| Summary: | 碩士 === 國立臺灣大學 === 植物病理學研究所 === 91 === The genus Phytophthora is an important plant pathogen worldwise. They cause a wild range of disease on a large variety of plants. Current routine methods for the detection and identification of Phytophthora species involve isolation, and is still based primarily on morphology and grow characteristics. These procedures are time and labour-consuming. In other to improve efficiency and accuracy for the detection of Phytophthora, molecular techniques can be a useful tools. In order to establish a method for rapid identification of important Phytophthora spp. in Taiwan, primers were designed based on the ribosomal internal transcribed spacer 1 (ITS1) and ITS2 sequences of these fungi. Analysis of the specificity of these primers by PCR demonstrated that a total of 7 primer sets amplified DNA fragments of expected size from DNA of specific fungal species but not other fungi, indicating that these primers are useful for identification of P. cactorum, P. capsici, P. cinnamomi, P. cryptogea, P. drechsleri, P. palmivora, and P. parasitica, respectively. PCR analysis demonstrated the DNA fragments of predicted size and specificity for target species. In addition, PCR primers for Phytophthora genus specific were designed based on multiple sequence alignment, which included ITS 1 and 28S rRNA sequence. Analysis by PCR showed that the sensitivity of each primers sets varied, very likely due to differences in the nucleotide composition of the amplicons. Moreover, in order to enhance the specificity and sensitivity of the detection method, the primer set Phy1S-1 / Phy2A-1 and Phytophthora species-specific primers as the first and second primer sets, respectively, provides more reliable methods for rapid detection of Phytophthora species. Moreover, we develop Nested-Multiplex PCR assay for infect tomatoes. This PCR assay provide tools which may be used to detection Phytophthora blight in naturally infected tomato. The assay we describe allows rapid, sensitive detection and identification for Phytophthora spp. in pure culture or disease plant tissue.
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