| Budget Amount *help |
¥16,970,000 (Direct Cost: ¥15,800,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2007: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2006: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2005: ¥7,600,000 (Direct Cost: ¥7,600,000)
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| Research Abstract |
Fungicides have greatly contributed to sustaining quality food production through protecting a variety of crops from fungal diseases. However, such production is often threatened by the occurrence of pathogen strains resistant to fungicides.
Carpropamid, an inhibitor of scytalone dehydratase in melanin biosynthesis (MBI-D fungicide), has been developed and widely used for the control of rice blast disease caused by Magnaporthe oryzae (= Pyricularia oryzae) in Japan. The use of carpropamid in rice seedling box treatment greatly contributed to diminishing fungicide applications at later stages in paddy fields as this fungicide exhibits long-lasting control efficacy against the most important blast disease. However, in 2001, the efficacy of carpropamid against leaf blast was lost suddenly. Results from extensive studies indicated that resistant strains appeared and played a significant role in the decrease of the fungicide efficacy (Yamaguchi, et. al. 2002; Sawada, et. al. 2004). The molec
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ular mechanism of MBI-D resistance has been studied and found that resistance was caused by a single point mutation in the fungicide target scytalone dehydratase (SCDH) gene (Takagaki, et. al. 2004; Yamada, et. al. 2004).
In this research project, PCR (polymerase chain reaction)-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs), was successfully introduced for the diagnosis of MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. This system is based on the hybridization between a biotin labeled PCR product and an oligonucleotide probe coupled with distinct beads which specifically recognize the difference of nucleotide sequence. So far, this is the first case in which DNA-based PCR-Luminex system was used in the field of crop protection and/or agricultural sciences.
QoI fungicides (strobilurin fungicides), which inhibit mitochondrial respiration at the Qo site of cytochrome bc1 enzyme complex, are the second most important class of fungicides at present and azoxystrobin is the world's biggest-selling fungicide. They have a broad spectrum of control activity against a large number of pathogens on various crops. Three QoI fungicides (azoxystrobin, metominostrobin, and orysastrobin) were registered for the control of rice blast in Japan but QoI fungicides generally carry a high risk of resistance development in pathogens. QoI fungicide resistance has been reported in over 20 pathogens in the world.
It is possible that SNP (G143A or F129L mutation) in the cytochrome b gene could confer resistance to QoI fungicides in rice blast fungus because these mutations caused different levels of resistance to QoI fungicides in Pyricularia grisea and other fungal plant pathogens. The introduction of G143A and F129L mutations into the plasmids containing cytochrome b sequence of rice blast fungus was achieved by site-directed mutagenesis in this project. Subsequently, molecular diagnosis methods were also developed for identifying QoI resistance in rice blast fungus using the constructed plasmids either with PCR-RFLP (restriction fragment length polymorphism) or real-time PCR. Mutation of cytochrome b gene of rice fungus was able to be specifically detected from disease leaves and seeds using the molecular methods. Finally, high-throughput and rapid diagnostic methods to simultaneously identify resistance to MBI-D and QoI fungicides in rice blast fungus have been developed using PCR-Luminex system. These results will be used for early detection of fungicide-resistant strains of rice blast fungus so that rational the design of control programs could be designed in near future. Less
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