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.

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Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA.

The genetic variation and evolutionary mechanisms of broad bean wilt virus 2 (BBWV-2) were studied by nucleotide sequence analysis of four genomic regions of 30 isolates from different countries. Nucleotide diversity was high (0.198) for a plant virus. Phylogenetic and genetic structure analyses showed low population subdivision, suggesting a significant gene flow between distant geographic areas. Analysis of synonymous and nonsynonymous substitutions showed different negative selection pressures for different parts of the coding regions, but no positive selection was found. Several recombination detection methods showed that some BBWV-2 genomes might have originated from recombination or reassortment.

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The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

Viruses cause significant damage in agricultural crops worldwide. Disease management requires sensitive and specific tools for virus detection and identification. Also, detection techniques need to be rapid to keep pace with the continuous emergence of new viral diseases. The genus Fabavirus is composed of five viruses infecting many economically important crops worldwide. This research describes the development of a procedure based on flow-through hybridisation (FTH), which is faster than and as sensitive as conventional hybridisation for virus detection in tissue-prints from infected plants. Six digoxigenin-labelled RNA probes were synthesised with two levels of specificity: (a) five specific for each viral species within this genus, and (b) a genus-specific probe that hybridises with a nucleotide sequence signature only found in the 5′-untranslated region of the genus Fabavirus, which is the first of this type reported for plant viruses. The new procedure developed is useful for rapid detection and discrimination of the five fabaviruses identified so far and opens the possibility of discovering new species of this genu

Tomato apex necrosis virus (ToANV, species Tomato marchitez virus, genus Torradovirus, family Secoviridae) causes a severe tomato disease in Mexico. One distinctive feature of torradoviruses compared with other members of the family Secoviridae is the presence of an additional open reading frame (ORF) in genomic RNA2 (denominated RNA2-ORF1), located upstream of ORF2. RNA2-ORF2 encodes a polyprotein that is processed into a putative movement protein and three capsid proteins (CPs). The RNA2-ORF1 protein has homologues only amongst other torradoviruses and, so far, no function has been associated with it. We used recombinant and mutant ToANV clones to investigate the role of the RNA2-ORF1 protein in various aspects of the virus infection cycle. The lack of a functional RNA2-ORF1 resulted in an inability to systemically infect Nicotiana benthamiana and tomato plants, but both positive- and negative-strand RNA1 and RNA2 accumulated locally in agroinfiltrated areas in N. benthamiana plants, indicating that the RNA2-ORF1 mutants were replication competent. Furthermore, a mutant with a deletion in RNA2-ORF1 was competent for virion formation and cell-to-cell movement in the cells immediately surrounding the initial infection site. However, immunological detection of the ToANV CPs in the agroinfiltrated areas showed that this mutant was not detected in the sieve elements even if the surrounding parenchymatic cells were ToANV positive, suggesting a role for the RNA2-ORF1 protein in processes occurring prior to phloem uploading, including efficient spread in inoculated leaves.