Background and Objectives Beet necrotic yellow vein virus (BNYVV) and Beet black scorch virus (BBSV) have been observed in infected sugar beet roots from Iranian fields. This study aimed to detect the two viruses simultaneously to save time and energy during the infection detection process. Materials and Methods Three methods were utilized in this study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect two viruses following total RNA extraction from the farm sample. In the first method (the standard method), the mentioned viruses were identified by amplifying 545 bp fragments from the TGB region of the RNA2 genome of the BNYVV virus and 305 bp fragments from the 3\\\\\\\'UTR region of the BBSV virus using specific primers. Viruses on an infected root were identified using RT-PCR with primers specific to each virus and performed independently. In the second and third methods, we employed simplex and duplex RT-PCR gradient methods to simultaneously identify two viruses at annealing temperatures of 52, 54, and 56 °C. To this end, virus-specific primers (designed from a part of the gene encoding the coat protein) were used separately but simultaneously with a common PCR program in a thermocycler. These specific primers amplified 391 and 453 bp fragments of the BNYVV and BBSV coat proteins, respectively. Results The study\\\\\\\'s findings show that by performing simplex and duplexRT-PCR simultaneously and optimizing the annealing temperature of primers from the gene encoding the protein coat, target fragments of 453 and 391 bp from BBSV and BNYVV, respectively, are amplified. This study found that an annealing temperature of 56 °C is the optimal temperature for duplex RT-PCR to separate the two viruses. Consequently, simultaneous identification of the BNYVV and BBSV was achieved through this method. Discussion The present study demonstrates that simplex and duplexRT-PCR are suitable for simultaneous detection and are cost-effective in terms of saving time and energy. They can Plant Protection (Scientific Journal of Agriculture) 46(1), Spring, 2023 also be used to prepare distribution maps in sugar beet cultivation areas and to evaluate and identify resistant sugar beet lines in breeding programs under greenhouse conditions.

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