Application of New Diagnostic Reagents for Identification of Diverse and Newly Identified Strains of Potato Virus Y
Fruit and Vegetable Insect Research
2013 Annual Report
1a.Objectives (from AD-416):
Develop and test new diagnostic antibody and molecular reagents for detection and identification of strains of potato virus Y.
1b.Approach (from AD-416):
Purified nucleoproteins of newly identified strains of potato virus Y will be purified in the laboratory of the SY with the U. of Idaho. New antibodies will be produced and screened. Promising materials will be sent to the ARS laboratory in Prosser for additional tests with the numerous isolates of potato virus Y maintained by the ARS researcher. Results will be relayed back to Un of Idaho. Similarly, genomic sequence information will be used to design new sets of RT-PCR primers. These will be tested for detection and identification of diverse strains of potato virus Y by both Un of Idaho and ARS.
This project studies the variability in isolates of Potato virus Y and develops improved diagnostic methods for detection of this economically important pathogen of potatoes. This research contributes to Objective 2 of the in-house project, "Determine host resistance options, epidemiological parameters and develop diagnostic tests for emerging pests and pathogens of potato." A multiplex RT-PCR assay was previously developed to identify a group of PVY isolates with unusual recombinant structures, e.g. PVYNTN-NW and SYR-III, and to differentiate them from other PVY strains. The efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. When applied to a panel of well-characterized reference isolates, this multiplex RT-PCR assay accurately identified and differentiated nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure described a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field. These new procedures make it possible to identify novel isolates of the virus that are currently present in potato production around the world.