The proposed phylogenetic structure of the genus Tobamovirus supports the idea that these viruses have codiverged with their hosts since radiation of the hosts from a common ancestor. The determinations of genome sequence for two strains of Passion fruit mosaic virus (PafMV), a tobamovirus from plants of the family Passifloraceae (order Malpighiales) from which only one other tobamovirus (Maracuja mosaic virus; MarMV) has been characterized, combined with the development of Bayesian analysis methods for phylogenetic inference, provided an opportunity to reassess the co-divergence hypothesis. The sequence of one PafMV strain, PfaMV-TGP, was discovered during a survey of plants of the Tallgrass Prairie Preserve for their virus content. Its nucleotides are only 73 % identical to those of MarMV. A conserved ORF not found in other tobamovirus genomes, and encoding a cysteine-rich protein, was found in MarMV and both PafMV strains. Phylogenetic tree construction, using an alignment of the nucleotide sequences of PafMV-TGP and other tobamoviruses resulted in a major clade containing isolates exclusively from rosid plants. Asterid-derived viruses were exclusively found in a second major clade that also contained an orchid-derived tobamovirus and tobamoviruses infecting plants of the order Brassicales. With a few exceptions, calibrating the virus tree with dates of host divergence at two points resulted in predictions of divergence times of family specific tobamovirus clades that were consistent with the times of divergence of the host plant orders.
Next generation sequencing (NGS) is not used commonly in diagnostics, in part due to the large amount of time and computational power needed to identify the taxonomic origin of each sequence in a NGS data set. By using the unassembled NGS data sets as the target for searches, pathogen-specific sequences, termed e-probes, could be used as queries to enable detection of specific viruses or organisms in plant sample metagenomes. This method, designated e-probe diagnostic nucleic acid assay, first tested with mock sequence databases, was tested with NGS data sets generated from plants infected with a DNA (Bean golden yellow mosaic virus, BGYMV) or an RNA (Plum pox virus, PPV) virus. In addition, the ability to detect and differentiate among strains of a single virus species, PPV, was examined by using probe sets that were specific to strains. The use of probe sets for multiple viruses determined that one sample was dually infected with BGYMV and Bean golden mosaic virus.
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