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<p>Tomato (<i>Solanum lycopersicum</i> L.) is an economically important vegetable crop whose production is severely affected by tomato leaf curl disease. During field surveys in Meerut district, Uttar Pradesh, India, tomato plants exhibiting leaf curl symptoms were observed; however, symptom expression overlapped with those of other viral diseases and abiotic disorders, making diagnosis based on visual symptoms alone unreliable. Therefore, molecular characterization was undertaken to identify the virus associated with the disease. PCR amplification of genomic DNA using coat protein (AV1) gene-specific primers produced a distinct 771&#xa0;bp amplicon exclusively from symptomatic plants, whereas no amplification was obtained from healthy plants. Sequence analysis revealed 95–98% nucleotide identity with Tomato leaf curl New Delhi virus (ToLCNDV) isolates and phylogenetic analysis confirmed the Meerut isolate as ToLCNDV. The sequence was deposited in GenBank under accession number PZ404403. A previously reported TaqMan probe-based quantitative real-time PCR assay was validated using naturally infected tomato plants, alternate weed hosts, chilli cultivars and <i>Bemisia tabaci</i>. The assay successfully detected ToLCNDV in all conventionally PCR-positive samples, including individual and pooled adults and nymphs of <i>B. tabaci</i>, whereas no amplification was observed in healthy plant samples, non-viruliferous <i>B. tabaci</i> or the no-template control (NTC), demonstrating high analytical specificity and broad applicability for detecting ToLCNDV in cultivated tomato, chilli cultivars, alternate weed hosts and its insect vector (<i>B. tabaci</i>). In pot experiments, Diafenthiuron 50 WP (0.12%) was the most effective treatment for reducing whitefly populations and disease severity, followed by Acetamiprid 20 SP (0.02%), whereas <i>Lecanicillium lecanii</i> was the most effective biological treatment. Overall, symptom-based diagnosis alone is inadequate for reliable detection of tomato leaf curl disease. The validated TaqMan probe-based qPCR assay provides a rapid, sensitive and reliable method for routine diagnosis, early detection and epidemiological surveillance of ToLCNDV in host plants and its insect vector.</p>

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Molecular characterization and validation of a TaqMan probe-based qPCR assay for detection of tomato leaf curl New Delhi virus in host plants and Bemisia tabaci

  • 期刊论文

  • 发表日期：

    2026年7月16日

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  • Shivanshu Mishra,
  • Ramesh Singh,
  • Ankit Kumar Chaurasia,
  • Shashank Shekhar,
  • Rinku Bhaskar

摘要

Tomato (Solanum lycopersicum L.) is an economically important vegetable crop whose production is severely affected by tomato leaf curl disease. During field surveys in Meerut district, Uttar Pradesh, India, tomato plants exhibiting leaf curl symptoms were observed; however, symptom expression overlapped with those of other viral diseases and abiotic disorders, making diagnosis based on visual symptoms alone unreliable. Therefore, molecular characterization was undertaken to identify the virus associated with the disease. PCR amplification of genomic DNA using coat protein (AV1) gene-specific primers produced a distinct 771 bp amplicon exclusively from symptomatic plants, whereas no amplification was obtained from healthy plants. Sequence analysis revealed 95–98% nucleotide identity with Tomato leaf curl New Delhi virus (ToLCNDV) isolates and phylogenetic analysis confirmed the Meerut isolate as ToLCNDV. The sequence was deposited in GenBank under accession number PZ404403. A previously reported TaqMan probe-based quantitative real-time PCR assay was validated using naturally infected tomato plants, alternate weed hosts, chilli cultivars and Bemisia tabaci. The assay successfully detected ToLCNDV in all conventionally PCR-positive samples, including individual and pooled adults and nymphs of B. tabaci, whereas no amplification was observed in healthy plant samples, non-viruliferous B. tabaci or the no-template control (NTC), demonstrating high analytical specificity and broad applicability for detecting ToLCNDV in cultivated tomato, chilli cultivars, alternate weed hosts and its insect vector (B. tabaci). In pot experiments, Diafenthiuron 50 WP (0.12%) was the most effective treatment for reducing whitefly populations and disease severity, followed by Acetamiprid 20 SP (0.02%), whereas Lecanicillium lecanii was the most effective biological treatment. Overall, symptom-based diagnosis alone is inadequate for reliable detection of tomato leaf curl disease. The validated TaqMan probe-based qPCR assay provides a rapid, sensitive and reliable method for routine diagnosis, early detection and epidemiological surveillance of ToLCNDV in host plants and its insect vector.