PCR Detection of the Three <i>Neofabraea</i>Pathogenic Species Responsible for Apple Bull’s Eye Rot

Apple bull’s eye rot is caused by pathogenic Neofabraea species including N. malicorticis, N. perennans and N. alba. Fruits carrying this fungal quarantine disease are prohibited from entering China. The host plants for the pathogens include several Rosaceae fruits including apple and pear. Disease symptoms and pathogen morphology are often insufficient to determine the identity of the pathogen, particularly at the species level. In the current study, we analyzed the inter-species sequence variations in the β-tublin gene, and designed specific primers to allow PCR amplification of 554 bp fragments from pathogenic Neofabraea species. The PCR products were recovered and sequenced, and Blast search was conducted using the DNA sequences in the Genbank database. The results indicated precise PCR amplification of the target sequences from the host pathogen, which allowed unambiguous identification of the species.

TaqMan探针实时荧光PCR快速检测苹果牛眼果腐病菌方法的建立

苹果牛眼果腐病菌(Neofabraea malicorticis、N.perennans、N.alba和N.kienholzii)是我国检疫性植物病原真菌,为建立该病菌的实时荧光PCR检测法,根据病菌及其近缘种的翻译延伸因子(EF-1α)的保守序列设计了特异性探针,分别以苹果牛眼果腐病菌菌株和本研究构建的EF-1α重组质粒DNA为阳性标准品检验探针的特异性和灵敏度。结果显示探针MAL-P、PER-P、ALB-P和KIE-P分别对N.malicorticis、N.perennans、N.alba和N.kienholzii表现特异性阳性扩增,而与近缘种及其他常见的果腐病菌无交叉反应,单重探针和4种探针混合液的灵敏度分别达1 fg/μL和10 fg/μL DNA。总计从美国、智利、新西兰、法国进境截获的29批可疑病果的分离物中检测到PCR阳性荧光信号,包括20批N.perennans、8批N.alba和1批N.kienholzii。该方法在6 h内即可完成整个检测流程,其特异性强、灵敏度高,适用于实际样品中苹果牛眼果腐病菌的快速检测。