Plant viruses cause considerable economic losses and are a threat for sustainable agriculture. The frequent emergence of new viral diseases is mainly due to international trade, climate change, and the ability of viruses for rapid evolution. Disease control is based on two strategies: i) immunization (genetic resistance obtained by plant breeding, plant transformation, cross-protection, or others), and ii) prophylaxis to restrain virus dispersion (using quarantine, certification, removal of infected plants, control of natural vectors, or other procedures). Disease management relies strongly on a fast and accurate identification of the causal agent. For known viruses, diagnosis consists in assigning a virus infecting a plant sample to a group of viruses sharing common characteristics, which is usually referred to as species. However, the specificity of diagnosis can also reach higher taxonomic levels, as genus or family, or lower levels, as strain or variant. Diagnostic procedures must be optimized for accuracy by detecting the maximum number of members within the group (sensitivity as the true positive rate) and distinguishing them from outgroup viruses (specificity as the true negative rate). This requires information on the genetic relationships within-group and with members of other groups. The influence of the genetic diversity of virus populations in diagnosis and disease management is well documented, but information on how to integrate the genetic diversity in the detection methods is still scarce. Here we review the techniques used for plant virus diagnosis and disease control, including characteristics such as accuracy, detection level, multiplexing, quantification, portability, and designability. The effect of genetic diversity and evolution of plant viruses in the design and performance of some detection and disease control techniques are also discussed. High-throughput or next-generation sequencing provides broad-spectrum and accurate identification of viruses enabling multiplex detection, quantification, and the discovery of new viruses. Likely, this technique will be the future standard in diagnostics as its cost will be dropping and becoming more affordable.

Tomato spotted wilt virus (TSWV) causes severe economic losses in many crops worldwide and often overcomes resistant cultivars used for disease control. Comparison of nucleotide and amino acid sequences suggested that tomato resistance conferred by the gene Sw-5 can be overcome by the amino acid substitution C to Y at position 118 (C118Y) or T120N in the TSWV movement protein, NSm. Phylogenetic analysis revealed that substitution C118Y has occurred independently three times in the studied isolates by convergent evolution, whereas the substitution T120N was a unique event. Analysis of rates of non-synonymous and synonymous changes at individual codons showed that substitution C118Y was positively selected.Tomato spotted wilt virus (TSWV) is one of the most economically important plant viruses and causes damage in many agronomic crops worldwide (Adkins, 2000). TSWV is the type member of the genus Tospovirus, which contains the only plant-infecting members of the family Bunyaviridae. The genome consists of three negative-sense or ambisense RNA segments: segment L encodes a putative RNAdependent RNA polymerase (de Haan et al., 1991); segment M encodes the cell-to-cell movement protein, NSm (Li et al., 2009), and the precursor of the surface glycoproteins, G N / G C , involved in TSWV transmission by thrips (Sin et al., 2005;Naidu et al., 2008); and segment S encodes a silencing suppressor, NSs (Takeda et al., 2002), and the nucleocapsid, N (de Haan et al., 1990).Controlling TSWV in crop plants has proven difficult because of the wide range of plant hosts (.1000 species) and effective spread of TSWV by thrip vectors (Hanssen et al., 2010). Intense efforts have been made around the world to obtain genetically resistant cultivars against TSWV. Only the dominant genes Sw-5 and Tsw introgressed in tomato (Lycopersicon esculentum, 5Solanum lycopersicum) and pepper (Capsicum annuum) cultivars, respectively, have been found to confer resistance to a wide spectrum of TSWV isolates; they have been deployed in commercial cultivars worldwide. Sw-5 is also effective against two other tospoviruses, Tomato chlorotic spot virus (TCSV) and Groundnut ringspot virus (GRSV) (Soler et al., 2003). However, Sw-5 resistance-breaking (SRB) isolates have been detected in Hawaii, Australia, South Africa, Spain and Italy (Cho et al., 1996;Thompson & van Zijl, 1996;Latham & Jones, 1998;Aramburu & Martí, 2003;Ciuffo et al., 2005) and Tsw resistance-breaking (TRB) isolates have been detected in Brazil, USA, Italy, Spain and Australia (Boiteux et al., 1993;Hobbs et al., 1994;Roggero et al., 2002; Margaria et al., 2004;Sharman & Persley, 2006).Understanding the molecular mechanisms linked to resistance breakdown and the evolutionary processes involved in the emergence of resistance-breaking isolates is a major challenge. This is relevant to the development of more durable and efficient resistance, which would certainly have a considerable economic impact on agriculture. Since a reverse-genetics system based on infectious cDNA clones is not availab...

A graft-transmissible pathogen causing bud union crease of Nagami kumquat SRA-153 on Troyer citrange was characterized for host range and symptomatology. Buds of Marsh grapefruit, Nules clementine, Eureka lemon and Pineapple sweet orange preinoculated with kumquat SRA-153 were propagated on citrange rootstocks. Some plants of Nules clementine and Eureka lemon had developed bud union crease six months after propagation, whereas all Marsh grapefruit and Pineapple sweet orange plants still showed normal bud union after one year. On indexing these preinoculated species, Nules clementine and Eureka lemon caused vein clearing in Pineapple sweet orange and Dweet tangor, chlorotic blotching in Dweet tangor and stem pitting in Etrog citron, whereas Marsh grapefruit and Pineapple sweet orange caused only chlorotic blotching in Dweet tangor and stem pitting in Etrog citron. Following shoot-tip grafting in vitro of kumquat SRA-153, kumquats 38-1 and 497-2 obtained from it caused chlorotic blotching in Dweet tangor and stem pitting in Etrog citron, but not vein clearing in Pineapple sweet orange and Dweet tangor or bud union crease when propagated on citrange. These results suggest the presence of at least two pathogens or pathogen strains in kumquat SRA-153 and the elimination of one of them after shoot-tip grafting in vitro or inoculation on Marsh grapefruit or Pineapple sweet orange. They also indicate that the pathogens in kumquat SRA-153 can be detected by indexing on Dweet tangor or Etrog citron.

The complete nucleotide sequence of Citrus leaf blotch virus (CLBV) was determined. CLBV genomic RNA (gRNA) has 8747 nt, excluding the 3'-terminal poly(A) tail, and contains three open reading frames (ORFs) and untranslated regions (UTR) of 73 and 541 nucleotides at the 5' and 3' termini, respectively. ORF1 potentially encodes a 227.4-kDa polypeptide, which has methyltransferase, papain-like protease, helicase, and RNA-dependent RNA polymerase motifs. ORF2 encodes a 40.2-kDa polypeptide containing a motif characteristic of cell-to-cell movement proteins. The 40.7-kDa polypeptide encoded by ORF3 was identified as the coat protein. The genome organization of CLBV resembles that of viruses in the genus Trichovirus, but they differ in various aspects: (i) in trichoviruses ORF2 overlaps ORFs 1 and 3, whereas in CLBV, ORFs 2 and 3 are separated and ORFs 1 and 2 overlap in one nucleotide; (ii) CLBV gRNA and CP are larger than those of trichoviruses; and (iii) the CLBV 3' UTR is larger than that of trichoviruses. Phylogenetic comparisons based on CP amino acid signatures clearly separates CLBV from trichoviruses. Also contrasting with trichoviruses, CLBV could not be transmitted to Chenopodium quinoa Willd. Considering these singularities, we propose that CLBV should be included in a new virus genus.

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Parietaria mottle virus (PMoV-T) infecting tomatoes from different commercial fields in Catalonia, Spain was detected by dot-blot and tissue-printing hybridization and one-step reverse transcriptase PCR (RT-PCR). Specific cDNA was amplified by RT-PCR from mechanically infected Chenopodium quinoa plants and cloned into a plasmid vector. A digoxigenin-labelled riboprobe was synthesized for molecular hybridization, and a specific set of primers (PMoV-1F/ PMoV-1R) were designed for one-step RT-PCR assays. Material from different parts of PMoV-T-infected tomato plants was used for the analysis. The virus was detected on necrotic apical shoots, fruits with symptoms, and symptomless shoots grown from old necrotic branches, but not on the old necrotic branches themselves. Both molecular hybridization and one-step RT-PCR procedures were highly specific, and no cross-reactions were observed with other related ilarviruses. PMoV-T was detected successfully by molecular techniques on all infected tomato plants collected from commercial fields, although one-step RT-PCR was less robust than molecular hybridization. The observed reliability of tissue-printing hybridization could greatly facilitate both the large-scale analysis and epidemiological studies of this important pathogen.

Citrus leaf blotch virus (CLBV) has a single-stranded, positive-sense, genomic RNA (gRNA) organized in three ORFs, which encode a polyprotein involved in replication (RP), a potential movement protein (MP), and coat protein (CP). Northern blot hybridization of total, virion, or double-stranded RNA with probes of different gRNA regions revealed that CLBV produces two 3'-coterminal and two 5'-coterminal subgenomic RNAs (sgRNAs). The 3'-coterminal sgRNAs contain the MP (3'MP sgRNA) and CP (3'CP sgRNA) genes and untranslated regions (UTRs) of 123 and 284 nt, respectively, at their 5' end. These sgRNAs start with a hexanucleotide which is also present at the 5' terminus of the gRNA. The 5'-coterminal sgRNAs have 6795 and 5798 nt, colinear with the gRNA, and contain ORF1 and most MP gene (5'RPMP sgRNA) and most ORF1 (5'RP sgRNA), respectively. Their 3' termini map 35 and 40 nt upstream of the transcription initiation of the 3'CP and 3'MP sgRNAs, respectively, next to a potential promoter element. Our results suggest that, as in alphaviruses, CLBV internal genes are expressed via 3'-coterminal sgRNAs transcribed from the minus gRNA strand. The 5'-coterminal sgRNAs may result from early termination of the gRNA during the plus-strand synthesis.

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