Ecogenomic survey of plant viruses infecting Tobacco by Next generation sequencing

Abstract

Background: The invasion of plant by viruses cause major damage to plants and reduces crop yield and integrity. Devastating plant virus infection has been experienced at different times all over the world, which are attributed to different events of mutation, re-assortment and recombination occurring in the viruses. Strategies for proper virus management has been mostly limited to eradicating the vectors that spreads the plant viruses. However, development of prompt and effective diagnostic methods are required to monitor emerging and re-emerging diseases that may be symptomatic or asymptomatic in the plant as well as the genetic variation and evolution in the plant viruses. A survey of plant viruses infecting field-grown Tobacco crop was conducted in Anhui Province of China by the deep sequencing of sRNAs.

Methods: Survey of plant viruses infecting Tobacco was carried based on 104 samples collected across the province. Nine different sRNA libraries was prepared and custom-made bioinformatics pipeline coupled with molecular techniques was developed to sequence, assemble and analyze the siRNAs for plant virus discovery. We also carried out phylogenetic and recombination analysis of the identified viruses.

Results: Twenty two isolates from eight different virus species including Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, Tobacco vein banding Mosaic virus, Pepper mottle virus, Brassica yellow virus, Chilli venial mottle virus, Broad bean wilt virus 2 were identified in tobacco across the survey area. The near-complete genome sequence of the 22 new isolates were determined and analyzed. The isolates were grouped together with known strains in the phylogenetic tree. Molecular variation in the isolates indicated the conserved coding regions have majorly a nucleotide sequence identity of 80-94 % with previously identified isolates. Various events of recombination were discovered among some of the isolates indicating that two or more viruses or different isolates of one virus infect the same host cell.

Conclusion: This study describes the discovery of a consortium of plant viruses infecting Tobacco that are broadly distributed in Anhui province of China. It also demonstrates the effectiveness of NGS in identifying plant viruses without a prior knowledge of the virus and the genetic diversity that enhanced mixed infection.

Keywords: Assembly; Bioinformatics; Next generation sequencing; Plant virus; sRNA.

Fig. 1

Schematic representation of virus detection and discovery pipeline using next generation sequencing

Fig. 2

Phylogenetic analyses of Cucumber mosaic virus Isolates and selected strains, with aligned nucleotide sequences, generated using the neighbor-joining method and MEGA6 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Peanut stunt virus ER RNA 1 was used as an out group for the tree construction

Fig. 3

Phylogenetic analyses of Potato virus Y isolates and selected strains, with aligned nucleotide sequences, generated using the neighbor-joining method and MEGA6 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree of the Potyvirus was rooted using Sunflower chlorotic mottle virus (SuCMoV) as an out group sequence for the phylogenetic analysis

Fig. 4

Phylogenetic analyses of Tobacco mosaic virus isolates and selected strains, with aligned nucleotide sequences, generated using the neighbor-joining method and MEGA6 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree was rooted using Odontoglossum ring spot virus (ORSV) as an out group sequence for the phylogenetic analysis

Fig. 5

a Sampling areas of infected tobacco plants in Anhui Province of China. b Typical view representation of yellow mosaic mosaic, stunting, Shoestring and deformation symptom of the overall Tobacco leaf samples collected across Anhui Province