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.</em

ABC transporters and azole susceptibility in laboratory strains of the wheat pathogen Mycosphearella graminicola

Laboratory strains of Mycosphaerella graminicola with decreased susceptibilities to the azole antifungal agent cyproconazole showed a multidrug resistance phenotype by exhibiting cross-resistance to an unrelated chemical, cycloheximide or rhodamine 6G, or both. Decreased azole susceptibility was found to be associated with either decreased or increased levels of accumulation of cyproconazole. No specific relationship could be observed between azole susceptibility and the expression of ATP-binding cassette (ABC) transporter genes MgAtr1 to MgAtr5 and the sterol P450 14-demethylase gene, CYP51. ABC transporter MgAtr1 was identified as a determinant in azole susceptibility since heterologous expression of the protein reduced the azole susceptibility of Saccharomyces cerevisiae and disruption of MgAtr1 in one specific M. graminicola laboratory strain with constitutive MgAtr1 overexpression restored the level of susceptibility to cyproconazole to wild-type levels. However, the level of accumulation in the mutant with an MgAtr1 disruption did not revert to the wild-type level. We propose that variations in azole susceptibility in laboratory strains of M. graminicola are mediated by multiple mechanisms

Clinical characteristics of women captured by extending the definition of severe postpartum haemorrhage with 'refractoriness to treatment': a cohort study

Background: The absence of a uniform and clinically relevant definition of severe postpartum haemorrhage hampers comparative studies and optimization of clinical management. The concept of persistent postpartum haemorrhage, based on refractoriness to initial first-line treatment, was proposed as an alternative to common definitions that are either based on estimations of blood loss or transfused units of packed red blood cells (RBC). We compared characteristics and outcomes of women with severe postpartum haemorrhage captured by these three types of definitions. Methods: In this large retrospective cohort study in 61 hospitals in the Netherlands we included 1391 consecutive women with postpartum haemorrhage who received either ≥4 units of RBC or a multicomponent transfusion. Clinical characteristics and outcomes of women with severe postpartum haemorrhage defined as persistent postpartum haemorrhage were compared to definitions based on estimated blood loss or transfused units of RBC within 24 h following birth. Adverse maternal outcome was a composite of maternal mortality, hysterectomy, arterial embolisation and intensive care unit admission. Results: One thousand two hundred sixty out of 1391 women (90.6%) with postpartum haemorrhage fulfilled the definition of persistent postpartum haemorrhage. The majority, 820/1260 (65.1%), fulfilled this definition within 1 h following birth, compared to 819/1391 (58.7%) applying the definition of ≥1 L blood loss and 37/845 (4.4%) applying the definition of ≥4 units of RBC. The definition persistent postpartum haemorrhage captured 430/471 adverse maternal outcomes (91.3%), compared to 471/471 (100%) for ≥1 L blood loss and 383/471 (81.3%) for ≥4 units of RBC. Persistent postpartum haemorrhage did not capture all adverse outcomes because of missing data on timing of initial, first-line treatment. Conclusion: The definition persistent postpartum haemo