Virulence and Aggressiveness of Parasexual Recombinants of the Rice Blast Fungus Magnaporthe oryzae

Blast disease, caused by Magnaporthe oryzae B.Couch, is the most serious diseases of rice. Multilines, which are mixtures of lines that are genetically uniform except for a single trait such as disease resistance, have been used to prevent rice blast; however, their usefulness may be nullified when pathogenic variants (super races) arise that can attack all of lines in a multiline field. Parasexual recombination is considered one source of variation in the pathogenicity of M.oryzae. To understand the mechanism of pathogenic variation through parasexual recombination with a potential for new variants to cause severe damage in a multiline field, I analyzed the ploidy, number of nuclei per cell, and the segregation of pathogenicity in parasexual recombinants of M.oryzae. In addition, I determined the mating type and karyotype of the recombinants. The pathogenic stability of recombinants was evaluated by successive inoculation. The aggressiveness of recombinants were determined by measurements of lesion size, amount of sporulation, and blast development in a field planted with a single cultivar or a cultivar mixture. To demonstrate genetic exchange through parasexual recombination, a selectable marker gene conferring resistance to bialaphos(BI) was introduced into M.oryzae isolate Y90-71BI, whereas another to conferring resistance to blasticidin S (BS) was introduced into the isolate 3514-R-2BS.Colonies obtained from co-cultures of the two isolates were resistant to both BI and BS, and presence of the resistance genes was confirmed by southern hybridization using genomic DNA. These results indicated genetic exchange by parasexual recombination. The staining of conidia from the BI-BS-resistant parasexual recombinants with 4', 6-diamine-2-phenylindole (DAPI) revealed only one nucleus per cell. Flow cytometric analysis indicated that the recombinants were haploid. Thus, the number of nucleic per cell and the ploidy of the recombination were identical to that of the parental isolates. To examine the heritability of pathogenicity, I compared the segregati on of avirulence and virulence in the 49 BI-BS-resistant parasexual recombinants with that in the 70 BI-BS-resistant progeny derived from a cross of Y90-71BI and 3514-R-2BS. The segregation of avirulence and virulence on cv. Hattan 3 and a line K59-1in the parasexual recombinants were consistent with that in sexual progeny. The mating types of the parasexual recombinants were also consistent with those of sexual progeny. Using pulse-field gel electrophoresis (PFGE), the karyotype variation in the parasexual recombinants appeared corresponding with that in the sexual progeny. The karyotype segregation of the parasexual recombinants was consistent with that of the sexual progeny. Thus, the genetic characters of the parasexual recombinants segregated in a manner similar to those of the sexual recombinants. To determine the virulence and aggressiveness of the parasexual recombinants, I obtained pathogenic variants derived from co-cultures of two M.oryzae isolates, i.e., NAO-02, race 133.1 and TH77-1, race 047.0 in liquid yeast extract medium. The cocultured isolates were transferred to oatmeal agar to produce a conidial inoculum, and the suspension was sprayed onto cv. Akiyutaka, which has two rice blast resistance genes;Pik and Piz. Three pathogenic variants isolated from typical leaf blast lesions were examined randomly amplified polymorphic DNA(RAPD) to determine whether they were the result of parasexual recombination. Based on the RAPD patterns, the variants possessed genomic DNA from both parents. After successive seven inoculations onto cv. Akiyutaka, the variants maintained their original level of pathogenicity. The parasexual recombinants produced more conidia and larger lesions on the host than one parent, TH77-1; however, they produced fewer conidia and smaller lesions than other parent, NAO-02. The disease development in the rice plants inoculated with the variants was more severe than in those inoculated with TH77-1, but less severe than in those inoculated with NAO-02. Thus, genetic exchange in the blast fungus occurs via parasexual recombination, and the resulting recombinants are haploid and mononuclear. Parasexuality is similar to sexuality in terms of the heritability of virulence, mating type, and karyotype. In addition, a biparental pathogenic race that is capable of causing severe losses in multiline fields can arise through parasexual recombination.