2011 Annual Report
1a.Objectives (from AD-416)
To demonstrate the ability of an oligonucleotide microarray to detect and differentiate plant viruses from random amplification of plant total nucleic acid extracts, and to provide education, through training, of both undergraduate interns and a graduate student, and the incorporation of microarray techniques into curricula.
1b.Approach (from AD-416)
ARS will acquire the lists of viral taxa to be represented on the viral detection microarray, and virus-infected samples from which to amplify nucleic acids to validate the microarray. This information and material will be utilized by both ARS and the Cooperator to jointly develop and validate the microarray for detection of target viruses, and to make validation results available to collaborators via a web server. The COOPERATOR will perform analysis of viral sequences to identify suitable sequences for the development of oligonucleotides, and participate in analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples. The COOPERATOR will also integrate microarray techniques into curricula, and educate students on the theory and application of microarrays.
Samples infected with various viruses that had been identified by the Oklahoma State University Plant Disease and Insect Diagnostic using diagnostic PCR and ELISA were extracted, labeled, and hybridized to the Universal Plant Virus Microarray (UPVM). Additional samples from the Tallgrass Prairie Reserve (TPR) in northeastern OK and known to be infected with previously uncharacterized viruses that had been classified to family or genus by sequencing, were similarly prepared and tested. Probes representing the appropriate families, genera, and species are present on the UPVM. Two samples of previously uncharacterized partitiviruses (dsRNA) from the TPR yielded signal with probes representing the genus Partitiviridae, whereas an uncharacterized caulimovirus (dsDNA) and an uncharacterized reovirus (dsRNA) were not detected. The detection of signal for two previously uncharacterized partitiviruses demonstrates the potential of the UPVM to identify such viruses at least to family or genus level. Among the characterized virus samples, one Wheat streak mosaic virus (WSMV) sample was detected by species-, genus-, and family-level specific probes. One Barley yellow dwarf virus (BYDV; Luteovirus; Luteoviridae) infected sample yielded signal from three species-specific probes, but a second BYDV sample did not yield significant signal. An Impatiens necrotic spot virus sample was detected by a tospovirus genus-specific probe, but not species-specific probes. Various other samples infected with: Wheat spindle streak virus (Bymovirus, Potyviridae); Tobacco mosaic virus (Tobamovirus, Virgaviridae); Hosta virus X (Potexvirus, Alphaflexiviridae); Tobacco ringspot virus (Nepovirus, Secoviridae); and Oat necrotic mottle virus (Tritimovirus, Potyviridae) did not yield specific signals. The effects of target concentration on detection were examined for WSMV. Purified WSMV cDNA was diluted in a ten-fold series and solutions containing 4.5, 0.45, and 0.045 pg of WSMV cDNA subjected to dye coupling, hybridization to the UPVM, and microarray scanning. All three WSMV-reactive positive-sense probes (WSMV-, Tritimovirus-, and Potyviridae-specific), were observed to hybridize for all three sample dilutions, with decreasing signal intensity corresponding to increased sample dilution. This information will be of most immediate application to the UPVM collaborators, but will also be of value to regulatory agencies, plant diagnostic clinics, germplasm repositories, and producers operating plant certification schemes.
Communications to monitor progress were carried out by e-mail and conference calls between the various partners, by a meeting during the Annual meeting of the American Phytopathological Society, and by written and oral reports to the NRI Plant Biosecurity Program.