2010 Annual Report
1a.Objectives (from AD-416)
To develop a universal plant virus micro array for detection and identification of plant viruses.
1b.Approach (from AD-416)
Develop microarrray containing probes representing all known plant viruses.
This research was carried out under a National Research Initiative Plant Biosecurity Program grant, in collaboration with scientists at the Donald Danforth Plant Science Center, Washington University, the University of Utah, Oklahoma State University, and Cornell University. The goals are to able to detect any plant virus in extracts of infected plants, and to identify previously characterized viruses to the species level, or previously uncharacterized virus to at least the viral family or genus level.
Progress was made towards development of a robust and reliable method for total nucleic acid extraction applicable to diverse plant species. The best strategy to control viral pathogens in plants is rapid identification and detection in quarantine, breeding programs, certification, and production. The various genera of plant viruses have different types of nucleic acid as their genome; either single-stranded or double-stranded DNA, or single-stranded or double stranded RNA. ARS researchers at Beltsville, MD are working with collaborators to develop a Universal Plant Virus Microarray for the detection and identification of plant viruses and viroids. Molecular techniques such as polymerase chain reaction (PCR)-based tests, and microarrays, can be fast and sensitive, and are increasingly used to detect genetic materials of these pathogens. A significant challenge in using these techniques, however, is the need to prepare plant samples or extracts containing these nucleic acids, but free of other compounds that occur in plant cells, such as oligosaccharides and polyphenols, that can interfere with the pathogen detection technique. To enable rapid detection of plant viruses and viroids from any potential plant host, a method for preparing total nucleic acids is required, which will yield both RNA and DNA of adequate yield and purity for detection techniques. ARS scientists at Beltsville, MD have developed a robust and reliable method of nucleic acid extraction for the microarray-based detection of diverse plant pathogens, including the viruses and viroids that are the target of the Universal Plant Virus Microarray. The method developed has been optimized for speed and purity of the nucleic acids in order to facilitate sample throughput. The method has been tested successfully against a diverse range of plant material known to contain significant levels of compounds that potentially inhibit nucleic acid amplification and labeling. The resulting total nucleic acid extracts were evaluated for purity, and tested by amplification of viral sequences known to be present. Samples prepared by this method will be utilized for validation of the Universal Plant Virus Microarray. This information will also be useful to those who work in quarantine and certification programs that need to test valuable plant material for pathogens.
Communications to monitor progress were carried out by e-mail and conference calls between the various partners, by a group meeting during the Annual meeting of the American Phytopathological Society, and by written and oral reports to the NRI Plant Biosecurity Program.