Copy number variation of two begomovirus acquired and inoculated by different cryptic species of whitefly, Bemisia tabaci in Okra

Abstract

The whitefly, B.tabaci is a major pest of agricultural crops which transmits begomovirus in a species-specific manner. Yellow vein mosaic disease (YVMD) and okra leaf curl disease (OLCD) caused by distinct begomovirus are a major limitation to production of okra in India. In this framework the present investigation reports, for the first time, comparative study of begomovirus species viz. yellow vein mosaic virus (YVMV) and okra enation leaf curl virus (OELCuV) ingested and egested by two cryptic species (Asia I and Asia II 5) of B.tabaci at different time interval using detached leaf assay. A gradual increase of both virus copies were observed with increased feeding exposure in Asia I and Asia II 5. Both the genetic groups of whitefly could acquire the viruses within just 5 minutes of active feeding however, a significant amount of variation was noted in virus uptake by the both. At 24 hours of active feeding Asia II 5 acquired more of YVMV whereas, Asia I ingested more OELCuV. Similarly, the genetic group acquiring higher titre of virus egested higher amount during inoculation period. On the whole, it can be presumed that Asia I is a more effective transmitter of OELCuV whereas, Asia II 5 of YVMV further suggesting increased risk of virus pandemics (both YVMV and OELCuV) in regions where Asia I and Asia II 5 is dominant.

Fig 1. Phylogenetic tree of B. tabaci cryptic species identified based on cytochrome oxidase subunit I (COI) sequences.

The samples from the study are indicated by bold text in the tree; all other sequences were obtained from the GenBank database. Pink and blue coloured shade represent sequences of Asia I and Asia II 5 respectively, whereas, yellow shade represent the other cryptic species of B.tabaci. Bemisia afer sequences (green shade) was taken as an out-group.

Fig 2

Real-time PCR analysis of YVMV indicate the specificity of the reactions (A) Thermal profile (B) PCR amplification plots (C) dissociation curve and (D) standard curves of serially diluted linearized plasmids obtained using SYBR^® Green chemistry. The specific melting temperature for YVMV products was around 78°C. Standard curves show a linear relationship between initial viral copy number per microliter on X-axis and Ct values on Y-axis. Each concentration was replicated twice. The equation of the straight line and the coefficient of correlation (R²) are mentioned on the graph.

Fig 3

Real-time PCR analysis of OELCuV indicate the specificity of the reactions (A) Thermal profile (B) PCR amplification plots (C) dissociation curve and (D) standard curves of serially diluted linearized plasmids obtained using SYBR^® Green chemistry. The specific melting temperature for YVMV products was around 84°C. Standard curves show a linear relationship between initial viral copy number per microliter on X-axis and Ct values on Y-axis. Each concentration was replicated twice. The equation of the straight line and the coefficient of correlation (R²) are mentioned on the graph.

Fig 4

Dynamics of YVMV and OELCuV copies ingested (A and B) and egested (C and D) by individual Asia I and Asia II 5 genetic group of B. tabaci. Acquisition/ Inoculation feeding time is expressed on X-axis and log of virus copy number is plotted on Y-axis. YVMV and OELCuV copies were estimated at 1 min, 5 min, 15 min, 1 hr, 2 hrs, 6 hrs, 12 hrs, and 24 hrs of feeding. The different letters indicate statistically significant differences between the treatments. The bars represent the standard error of mean (± SEM).