Screening and identification of mutants of Magnaporthe grisea by REMI

Abstract

The plasmid pUCATPH was used to establish a transformation system in wild-type isolate M131 of Magnaporthe grisea. Six hundred and thirty-nine transformants were obtained by restriction enzyme-mediated integration (REMI) with hygromycin B (hyg B) resistance as a tag. Morphological analysis of two of the REMI mutants confirmed that they produced little melanin under black light and continued for three generations. Pathogenicity identification of six mutants screened proved that they made pathogenicity changes on three sets of differential varieties with different resistance genes. Rep-PCR analyses showed that two morphological mutants and two pathogenicity mutants differed from wild-type isolate M131 at the molecular level. RFLP analyses were performed to study the four mutants at the molecular level and the integration sites of the plasmid DNA. The results showed that the plasmid was inserted into all four mutants and that the insertion sites were random.