Mechanisms of action of the organophosphorus fungicide pyrazophos

In plant protection organophosphorus compounds are well-known for their insecticidal and acaricidal action. Since 1960, a number of organophosphorus fungicides have also been introduced. In an in vivo screening of these and some related insecticidal compounds against Sphaerotheca fuliginea on cucumber, pyrazophos (O,O-diethyl O-(5-methyl-6-ethoxycarbonylpyrazolo[1,5-a]pyrimidin-2-yl) phosphorothioate) proved to be most active. The chemical displayed both a protective and curative action against the disease; in addition, considerable toxicity to Pyricularia oryzae on barley was also found. Pyrazophos proved to be rather selective because hardly any other fungi sensitive to the chemical were found in a study on the in vitro spectrum of antifungal activity.The organophosphorus fungicides Hinosan (O-ethyl S,S-diphenyl phosphorodithioate) and Kitazin (O,O-diethyl S-benzyl phosphorothioate) were about as toxic as pyrazophos to P. oryzae, but were much less toxic than pyrazophos to S. fuliginea. In view of these results, investigations on mechanisms of fungitoxic action of orianophosphorus fungicides were mainly focused on pyrazophos.Both pyrazophos and its phosphate analogue (PO-pyrazophos) were found to inhibit the activity of carboxylesterases of S.fuliginea. However, because no correlation could be established between in vivo inhibition of the activity of these enzymes by pyrazophos and PO-pyrazophos, and their fungitoxicity, this effect probably does not account for the mechanism of action of pyrazophos.Neither could the toxicity of pyrazophos, in all other experiments studied with P. oryzae, be attributed to an effect on cell membrane permeability, as was shown to be present upon incubation of mycelium of the fungus with Hinosan and Kitazin. In addition, pyrazophos hardly affected nucleic acid and protein synthesis, and only slightly inhibited oxygen uptake.In short-term experiments, using an incubation time of 2 hours, pyrazophos was 100-1000 x less toxic to fungal growth in mycelial suspensions than to radial growth on agar and growth in liquid media inoculated with conidia. In the latter tests, growth was assayed after I week of incubation. These results can be partly ascribed to the fact that pyrazophos is metabolically converted in the fungus into two fungitoxic breakdown products, PO-pyrazophos and 2-hydroxy-5-methyl-6-ethoxycarbonylpyrazolo(1,5-a)pyrimidine (PP). In short-term experiments the toxicity of PP for mycelial growth in suspensions buffered at pH 4.0 even proved to be considerably higher than that of pyrazophos and PO-pyrazophos. PP might, therefore, be regarded as the actual fungitoxic principle of pyrazophos. This hypothesis is supported by the finding that PP, in contrast to pyrazophos, also displayed an inhibitory activity towards nucleic acid and protein synthesis and towards oxygen uptake of the fungus. The weak effects of pyrazophos on these processes and on mycelial growth in short-term experiments can probably be ascribed to an insufficient conversion of pyrazophos into PP under these conditions.Regarding the site of fungitoxic action of PP in P. oryzae, two hypotheses could be suggested. First, PP could inhibit specifically oxygen uptake and hence indirectly cellular synthetic processes like nucleic acid and protein synthesis. Secondly, PP might react aspecifically with cellular components and hence, directly affect both oxygen uptake and biosynthetic processes.Pythium debaryanum and Saccharomyces cerevisiae are practically insensitive to pyrazophos. Upon incubation of these fungi with the fungicide no breakdown products could be detected. Therefore, sensitivity of fungi for pyrazophos seems to be the result of a selective uptake of pyrazophos and/or of its conversion into PO pyrazophos and PP as has been demonstrated for P.oryzae.