2013 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.
The funding for this project originates from project 6402-21310-003-09R where additional details can also be found. This project deals with monitoring cotton leaf curl virus (CLCuV) in Pakistan and its white fly vector.
CLCuV is known to change its DNA sequence, mainly by recombination with other viruses, and has a broad plant host range. Therefore samples are being processed to monitor the virus for DNA changes and its presence in various plant species. There are several species of white fly and only one has been confirmed to act as a vector for CLCuV. However there is concern that other species or specific sub-species might act as a vector.
Samples from Pakistan are processed to isolate DNA which is then sent to the University of Arizona for cloning. Clones are then sequenced in the ARS’ Genomics and Bioinformatics Research Unit in Stoneville, MS. The data is then transferred back to partner institutes for analysis. To date this whole process has been successful and has examined over 700 samples. However, this procedure is very time consuming and next generation DNA sequencing methods are now being explored to see if they are quicker, more informative, limited in sampling bias and more cost efficient.
In addition to using this data to monitor CLCuV and its vector, the sequence data is being used to generate rapid and efficient methods for testing for specific strains of CLCuV or white fly in field samples. Presently the approach being taken is based polymerase chain reaction (PCR) so it could be easily employed by numerous labs. Ideally the final method could act as a detection method that could be used by the USDA Animal and Plant Health Inspection Service (APHIS) to protect the U.S.A. borders from accidental introduction of CLCuV by importation of infected plants.