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<p>Tomato leaf curl disease, primarily caused by begomoviruses, is a major limiting factor in tomato (<i>Solanum lycopersicum</i>) cultivation across India. Under North Indian conditions, tomato leaf curl Palampur virus (ToLCPalV), a begomovirus, has been identified as one of the predominant causal agent. While whiteflies are established vectors of begomoviruses, recent reports indicate the potential for seed transmission in some species. This study aimed to identify suitable DNA isolation protocols for reliable molecular diagnosis and to evaluate the seed borne nature of ToLCPalV by detecting its presence in tomato seeds. Five DNA isolation methods -standard CTAB (M1), modified CTAB (M2), CTAB by Murray and Thompson (M3), SGS buffer (M4), and Doyle and Doyle (M5) – were compared for their efficiency. Among these, M2 and M5 yielded high-quality genomic DNA suitable for PCR and qPCR analyses from both surface-sterilized and unsterilized seeds. Amplification of a ~ 647&#xa0;bp fragment using ToLCPalV-specific primers, followed by sequencing, confirmed the virus’s presence in seeds of the cultivar Pusa Sheetal. Real-time PCR analysis revealed low cycle threshold (Ct) values (16.40 to 20.06) and high viral copy numbers (1.96 × 10⁷ to 3.878 × 10⁸), suggesting high detection sensitivity. The presence of ToLCPalV was further confirmed from tomato seed in additional cultivars -Punjab Chhuara, Pusa Ruby, and Pusa Prasanskit -using M2 and M5. These findings provide molecular evidence supporting the seed borne nature of ToLCPalV and highlight effective, promising protocols for downstream virus detection. The results warrant further exploration of vertical transmission and its epidemiological implications in tomato virus management.</p>

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Comparative evaluation of DNA isolation protocols for efficient molecular detection of tomato leaf curl Palampur virus in tomato (Solanum lycopersicum) seeds

  • 期刊论文

  • 发表日期：

    2026年5月29日

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  • Rashmitha Monappa,
  • Nagamani Sandra,
  • H. M. Heena Kouser,
  • Zakir Hussain

摘要

Tomato leaf curl disease, primarily caused by begomoviruses, is a major limiting factor in tomato (Solanum lycopersicum) cultivation across India. Under North Indian conditions, tomato leaf curl Palampur virus (ToLCPalV), a begomovirus, has been identified as one of the predominant causal agent. While whiteflies are established vectors of begomoviruses, recent reports indicate the potential for seed transmission in some species. This study aimed to identify suitable DNA isolation protocols for reliable molecular diagnosis and to evaluate the seed borne nature of ToLCPalV by detecting its presence in tomato seeds. Five DNA isolation methods -standard CTAB (M1), modified CTAB (M2), CTAB by Murray and Thompson (M3), SGS buffer (M4), and Doyle and Doyle (M5) – were compared for their efficiency. Among these, M2 and M5 yielded high-quality genomic DNA suitable for PCR and qPCR analyses from both surface-sterilized and unsterilized seeds. Amplification of a ~ 647 bp fragment using ToLCPalV-specific primers, followed by sequencing, confirmed the virus’s presence in seeds of the cultivar Pusa Sheetal. Real-time PCR analysis revealed low cycle threshold (Ct) values (16.40 to 20.06) and high viral copy numbers (1.96 × 10⁷ to 3.878 × 10⁸), suggesting high detection sensitivity. The presence of ToLCPalV was further confirmed from tomato seed in additional cultivars -Punjab Chhuara, Pusa Ruby, and Pusa Prasanskit -using M2 and M5. These findings provide molecular evidence supporting the seed borne nature of ToLCPalV and highlight effective, promising protocols for downstream virus detection. The results warrant further exploration of vertical transmission and its epidemiological implications in tomato virus management.