Developing of specific nucleic acid probes for the detection of causal agents of banana Panama disease（Fusarium oxysporum f.sp. cubense） and citrus stem blight disease（Phoma tracheiphila）

• Main Authors: Chiao-yi Dai, 戴巧宜
• Other Authors: Shean-Shong Tzean
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/21731152379263003231

碩士 === 國立臺灣大學 === 植物病理學研究所 === 91 === Panama disease of banana caused by the fungus Fusarium oxysporum f. sp. cubense, and stem blight of citrus caused by the fungus Phoma tracheiphila, both are the crucial fungal pathogens and must be curtailed in qurantine to prevent accidentally introduced into Taiwan. Universal primes ITS4 and ITS5 were used to amplifify the ITS1-5.8S-ITS2 rDNA of 39 Fusarium spp. and 22 Phoma spp. by Polymerase Chain Reaction（PCR）. The amplified DNA products were sequenced, aligned, and analyzed with Vector NTI. Two sets of specific primers, RFP4-RFP5 and Ph1-Ph2, were thereby designed. In PCR, the primers RFP4 and RFP5 could specifically amplify a 300 bp DNA fragment from F. oxysporum f.sp. cubense Race 4 at annealing temperature of 66℃, with a sensitivity of 509 pg. Likewise, in PCR, the primers Ph1 and Ph2 could specifically amplify a 390 bp DNA fragment from P. tracheiphila at annealing temperature of 62℃, with a sensitivity of 102 pg. The 390 bp fragment was further ligated to plasmid pGEM-T Easy and being used to transform E. coli JM109, and obtained four subclones: Phc1, Phc2, Phc3 and Phc4. The inserted 390 bp DNA fragment in these subclones were verified by PCR using the Ph1 and Ph2 primers. It was concluded that the specific RFP4, RFP5 primers can be used to identify the banana Panama disease caused by F. oxysporum f. sp. cubense Race 4 and Ph1, Ph2 primers can be used to identify the citrus stem blight caused by P. tracheiphila, respectively. The inference of the phylogeny of the Phoma spp. is ongoing.