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 undergraduate interns, and the incorporation of macroarray and 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 macroarray and microarray techniques into curricula, and educate students on the theory and application of macroarrays and microarrays.
We have demonstrated that the Universal Plant Virus Microarray (UPVM) can detect a number of characterized plant viruses, including differentiation of the components of mixed infections, and work to validate the UPVM for a larger number of viruses continues. Detection of high-titered viruses is possible without sample amplification, and work is in progress to develop suitable non-specific amplification techniques to allow detection of viruses occurring at low concentration in host tissues. Random amplification for sample labeling yielded poor results with Potato spindle tuber viroid, possibly due to the high secondary structure of the viroid RNA. Including a generic viroid-specific primer in the amplification improved labeling of the sample, but may also lead to interaction of the labeled DNA with other viroid probes. We are currently seeking to determine whether there is any correlation between the levels of dsRNA produced by different virus groups and the efficiency of sample labeling; virus groups that will be tested to examine this possibility include ilarviruses and potyviruses (low dsRNA) in comparison to tobamoviruses and cucumoviruses (abundant dsRNA). Results also suggest that RNA quality and concentration are both critical for efficient sample amplification and labeling, and that extracts from frozen or senescent material of some plants, such as grapevine, are of lower quality than fresh tissue.
Communications to monitor progress were carried out by e-mail and conference calls between the various partners, and by written and oral reports to the NRI Plant Biosecurity Program. The Cornell scientist made a site visit to Beltsville in May, and the PIs also met and discussed the project in person during the NRI project directors meeting in July, and at the American Phytopathological Society annual meeting in August.