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United States Department of Agriculture

Agricultural Research Service

Research Project: Development of Cotton Leaf Curl Virus (Clcuv) Diagnostic Tools and Monitoring of Clcuv

Location: Genomics and Bioinformatics Research Unit

2012 Annual Report

1a.Objectives (from AD-416):
The vulnerability of the cotton crop to near complete failure owing to infection by whitefly-transmitted geminiviruses has been of concern for some time, both before and since the release of Bt varieties. The cotton crop has limited genetic variability and therefore low versatility in traits. Plant viruses are capable of rapid diversification to pressures imposed by a static genetic structure of the host, a type of pathogen that is referred to as ‘emergent.’ In the major cotton producing regions of Pakistan and India (Punjab region), enormous yield losses have been associated with twelve or more emergent begomovirus species (and additional strains). Additional strains likely exist within the population that have not been discovered but that could emerge under changing selection pressures, including the continued increase of Bt cotton cultivation. A recent example is the emergence in Pakistan of the Burewala strain of Cotton leaf curl virus (CLCuV), which overcame the genetic resistance in newly released cotton lines in less than two years. Stable and durable resistance to these highly virulent viruses has become essential. To develop durable resistant varieties, new tools will be needed to assess the genetic diversity within the virus population and to understand how selection pressure influences population structure. In addition the adaptation of high throughput sequencing methods will be needed to assess virus population composition and dynamics, and new approaches will be required to quantify the viral load in germplasm assessed for susceptibility and resistance to the cotton leaf curl complex. Optimized virus detection methods will allow monitoring of virus population dynamics in relation to the performance of cotton germplasm lines and cultivars under natural field conditions where selection pressures are unknown and dynamic from year to year.

1b.Approach (from AD-416):
To preserve the effectiveness of resistant cotton lines, it is essential to be able to identify, characterize and monitor the different strains of CLCuV. New tools (predominately based on molecular biology approaches) will be needed to determine the range of different viral strains, track their changes and study how these changes affect virulence. Assessing the range of CLCuV strains, rates of change, and the effect on virulence will require the expertise of a virologist(s) well versed in the area of molecular biology.

3.Progress Report:

Objectives: Leaf curl virus disease of cotton is caused by a complex of whitefly-transmitted geminiviruses (circular, single-stranded DNA viruses), referred to as Cotton leaf curl virus (CLCuV) complex, and one of several associated DNA satellites that contribute to ‘helper virus’ virulence. The leaf curl disease was first observed near Multan in the 1967, and became overwhelmingly damaging during the 1990’s when it was distributed by the whitefly vector into all cotton growing areas of Pakistan (and into the Indian Punjab). Resistance was identified in cotton germplasm and introgressed into the main Pakistani commercial cotton varieties, resulting in disease abatement. However, during 2001 a new strain of virus emerged in the Burewala, Vehari district, referred to as the Burewala strain, for which no durable genetic resistance in cotton was available. The purpose of this project objective is to develop molecular epidemiological tools and approaches for understanding the complex population structure of the leaf curl virus complex and its satellites, and the extent of virus diversity in commercial cotton crops and in germplasm and other breeding materials, as well as in cultivated non-cotton hosts, and uncultivated hosts (endemic or weedy hosts) that could serve as sources of viral genetic diversity possibly contributing to the evolution of new, more damaging strains of leaf curl virus that are capable of overcoming subsequently introgressed resistance genes. In addition the apparent, importance of the role of different whitefly vector haplotypes in the emergence of new viral strains is under study to map the distribution of distinct mitochondria-cytochrome oxidase subunit 1 (mt-COI) haplotypes in Pakistan cotton growing regions, and the whitefly transcriptome has been determined and used as a source of lucrative gene targets that if silenced using Ribonucleic acid (RNA)-interference could feasibly lead to gene silencing of genes crucial for whitefly survival. If expressed in transgenic plants, together with key viral transgene sequences, resistance to both the whitefly vector and the leaf curl complex is feasible. Toward these goals, we have developed a high throughput DNA isolation, genome amplification, cloning, and sequencing pipeline and processed 74 samples for 2011, respectively, and are currently processing 189 plant samples and a total of 107 whitefly samples, received January and June 2012 from our collaborators at the University of Punjab, Lahore. DNA sequencing was carried out using Sanger sequencing (bidirectional) facilitated by primer walking for cloned amplicons produced by rolling circle amplification to reveal the extent of genetic diversity among the most abundant variants occurring in cotton, and non-cotton cultivated and uncultivated plant species. Sequencing and assembly of viral genomes and satellite DNA genomes has been completed for all 2011 samples, and editing and phylogenetic analysis is underway. Our second sequencing strategy is to employ Illumina technology to sequence total DNA extracts pooled from subsets of plant and whitefly samples, and of Rolling Circle Amplitications (RCA-amplicons) of those same samples (in progress), to facilitate a comparison of the three approaches in estimating viral diversity and population composition/dynamics in different hosts and geographic locales in the major Pakistan cotton-growing areas. Our goal in this aim is to obtain an understanding of the ability of each of the approaches to deliver a measure of genetic diversity, and at the same time elucidate the extent of diversity in plant samples, and in whitefly vector populations associated with plant samples.

Regarding whitefly vector diversity, we are using Polymerase chain reaction (PCR) to amplify the mtCOI and the endosymbiont16S rDNA sequence from individual whiteflies. The complement of endosymbionts is expected to reflect potentially unique biological adaptations of the different haplotypes that may thereby directly or indirectly influence virus transmission parameters (competency, frequency) and life history traits (fecundity, survival, sex ratio). Finally, we annotated a partial transcriptome of the whitefly (Bemisia tabaci (biotype B))and mined the database to identify five candidate genes that if disrupted at the level of expression using dsRNA delivered to adult whiteflies in an artificial feeding system, would result in premature mortality. Promising results were obtained for at least two of the five candidates, based on quantitative PCR analysis of gene expression following dsRNA ingestion, combined with mortality data.

Approach: To preserve the effectiveness of resistant cotton lines, it is essential to be able to identify, characterize and monitor the different strains of CLCuV. New tools (predominately based on molecular biology approaches) will be needed to determine the molecular biology approaches) will be needed to determine the range of different viral strains and species, track the changes in predominance, and study how germplasm differences influence composition, rates of change, and effects on virulence.

Last Modified: 4/21/2014
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