Detection of Phytophthora nicotianae and P. palmivora in citrus roots using PCR-RFLP in comparison with other methods
Detection of Phytophthora nicotianae and P. palmivora in citrus roots using PCR-RFLP in comparison with other methods
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DOI：
10.1007/s10658-007-9135-7
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发表时间：
2007-08
影响因子：
1.8
通讯作者：
K. Bowman;U. Albrecht;J. Graham;D. Bright
中科院分区：
文献类型：
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作者：
K. Bowman;U. Albrecht;J. Graham;D. Bright
烟草疫霉（Phytophthora nicotianaeandP.棕榈类是佛罗里达柑橘最重要的土传病原菌。这两个物种的检测和鉴定单，双感染的植物，利用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）的核糖体DNA的内部转录间隔区（ITS）的区域。分析了PCR-RFLP的敏感性，并评价了该方法作为血清学方法和半选择性培养基回收的替代或补充的有用性。用通用引物ITS 4和ITS 6进行半巢式PCR，检测限为1 fg，比用引物ITS 4和DC 6进行一步法PCR的灵敏度高1000倍。检测P. nicotianae比P. palmivora，限制其检测与限制性内切酶谱在植物感染的真菌物种。烟草疫霉菌检测与种特异性引物在所有样品接种该物种，尽管没有种特异性模式的RFLP。与此相反，P. palmivorain在P.用平板法和形态学检测法测定烟草的致病性较低。PCR扩增核糖体ITS区具有较高的灵敏度，是检测和鉴定植锈菌的有效手段。在柑橘根中，只要在解释数据之前建立目标物种的反应条件的全面知识。
Phytophthora nicotianaeandP. palmivoraare the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection forP. nicotianaewas shown to be ten-fold lower than forP. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species.Phytophthora nicotianaewas detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection ofP. palmivorain the presence ofP. nicotianaewas considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifyingPhytophthoraspp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.