Separation of Slovenian isolates of PVY(NTN) from the North American isolates of PVY(N) by a 3-primer PCR.

The potato tuber ringspot necrosis isolate of potato virus Y (PVY(NTN)) is a recently recognized and highly aggressive isolate of the PVY(N) group of strains. In order to screen specifically sources of resistance to PVY(NTN) a method to separate PVY(NTN) from PVY(N) is needed. To achieve this, 61 isolates from 13 imported and locally developed potato cultivars in Slovenia were studied. On the basis of the reactions in indicator plants Nicotiana tabacum cv. Samsun and Solanum brachycarpum and with a PVY(N) specific monoclonal antibody (4E7), all Slovenian isolates (Sl-NTN) were identified as PVY(N). Using two primer pairs from the P1 gene of a Hungarian isolate of PVY(NTN) by a conventional single primer pair, reverse transcription polymerase chain reaction (RT-PCR) both PVY(NTN) and PVY(N) were amplified similarly. However, specific amplification of PVY(NTN) was achieved by a nested-PCR at an annealing temperature of 63 degrees C. A simplified form of the nested-PCR, termed 3-primer PCR was developed, which is applicable for large-scale testing of samples. Using the 3-primer PCR at annealing temperature of 63 degrees C, known mixtures of PVY(NTN) and PVY(N) were correctly separated. PVY(NTN) was detected in dormant tubers and leaves from all Sl-NTN isolates. The 3-primer PCR was specific to PVY(NTN) and did not react with nine isolates of PVY(N), 13 isolates of PVY(o), one isolate of PVY(C), six commonly occurring potato viruses and a viroid.