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<p>Direct in-planta molecular detection of <i>Aphelenchoides fragariae</i> in asymptomatic infected shallot (<i>Allium cepa</i>) tissue has been enabled by using a rapid preparation DNA protocol. The NaOH lysis protocol combined with the CTAB method and the Plant Mini Kit protocol were compared for nematode DNA extraction. The DNA template was amplified with species-specific primers AFrag. The 8/9 asymptomatic shallot bulb samples successfully produced the predicted 169-bp fragment by the NaOH lysis+CTAB method. On the other hand, the same DNA template sample from the Plant DNA mini kit was negative for <i>A. fragariae</i> by PCR. The nucleotide DNA extracted from the infected bulb was confirmed as <i>A. fragariae</i> DNA based on the BLAST analysis and the phylogenetic tree of its ITS1 rDNA gene. This approach, rather than the plant mini-kit protocol, can be tested on a large scale of field samples to advance efficient and effective diagnosis of <i>A. fragariae</i> in asymptomatically infected shallot bulbs.</p>

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Rapid response of in-planta molecular detection: optimizing DNA extraction and detection of Aphelenchoides fragariae in asymptomatic shallot bulbs

  • 期刊论文

  • 发表日期：

    2026年7月8日

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  • Siti Hardiyanti,
  • Setyowati Retno Djiwanti,
  • Miftakhurohmah,
  • Rubi Heryanto,
  • Rohimatun,
  • Sukamto

摘要

Direct in-planta molecular detection of Aphelenchoides fragariae in asymptomatic infected shallot (Allium cepa) tissue has been enabled by using a rapid preparation DNA protocol. The NaOH lysis protocol combined with the CTAB method and the Plant Mini Kit protocol were compared for nematode DNA extraction. The DNA template was amplified with species-specific primers AFrag. The 8/9 asymptomatic shallot bulb samples successfully produced the predicted 169-bp fragment by the NaOH lysis+CTAB method. On the other hand, the same DNA template sample from the Plant DNA mini kit was negative for A. fragariae by PCR. The nucleotide DNA extracted from the infected bulb was confirmed as A. fragariae DNA based on the BLAST analysis and the phylogenetic tree of its ITS1 rDNA gene. This approach, rather than the plant mini-kit protocol, can be tested on a large scale of field samples to advance efficient and effective diagnosis of A. fragariae in asymptomatically infected shallot bulbs.