Developing molecular makers for detection of Fusarium oxysporum , the causal agents of plant wilt

• Main Authors: Chia-Hung Chou, 周佳宏
• Other Authors: Chin-Long Lan
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/05237751313032125535

碩士 === 輔仁大學 === 生物學系 === 88 === Fusarium oxysporum are widely distributed in Taiwan. Totally 21 formae speciales of F. oxysporum causing wilt diseases have been reported. Fusarium species are known to be difficultly identified by their morphological characteristics, and are troublesome in crop quarantine and disease prevention. Twenty-four F. oxysporum isolates were collected and analyzed their genomic DNA sequence polymorphism by the RAPD PCR methodology. In the study, 500 random primers (each primer had 10 bases with GC ratio of 50 - 80 %) and 10 Fusarium - specific primers reported in the literatures (each primer had 10 - 16 bases) were used. After denaturing at 94℃, DNA template and primer were annealed at 32℃-50℃ depending on the sequence composition and subsequently elongated in 1% agarose gel and visualized on the UV - Tran illumination table. Little polymorphism was examined with those 510 primers. Twelve primers were found to be capable of amplifying specific bands in most F. oxysporum isolates. Representative primers were further tested to optimize their respective PCR conditions including the primer annealing temperatures, the cycle numbers and the DNA amount. The F. oxysporum specific bands were eluted from agarose gels and cloned to pucT vector. Nine clones were sequenced, and aligned with the published sequences in the GenBank database. Sequence specificity was also confirmed by the Southern dot blotting. It is intended to suggest that these specific primers which capable of amplifying specific bands from the F. oxysporum genomes can be invaluable tools for identification of F. oxysporum at the molecular level.