PCR-specific detection of a plum pox virus (PPV) isolate in Chile

Marlene Rosales V.1, Patricio Hinrichsen R.1 y Guido Herrera M.1

The potyvirus plum pox virus (PPV) is the causal agent of Sharkas disease. This virus severely affects stone fruit trees. Usually, the virus can be found at very low titers and unevenly distributed in different tissues of plants. Until recently, PPV was restricted to Europe and the northern part of Africa, but the virus was detected for the first time out of that region in Chile, in Los Tilos Regional Center of INIA (Acuña, 1993). Considering the great importance of this disease, highly sensitive detection techniques are required to control its spread. In this work a detection method was optimized based on the polymerase chain reaction (PCR), which amplified a specific segment of the viral genome. Viral RNA present in a crude plant extract was first processed with reverse transcriptase to produce a cDNA that was used as a template for the PCR reaction, using specific primers designed to anneal at the 3' end of the viral genome. With this procedure, the amplification of a 243 bp fragment, corresponding to the carboxi-terminal regio n of the coat protein gene (CP primers), and of a 220 bp fragment of the 3' non-coding regio n of PPV genome (3'-NCR primers) were achieved. This technique allowed the unequivocal detection of the virus in 24 out of 28 samples that were mostly sero-positive with a policlonal anti-PPV antibody. However, only 36% of these samples had visual Sharkas disease sym ptoms.

Keywords: RT-PCR, virus, PPV, Sharkas disease, molecular diagnostic, Prunus sp.
1 Centro Regional de Investigación La Platina (INIA), Casilla 439, Correo 3, Santiago, Chile.