Henan Provincial Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, 450009, Henan, China; College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
Fungal Biol. 2019 Sep;123(9):669-675. doi: 10.1016/j.funbio.2019.05.011. Epub 2019 May 30.
The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102-148 transformants per 10 conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.
引起靶斑病的真菌，即姜球腔菌（Corynespora cassiicola）（Berk. & M. A. Curtis）C. T. Wei，对中国种植的主要葫芦科作物，如西瓜（Citrullus lanatus）、甜瓜（Cucumis melo）和黄瓜（Cucumis sativus），构成了日益严重的威胁。了解姜球腔菌致病性的分子机制对于开发控制这种致病真菌的新策略至关重要。根癌农杆菌介导的转化（ATMT）可能有助于获得姜球腔菌的转化体，从而最终鉴定参与致病性的基因。在本研究中，我们建立并优化了使用携带载体 pATMT1 的根癌农杆菌 AGL-1 菌株对姜球腔菌进行 ATMT 的方案。ATMT 的效率为每 10 个分生孢子产生 102-148 个转化体，并且转化体在非选择性和选择性培养基上的连续继代培养表明，整合的转移（T）-DNA 在姜球腔菌转化体中稳定遗传。通过 PCR 和 Southern blot 分析验证了潮霉素 B 磷酸转移酶（hph）基因的整合，结果表明近 90%的转化体含有单拷贝 T-DNA。对表型改变的转化体进行了特征分析。其中 3 个转化体完全丧失了致病性，而另外 3 个转化体在甜瓜叶片上对 Cc-GX 菌株的致病性明显减弱。这些结果强烈表明，ATMT 可以作为一种分子工具，用于鉴定与中国几种重要农业植物病原菌姜球腔菌致病性相关的基因。
