Separation of Slovenian isolates of PVYNTN from the north American isolates of PVYN by a 3-primer PCR

The potato tuber ringspot necrosis isolate of potato virus Y (PVYNTN) is a recently recognized and highly aggressive isolate of the PVYN group of strains. In order to screen specifically sources of resistance to PVYNTN a method to separate PVYNTN from PVYN 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 PVYN specific monoclonal antibody (4E7), all Slovenian isolates (S1-NTN) were identified as PVYN. Using two primer pairs from the P1 gene of a Hungarian isolate of PVYNTN by a conventional single primer pair, reverse transcription polymerase chain reaction (RT-PCR) both PVYNTN and PVYN were amplified similarly. However, specific amplification of PVYNTN 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 PVYNTN and PVYN were correctly separated. PVYNTN was detected in dormant tubers and leaves from all S1-NTN isolates. The 3-primer PCR was specific to PVYNTN and did not react with nine isolates of PVYN, 13 isolates of PVY degrees, one isolate of PVYC, six commonly occurring potato viruses and a viroid. (C) 1998 Elsevier Science B.V. All rights reserved.