Location: Floral and Nursery Plants Research2011 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.
3. Progress Report
Most virus detection methods are specific for a single virus or closely-related viruses, and are unable to detect viruses that have not previously been characterized. It is thus possible to detect only the specific viruses tested for, and not possible to identify all components of the mixed infections common in vegetatively-propagated plants. It is highly desirable to have a method that can detect any virus, including previously uncharacterized viruses, and to identify all components of mixed infections. ARS researchers at Beltsville, MD have, with funding from the USDA NRI Plant Biosecurity program (#2009-55605-05023), and in collaboration with scientists at the Danforth Science Center, Washington University, University of Utah, Oklahoma State University, and Cornell University, developed a Universal Plant Virus Microarray (UPVM). The UPVM has 9,600 oligonucleotides representing sequences conserved among members of an individual virus species, among members of a viral genus, or among members of a viral family (from genomic sequences available in public databases as of December 2009); and 44 highly conserved plant genes as controls. Oligonucleotide design and selection was based on E-predict, by a University of Utah collaborator. Other software (UChip and T-Predict), adapted specifically for the UPVM, is used to analyze results from hybridization of nucleic acids extracted from infected plant samples. We have demonstrated that the UPVM can detect a number of high-titered characterized plant viruses, including differentiation of the components of mixed infections, and work to validate the UPVM for a larger number of viruses continues. Work is in progress to develop suitable non-specific amplification techniques to allow detection of viruses occurring at low concentration in host tissues. Some previously undescribed viruses were correctly assigned to the taxonomic group indicated by independent genome sequencing, demonstrating the potential of the UPVM to detect previously unknown plant viruses. Beltsville ARS researchers developed a method (the ‘CKC’ method) suitable for the rapid and routine extraction of total nucleic acids from a wide variety of plant tissues for use with the UPVM. The method has been tested on over 60 species from 35 diverse plant families, including species rich in polysaccharides, phenolics, and latex. The UPVM is expected to benefit regulatory agencies, germplasm repositories, and breeders or others introducing new plant materials. Communications to monitor progress were carried out by e-mail, conference calls, a site visit between the various partners, by a meeting during the Annual meeting of the American Phytopathological Society, a meeting at the NRI Plant Biosecurity program directors meeting, and by written and oral reports to the NRI Plant Biosecurity Program.