Rapid differentiation of Fusarium oxysporum isolates using PCR-SSCP with the combination of pH-variable electrophoretic medium and low temperature.
Differentiation of Fusarium oxysporum is significantly important for unraveling the pathogenetic mechanism of Fusaria wilts. In this study, isolates of F. oxysporum were screened from the soils in the rhizosphere of watermelon plant by Komada medium and differentiated by SSCP approach with the combination of pH-variable electrophoretic medium (Tris-MES-EDTA (TME), pH 6.1) and low temperature (9 degrees C). We found that TME was a good electrophoretic medium and its pH value was variable over the course of electrophoresis in our apparatus. The pH-variable electrophoretic medium made more contribution for the better differentiation of F. oxysporum isolates than low temperature. The combination of TME pH 6.1 and low temperature showed an improved effect on resolution of ssDNAs. Leaving partial nondenatured dsDNA for SSCP was advantageous for differentiation of F. oxysporum isolates. The SSCP patterns of F. oxysporum isolates proved to be highly reproducible. Sequencing data confirmed that this SSCP method could detect one single base change within the 550 bp PCR fragment from the ribosomal internal transcribed spacer region of F. oxysporum.
Dong
Dexian
D
Laboratory of Molecular Microbiology, College of Life Science and Biotechnology, Shanghai Jiao Tong University, PR China.
Liu
Haiming
H
Peng
Huasong
H
Huang
Xiangqing
X
Zhang
Xuehong
X
Xu
Yuquan
Y
D001483:Base Sequence; D003080:Cold Temperature; D004271:DNA, Fungal / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005670:Fusarium / Q000235:genetics* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D016415:Sequence Alignment
D004271:DNA, Fungal