玉米圆斑病病原的快速检测

Rapid detection of northern corn leaf spot pathogen

玉米生平脐蠕孢菌(Bipolaris zeicola)是引起玉米圆斑病的病原菌。本研究通过对玉米生平脐蠕孢菌及其近似种的EF-1α基因(elongation factor 1α)部分序列进行比对,设计出玉米生平脐蠕孢菌的特异性引物Y-EF-F和Y-EF-R,利用该引物可以从B.zeicola中扩增出137 bp的特异片段,而其余的17个参试菌株扩增结果为阴性。灵敏度实验表明该对引物可以检测到目标DNA的浓度为1 pg·μL^(-1)。用B.zeicola接种玉米叶片、苞叶以及玉米粒,然后以接种发病的病组织DNA为模板,利用引物Y-EF-F和Y-EF-R进行PCR扩增,可以扩增出137 bp的特异性条带,而健康玉米组织DNA中未能扩增出任何条带。用B.zeicola孢子悬浮液接种大田玉米叶片,接种第3 d可以检测到未发病组织中有B.zeicola病原菌,第5 d可以看到明显的病斑。研究结果表明该方法可用于快速、准确和灵敏地检测玉米组织中的潜伏期玉米生平脐蠕孢菌,为玉米圆斑病的快速检测,进而及早采取防治措施提供积极的指导。

Bipolaris zeicola is the pathogen of northern corn leaf spot. The species-specific PCR primers Y-EFF and Y-EF-R for B. zeicola were designed after multiple sequences alignment of elongation factor 1α of B.zeicola with the other allied species of Bipolaris. A 137 bp single DNA fragment from the isolates of B. zeicola was amplified by PCR approach and no product was amplified from other fungi. The detection sensitivity of the pathogen by the species-specific primers is 1 pg ·μL-1. The identical 137 bp bands were amplified using total DNA extracted from the diseased tissue including maize leaves,maize bracts and maize kernels inoculated with B. zeicola. The maize leaves were inoculated in the field with spore suspension,and the obvious disease symptoms could be seen in the fifth days post inoculation and the pathogen could be detected from the symptom-free leaves inoculated in the third days post inoculation. The PCR protocol provides a rapid and reliable tool for routine detection and identification of B. zeicola. In addition,it also provides an early detection approach for the latent pathogen in symptom-free maize,which is conductive for the disease control timely.