From the infected rice seeds with

Joint action between a phosphorothiolate (PTL) fungicide, iprobenfos, and a sterol demethylation inhibitor (DMI), pefurazoate, was tested by crossed paper technique on three types of field isolates of Pyricularia oryzae Cavara that differed in PTL sensitivity and metabolism. Mutual antagonism in anti‐fungal action between iprobenfos and pefurazoate was observed in a wild‐type field isolate of the fungus sensitive to PTL and in an isolate moderately resistant to PTL, but not in a PTL‐resistant isolate lacking the ability to metabolize PTL. Antagonism of the antifungal action of iprobenfos by pefurazoate seemed to be a result of inhibition of activation by cleavage of the P‐S bond of iprobenfos mediated by mixed‐function oxygenase (mfo) activity, while antagonism of the anti‐fungal action of pefurazoate by iprobenfos may be caused by the binding of pefurazoate by large amounts of an iprobenfos‐induced mfo which results in reduced inhibition of ergosterol biosynthesis. In the PTL‐resistant isolate, the mutually antagonistic action was not observed, presumably because the induction of the mfo‐metabolizing iprobenfos was lacking. Similar antagonism was also observed when another PTL, edifenphos, was used instead of iprobenfos, and when other DMIs, propiconazole, prochloraz and hexaconazole were used instead of pefurazoate. The results of the present experiment indicate that DMIs may also bind to and inhibit an inducible type of fungal mfo which metabolizes xenobiotics, and that PTLs may be activated by an mfo prior to their anti‐fungal action.

Mutants of Gibberella fujihuroi were obtained by selection from a large number of conidia of a wild type field isolate of the fungus on agar plates containing lethal concentration of a sterol demethylation inhibitor, pefurazoate.The mutants isolated from the colonies grown on the plates had higher minimal growth-inhibitory concentrations of pefurazoate and the EC95 values were higher than that for the mother isolate but only a small increase was observed in the EC5a values. Though the slope of the dosage-response curve was changed in the mutants, the mechanism of action of the fungicide in a mutant proved to be its inhibition of sterol 14cc-demethylation as shown by an experiment on inhibition of ergosterol biosynthesis using 14C-acetate. Gibberellin production in the mutants in liquid culture was tested by bioassay of gibberellins and assay of gibberellin A3 using HPLC. Mutants were also tested for their virulence to rice plants.Decreased virulence due to decreased production of gibberellins was observed in the mutants.A mutational change in a site related to 14c -demethylation in ergosterol biosynthesis and to kaurene oxidation in gibberellin biosynthesis in common is discussed.

Pefurazoate is a 14a-demethylation inhibitor of 24-methylenedihydrolanosterol (24-MDL) and has an asymmetric carbon atom in its structure. We previously reported that the antifungal activity of the (S)-(-)-isomer of pefurazoate against Gibberella fujikuroi was about thirty times that of the (R)-(+)isomer. This enantioselective antifungal activity was presumed to be due to an asymmetric molecular shape caused by the binding of a bulky 2-furanylmethyl group to a nitrogen atom adjacent to the asymmetric carbon atom. Therefore, to further elucidate the difference in antifungal activity between the two enantiomers of pefurazoate, we synthesized optically active N-methyl analogues of pefurazoate, and evaluated their antifungal activity. Optical (R)-(+)-and (S)-(-)-isomers of pefurazoate exhibited different activities against Cochliobolus miyabeanus and Pyricularia oryzae as well as Gibberella fujikuroi, but the N-methyl (R)-(+)-and (S)-(-)-isomers of the pefurazoate analogues had almost the same degree of activity against these three fungi and of inhibition of ergosterol biosynthesis in G. fujikuroi. These findings strongly suggest that the enantioselective antifungal activity of pefurazoate is due to its asymmetric molecular shape caused by substitution of the furan ring.

A new azole fungicide, pefurazoate (4-pentenyl (RS)-2-[(2-furylmethyl) (imidazol-lylcarbonyl)amino]butanoate, Healthied) has one Chiral carbon center in its structure. From optically active 2-aminobutanoic acid, we prepared two enantiomers of pefurazoate, (R)isomer and (S)-isomer, whose optical rotations in methanol were [aTh + 10. 7 and [a]D -9. 90, respectively, and studied their antifungal activity and ergosterol biosynthesis in Gibberella fujikuyoi.The antifungal activity of the (S)-isomer was about 30 times higher than that of the (R)-isomer. At 0.24ppm, the in vitro inhibition of ergosterol biosynthesis by the (S) isomer was four 'times higher than by the (R)-isomer. These results indicate that the (S)isomer of pefurazoate is an active form fungicidal to G. fujikuyoi.