Rapid Detection and Identification of Six Tomato yellow leaf curl virus Isolates from Different Regions Using Polymerase Chain Reaction and Restriction Enzyme Analysis

Abstract

The combinational analysis of polymerase chain reaction and restriction enzyme analysis (PCR‐RE) to distinguish six Tomato yellow leaf curl virus (TYLCV) isolates from five countries was developed. Tomato yellow leaf curl virus has spread from the Middle East to Western Europe, Central America and Eastern Asia, and occurs on infected crops such as tomatoes, peppers, cucurbits and beans. Tomato yellow leaf curl virus isolates from Jordan (TYLCV‐Mld[Jo:Cuc] and TYLCV‐IL[Jo:Cuc]), Israel (TYLCV‐IL[IL:Reo:86]), Spain (TYLCV‐Mld[ES72/97]), USA (TYLCV‐IL[US:F10:04]) and Korea (TYLCV‐KR) were collected, and the sequences of the six isolates were analysed to distinguish them by PCR‐RE combination analysis. Oligonucleotide primers for the six TYLCV isolates were designed to amplify approximately 740 base pairs including the intergenic region (IR) and parts of V1 and V2 ORF. Unique restriction enzyme sites were analysed to identify isolate‐specific restriction enzyme sites on the PCR products of each isolate. Three enzymes (DdeI, FauI and BssSI) were selected by in silico analysis, and then, the PCR products following the serial digestion of each restriction enzyme were separated by agarose gel electrophoresis to distinguish the TYLCV isolates. Taken together, the PCR‐RE combination analysis by serial digestion with three restriction enzymes could be a useful method for distinguishing the six isolates.

Tomato yellow leaf curl virus Can Overwinter in Stellaria aquatica, a Winter-Hardy TYLCV-Reservoir Weed

“…Viral DNA was amplified by polymerase chain reaction (PCR). Amplification was performed in a 20-p.l reaction mixture containing 2x Taq PCR MaslerMix (Bioneer, Daejeon, the Republic of Korea) and the TYLCVspecific primer set (F: 5'-TTTCCTGAATCTGTTCACGGATT-3' and 5VAACTAATGCCTGTTCCTTCATTC-3') designed in our previous study (18). The following conditions for PCR reaction were employed: preheating at 94°C (3 min); then, 30 cycles of 30 s at 94°C, 30 s at 55 °C, and 30 s at 72°C; followed by a final extension at 72°C (10 min).…”

Begomovirus diversity in tomato crops in Costa Rica

“…Three samples with mixed TYLCV and ToYMoV infections were chosen and included in the analysis: two samples were collected in Alajuela and one in Heredia. The PCR was performed using the primers IRTyKrF (5'-TTTCCTGAATCTGTT-CACGGATT-3 0 ) and IRTyKrR (5'-AACTAATGCCTGTTCCTTCATTC-3 0 ), located from position 1 to 740, respectively, in the genome (Park et al, 2014). The cycling protocol consisted of an initial denaturation at 94 C for 3 min, followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 30 s and an extension at 72 C for 30 s, and a final extension at 72 C for 5 min.…”

“…No amplification was obtained with primers for TYLCV-Mld or TYLCV-Sardinia. To corroborate this information, a total of 31 samples were selected, and PCR products were cloned (Park et al, 2014); a sequence of 675 nt was considered from each one of the samples. A total of 14 haplotypes were found in the sequences (GenBank accession numbers MW807604, OQ863512-OQ863518, OQ863521-OQ863526), also considering sequences obtained from full genome clones.…”

Molecular Characterization of a Leaf Curl Disease Infecting Zucchini Squash in Iraq

“…Dalal et al, 2009), bacteria (Babu et al, 2014 and phytoplasmas (Nejat et al, 2010). This approach was combined with conventional restriction digestion method to distinguish TYLCV isolates amplified by PCR (Park et al, 2014).…”