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<p>Bamboo witches’ broom disease (WBD), caused by <i>Aciculosporium take</i> Miyake, devastates bamboo forests. However, no genetic transformation system is available for <i>A. take</i>, which hinders research on its pathogenesis and biological properties. This study developed an <i>Agrobacterium tumefaciens-</i>mediated transformation (ATMT) system by constructing a knockout vector, pCAMBIA1300ura-EGFP-A-HYG-B, which included a hygromycin B (HYG) resistance gene, a promoter (PtrpC) for fungal gene expression, and an enhanced green fluorescent protein gene. This setup enabled <i>A. take</i> to express enhanced green fluorescence and HYG resistance. The vector was introduced into <i>A. take</i> using <i>A. tumefaciens</i> AGL1 under specific conditions: a spore concentration of 1 × 10<sup>7</sup>&#xa0;CFU/ml, a 6-h pre-induction, a 200&#xa0;µM acetosyringone in IM-inducing medium at pH 5.4, and a 3-day screening at 25&#xa0;°C on cellophane. Successful integration of the vector (21 per 1 × 10<sup>7</sup>&#xa0;CFU/ml spores) was confirmed through PCR, fluorescence microscopy, and genetic stability tests, validating the transformation method. The development of a complementary target further validated the feasibility and stability of the transformation method. This study lays the groundwork for further gene analysis linked to bamboo WBD.</p>

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Agrobacterium tumefaciens-mediated transformation approach of bamboo Witches’ broom disease agent Aciculosporium take

  • 期刊论文

  • 发表日期：

    2025年7月23日

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  • Yu Gu,
  • Sainan He,
  • Haoyue Yu,
  • Zhen Liu,
  • Yingchong Liao,
  • Huiru Meng,
  • Xiaoping Ma

摘要

Bamboo witches’ broom disease (WBD), caused by Aciculosporium take Miyake, devastates bamboo forests. However, no genetic transformation system is available for A. take, which hinders research on its pathogenesis and biological properties. This study developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system by constructing a knockout vector, pCAMBIA1300ura-EGFP-A-HYG-B, which included a hygromycin B (HYG) resistance gene, a promoter (PtrpC) for fungal gene expression, and an enhanced green fluorescent protein gene. This setup enabled A. take to express enhanced green fluorescence and HYG resistance. The vector was introduced into A. take using A. tumefaciens AGL1 under specific conditions: a spore concentration of 1 × 107 CFU/ml, a 6-h pre-induction, a 200 µM acetosyringone in IM-inducing medium at pH 5.4, and a 3-day screening at 25 °C on cellophane. Successful integration of the vector (21 per 1 × 107 CFU/ml spores) was confirmed through PCR, fluorescence microscopy, and genetic stability tests, validating the transformation method. The development of a complementary target further validated the feasibility and stability of the transformation method. This study lays the groundwork for further gene analysis linked to bamboo WBD.