plasmid positive control, in which the SalI restriction-enzyme region (GTCGAC) was inserted, verified PRMV contamination in a comparison with the control, enabling a more accurate diagnosis. It is expected that the developed method will continuously contribute to the plant-quarantine process in Korea.

Fanleaf degeneration is considered the most damaging viral disease of grapevine. The two major nepoviruses involved are Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) which are respectively and specifically transmitted by the dagger nematodes Xiphinema index and X. diversicaudatum. The methods described below are aimed at detecting four prevalent grapevine Xiphinema species: the vector species previously mentioned and two nonvector species X. vuittenezi and X. italiae.

Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host-TRSV interactions.

Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 μg/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus , with detection limits of 2 to 60 ng/ml.