Next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology

Abstract

A novel, unbiased approach to plant viral disease diagnosis has been developed which requires no a priori knowledge of the host or pathogen. Next-generation sequencing coupled with metagenomic analysis was used to produce large quantities of cDNA sequence in a model system of tomato infected with Pepino mosaic virus. The method was then applied to a sample of Gomphrena globosa infected with an unknown pathogen originally isolated from the flowering plant Liatris spicata. This plant was found to contain a new cucumovirus, for which we suggest the name 'Gayfeather mild mottle virus'. In both cases, the full viral genome was sequenced. This method expedites the entire process of novel virus discovery, identification, viral genome sequencing and, subsequently, the development of more routine assays for new viral pathogens.

Figure 1

Sequence coverage and gene positions along the lengths of the viral genomes (5′–3′): (A) Pepino mosaic virus; (B) Gayfeather mild mottle virus (GMMV) RNA 1; (C) GMMV RNA 2; (D) GMMV RNA 3.

Figure 2

Bootstrapped neighbour‐joining tree (1000 replicates) constructed from an alignment of 1a protein sequences from the Bromoviridae family. Gayfeather mild mottle virus (GMMV) is placed within the Cucumovirus genus.

Figure 3

Bootstrapped neighbour‐joining tree (1000 replicates) constructed using uncorrected P distances derived from an alignment of the full reference genomes of all known cucumoviruses [Cucumber mosaic virus (CMV), Peanut stunt virus (PSV) and Tomato aspermy virus (TAV)], Gayfeather mild mosaic virus and a Bromovirus, Broad bean mottle virus (BBMV). Reference genome accession numbers: PSV (NC_002038, NC_002039, NC_002040); CMV (NC_002035, NC_002034, NC_001440); TAV (NC_003837, NC_003838, NC_003836); BBMV (NC_004008, NC_004007, NC_004006).