Botrytis cinerea field isolates collected in Japan were screened for resistance to Qo inhibitor fungicides (QoIs). Of the 198 isolates screened, six grew well on a medium containing azoxystrobin, a QoI, when salicylhydroxamic acid, an alternative oxidase inhibitor, was present. The resistance mutation in the cytochrome b gene ( cytb ) was characterized. All QoI-resistant isolates had the same mutation (GGT to GCT) in cytb that led to the substitution of glycine by alanine at position 143 of cytochrome b , which is known to confer QoI resistance in plant pathogens. To detect this mutation, a hybridization probe assay based on real-time PCR amplification and melting curve analysis was developed. Using DNA samples prepared from aubergines coinfected with QoI-resistant and QoI-sensitive B. cinerea isolates, two similar peak profiles with their corresponding melting temperatures were obtained. This result suggests that QoI-resistant and QoI-sensitive isolates may compete equally in terms of pathogenicity, and the assay may be used to assess the population ratio of mutant and wild-type isolates. However, the hybridization probe did not anneal to PCR products derived from the DNA samples of some QoI-sensitive isolates. Structural analysis of cytb revealed that B. cinerea field isolates could be classified into two groups: one with three introns and the other with an additional intron (Bcbi-143/144 intron) inserted between the 143rd and 144th codons. All 88 isolates possessing the Bcbi-143/144 intron were azoxystrobinsensitive, suggesting that the QoI-resistant mutation at codon 143 in cytb prevents self-splicing of the Bcbi-143/144 intron, as proposed in some other plant pathogens.

The inhibitory activity of carpropamid on scytalone dehydratase (SDH) extracted from a carpropamid-resistant strain of Magnaporthe grisea (Hebert) Barr was dramatically reduced in comparison with that on SDH extracted from the sensitive strain. A single-point mutation (G to A) located at the upstream region (233 bp downstream from the ATG codon) resulting in a one-amino-acid substitution (valine [GTG] 75 to methionine [ATG]: V75M) was found in the resistant strain. To examine whether the V75M mutation is the primary reason for decreasing the sensitivity of SDH to carpropamid, the SDH cDNAs of both the sensitive and the resistant strain were cloned into a GST-fused protein expression vector-system. The recombinant SDHs of both strains exhibited the same sensitivities to carpropamid as those extracted from the mycelia of the respective strains. These data clearly revealed that the V75M mutation causes the low sensitivities of the SDHs of the carpropamid-resistant strains, and strongly suggests that the V75M mutation confers resistance of these strains to carpropamid.

We have established a simple diagnosis method for rice blast fungus resistant to MBI-D. This involves the preparation of PCR templates directly from the lesions in combination with primer-introduced restriction enzyme analysis PCR (PIRA-PCR).

Rice blast fungus isolates were collected in Kyushu to investigate resistance to scytalone dehydratase inhibitors of melanin biosynthesis (MBI-D). In 2001, failure of control of rice blast was reported in the Saga prefecture, where MBI-Ds have been used since 1998. At that time, the distribution of resistant isolates was mainly limited to that area. However, in 2002, resistant isolates were detected in all prefectures of Kyushu. DNA fingerprinting analysis showed that the mutation causing resistance to MBI-Ds had arisen independently in each area. These data suggest that resistant isolates may occur in any area and become dominant under continuous selection pressure for MBI-Ds. Nevertheless, resistant strains can be controlled by reductase inhibitors of melanin biosynthesis (MBI-R) or commercial rice seed disinfectants.

Pyribencarb is a novel benzylcarbamate-type fungicide, which is active against a wide range of plant pathogenic fungi. In this paper, the inhibitory effects of this fungicide on the electron transport system of fungi, plants, rat and carp were examined to elucidate its mode of action and selectivity. Pyribencarb potently inhibited succinatecytochrome c reductase (SCR) activities of Botrytis cinerea (cucumber gray mold), Corynespora cassiicola (leaf spot) and decylubiquinol-cytochrome c reductase (UCR) activity of B. cinerea. Pyribencarb inhibited the UCR of B. cinerea in an uncompetitive manner with respect to decylubiquinol, which was the same as strobilurin fungicides, and the substrate-dependent inhibition constant was found from calculation to be 13 nM. These results suggested that the target site of pyribencarb is cytochrome b of complex III in the electron transport system of the respiratory chain. On the other hand, the inhibitory potency of pyribencarb on SCR activities of plants, rats and carp was relatively weak compared with that of strobilurin fungicides, indicating that pyribencarb is a Qo inhibitor of cytochrome b, whose properties are superior to well-known Qo inhibitor fungicides in terms of target. The binding site of pyribencarb on cytochrome b was assumed to be a little different from that of strobilurin fungicides, because pyribencarb inhibited SCRs of strobilurin fungicide-resistant strains of B. cinerea and C. cassiicola with relatively low concentrations. The binding site was also discussed through comparison of amino acid sequences of plants, rats, carp, yeast and fungi, including B. cinerea, which was elucidated in this paper.