Department of Biology, Sunchon National University, Suncheon 540-950, Korea.
Department of Plant Medicine, Sunchon National University, Suncheon 540-950, Korea.
Plant Pathol J. 2013 Dec;29(4):357-63. doi: 10.5423/PPJ.OA.06.2013.0055.
The fungus Venturia nashicola is the causal agent of scab on Asian pears. For the rapid and reliable identification as well as sensitive detection of V. nashicola, a PCR-based technique was developed. DNA fingerprints of three closely related species, V. nashicola, V. pirina, and V. inaequalis, were obtained by random amplified polymorphic DNA (RAPD) analysis. Two RAPD markers specific to V. nashicola were identified by PCR, after which two pairs of sequence characterized amplified region (SCAR) primers were designed from the nucleotide sequences of the markers. The SCAR primer pairs, designated as D12F/D12R and E11F/E11R, amplified 535-bp and 525-bp DNA fragments, respectively, only from genomic DNA of V. nashicola. The specificity of the primer sets was tested on strains representing three species of Venturia and 20 fungal plant pathogens. The nested PCR primer pair specific to V. nashicola was developed based on the sequence of the species-specific 525-bp DNA fragment amplified by primer set E11F/E11R. The internal primer pair Na11F/Na11R amplified a 235-bp fragment from V. nashicola, but not from any other fungal species tested. The nested PCR assay was sensitive enough to detect the specific fragment in 50 fg of V. nashicola DNA.
生梨黑星菌是亚洲梨树疮痂病的病原菌。为了快速、可靠地鉴定和灵敏检测生梨黑星菌，开发了一种基于 PCR 的技术。通过随机扩增多态性 DNA（RAPD）分析，获得了三种密切相关的物种，即生梨黑星菌、梨黑星菌和梨黑斑病菌的 DNA 指纹。通过 PCR 鉴定出两个与生梨黑星菌特异的 RAPD 标记，然后从标记的核苷酸序列设计了两对序列特征扩增区（SCAR）引物。SCAR 引物对 D12F/D12R 和 E11F/E11R 分别扩增出 535-bp 和 525-bp 的 DNA 片段，仅从生梨黑星菌的基因组 DNA 中扩增出来。用代表三种黑星菌和 20 种真菌植物病原菌的菌株对引物对的特异性进行了测试。基于由引物对 E11F/E11R 扩增的 525-bp 物种特异性 DNA 片段的序列，开发了生梨黑星菌的嵌套 PCR 引物对。内部引物对 Na11F/Na11R 从生梨黑星菌中扩增出 235-bp 片段，但不能从任何其他测试的真菌物种中扩增出来。嵌套 PCR 检测法足够灵敏，可在 50 fg 的生梨黑星菌 DNA 中检测到特异性片段。
