The changes in fungicide resistance frequency and population structure of the rice blast fungus Pyricularia oryzae were monitored after the discontinuance of melanin biosynthesis inhibitor targeting scytalone dehydratase (MBI-D) fungicides use in Saga Prefecture, Japan. After discontinuance in 2003, the frequency of resistant isolates decreased from 71.8% in 2002 to 25% in 2003, and became undetectable in 2007. The initial marked decrease was due to a decline of isolates possessing the predominant haplotype, although the haplotypic diversity among resistant isolates remained high from 2003 to 2005. These results revealed that resistant isolates were less fit in comparison with sensitive isolates in the absence of MBI-D fungicide pressure under field conditions. Pairwise FST values indicated that the change in population structure after MBI-D discontinuance was explainable by a rapid change in the proportions of resistant and sensitive subpopulations. Depending upon the existence of fitness cost and rapid changes in population structure, it may be possible to reintroduce MBI-D fungicides in areas where resistance has already developed, although we speculate that fitness cost related to MBI-D resistance may be small based on our present results and previous findings.

We investigated the use of single primers complementary to sequences in the terminal inverted repeat (TIR) of either Pot2 or MGR586, transposable elements found in Pyricularia grisea, for DNA fingerprinting by repetitiveelement-based polymerase chain reaction (rep-PCR). Under standard amplification conditions, rep-PCR with each single primer generated distinct fingerprint patterns among rice-infecting P. grisea isolates collected in Japan. With the Pot2-TIR primer, bands ranging in size from 0.2 to 8 kb and in number from 8 to 13 per isolate were amplified. Although fewer bands were amplified with the MGR586-TIR primer, this molecular technique should be more reliable to identify and classify P. grisea isolates by combining the data of fingerprint patterns from each TIR primer. In a cluster analysis based on DNA fingerprints from this rep-PCR with the Pot2-TIR primer, 10 reference isolates and 12 field isolates from Saga Prefecture in 2002 were separated into six clonal lineages. We also demonstrated that the 12 field isolates belonged to one clonal lineage. Thus, this rep-PCR method using the single primer Pot2-TIR will be useful for the analysis of the population structure of rice blast pathogens.

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein.PCR-Luminex, a novel system developed for highthroughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.

In 2001, field isolates of Pyricularia grisea resistant to scytalone dehydratase inhibitors of melanin biosynthesis (MBI-D) were reported in Saga prefecture, Kyushu. Among 1,175 isolates collected from six prefectures of Kyushu in 2002 and 2003, 647 were resistant to MBI-D fungicides, each due to a single point mutation of the scytalone dehydratase (SDH) gene. On the basis of repetitive element-based polymerase chain reaction (rep-PCR) fingerprint data, the haplotypes of the resistant isolates showed high genetic diversity, indicating that the resistance existed in a multigenetic background. Three predominant haplotypes mainly contributed to the widespread resistance in Kyushu; haplotype Sa4 was observed frequently in Saga, Sa18 was predominant in Oita and Miyazaki, and Sa5 was widely distributed among all four prefectures. Also, phylogenetic analysis showed that both the resistant and sensitive isolates were clustered together in a closely related group. These results suggest that isolates possessing the SDH mutation would have been selected and then multiplied rapidly in each region of Kyushu as a result of the widespread introduction of MBI-D fungicides in a short period.

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