Reverse Transcriptase-PCR-based techniques for the detection and identification of potato virus Y and tobacco mosaic virus in tobacco.

Symptomatological assays have been traditionally relied upon for the detection of tobacco mosaic virus (TMV) and potato virus Y (PVY), the two major economic consequential viruses infecting tobacco in Zimbabwe and globally. However, morphological methods are subjective and unreliable, as they are affected by abiotic factors and phytotoxicity, leading to misdiagnosis. The advent of polymerase chain reaction (PCR)-based assays has transformed pathogen diagnostics by offering robust diagnostic techniques to support disease management strategies that can avert yield losses. Reverse transcriptase‒polymerase chain reaction (RT‒PCR) protocols were developed for the identification of TMV and PVY in tobacco in Zimbabwe. The internal specific primer pairs amplified 480 bp of the coat protein for PVY, 420 bp for the protein movement gene, and 496 bp for the TMV virus genome. The effectiveness and reliability of the assays were analysed via sensitivity comparisons of the double-stranded RNA extraction method (dsRNA), double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and RT‒PCR, which were conducted at weekly intervals after viral infection of tobacco plants. DsRNA was the least effective at detecting PVY after three weeks of infection. Compared with dsRNA, DAS-ELISA was more sensitive for detecting viruses after one week of infection for up to six weeks for PVY and after three weeks for TMV. However, RT‒PCR consistently detected viral infection throughout the duration of the study. The use of RT‒PCR is recommended for application since it has improved sensitivity and specificity.