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Adaptation of RT-PCR technique for the diagnosis of potato yellow vein virus in Venezuela

Abstract:

Potato yellow vein virus (PYVV) belongs to the genus Crinivirus, family Closteroviridae, and is transmitted by the greenhouse whitefly (Trialeurodes vaporariorum). It is an important problem for Andean countries because it causes loss of production by reducing the number and size of tubers. The diagnosis based on symptom expression is inaccurate because it can infect plants asymptomatically, and serological diagnosis is hampered by the lack of commercial kits available in the country. The objective of this study was the adaptation of the RT-PCR technique for the PYVV diagnosis and subsequent viral tracking in producing areas. Ten symptomatic samples were collected in Táchira and Trujillo States, Venezuela. RNA extraction was performed using the RNeasy Plant Mini kit (Qiagen). Deep freezing of the tissue (-60 °C) and addition of sodium metabisulfite (0,05 mg·g-1) were incorporated as additional steps. The retrotranscripción was executed by using the kit ImPron-II Reverse-Transcription System (Promega). Specific primers to amplify CP and CPm genes were utilized, as well as different anneling and extension temperatures, and various amplification mixtures. Such adaptations allowed the amplification of specific fragments, and confirmed virus infection in 100 % of the samples. It is the first time that PYVV is diagnosed by molecular techniques in Venezuela, thus it represents a diagnostic procedure that can be implemented to prevent the spread of the virus from seed tuber.