Molecular Plant Pathology
Detection of Andean Potato Virus X Isolates by Radioactive and Nonradioactive Nucleic Acid Spot Hybridization Tests. M. Querci,International Potato Center, Apartado 5969, Lima, Peru; L. F. Salazar, and E. N. Fernandez-Northcote. International Potato Center, Apartado 5969, Lima, Peru. Phytopathology 83:171-176. Accepted for publication 17 September 1992. Copyright 1993 The American Phytopathological Society. DOI: 10.1094/Phyto-83-171.
A cDNA probe, pX61, prepared from the Andean potato virus X (PVX) cp strain was tested against a broad spectrum of Andean PVX isolates grouped in two serotypes: 1) the PVXA Andean serotype detected only in Peru and Bolivia, which includes the cp and HB strains; and 2) the PVXO common serotype, which contains isolates serologically similar to those occurring elsewhere in the world. In radioactive nucleic acid spot hybridization tests (R-NASH) of virus in crude sap using the 32P-labeled RNA probe pX61, the PVXA isolates showed stronger hybridization signals and a detectability usually from two to six threefold dilution steps higher than those shown for PVXO isolates. The difference in the detectability of isolates from PVXA and PVXO serotypes was similar to that in nonradioactive nucleic acid spot hybridization tests (NR-NASH) with biotinylated DNA probe pX61. However, detectability in NR-NASH was lower than in R-NASH. When biotinylated DNA probes of pX61 (PVXA-specific) and pPVX19 (PVXO-specific), prepared from a British PVX isolate of the common European strain-group 3, were compared in NR-NASH, pPVX19 hybridized much more strongly to most isolates from the PVXO serotype than to those from the PVXA serotype. Several PVXO isolates, mostly from Bolivia, reacted as weakly with pPVX19 as the PVXA isolates. For R-NASH and NR-NASH tests, virus concentration in crude sap was checked by the double-antibody sandwich form of enzyme-linked immunosorbent assay, confirming that the differences obtained were not because of differences in virus concentration. These results were reconfirmed in R-NASH tests with DNA probes pX61 and pPVX19 using known concentrations of purified RNA from selected isolates. The differences shown between PVXA and PVXO isolates stress the importance of using the appropriate probes to detect PVX in breeding programs for resistance and quarantine purposes.
Additional keywords: dot blot hybridization, potato, potexvirus variability.