Mode of resistance to respiration inhibitors at the cytochrome bc1 enzyme complex of Mycosphaerella fijiensis field isolates

Field isolates of Mycosphaerella fijiensis, causing black Sigatoka of banana, were characterised for their sensitivity to different inhibitors of the cytochrome bc₁ enzyme complex (Qo respiration inhibitors, strobilurin fungicides), using physiological, biochemical and molecular genetic methods. Strobilurin‐resistant isolates exhibited very high resistance factors both in mycelial growth inhibition and NADH consumption assays. Cross‐resistance was observed among all Qo inhibitors, including trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. However, the Qi and the cytochrome aa₃ inhibitors, antimycin A and potassium cyanide, respectively, were not cross‐resistant to Qo inhibitors. In sensitive but not in resistant isolates, mixtures of Qo inhibitors and SHAM, an inhibitor of the alternative oxidase (AOX), were more active than the components alone, indicating that the alternative pathway is essential in metabolism, but not causal for resistance. In the cell‐free NADH‐consumption assay, the Qo inhibitors affected the sensitive but not the resistant isolates, suggesting that AOX was not active in sub‐mitochondrial particles. In whole cells, however, the AOX has a basic expression level and is probably not inducible by trifloxystrobin. Sequencing of the cytochrome b gene of sensitive and resistant M fijiensis isolates revealed a difference in the nucleotide sequence leading to a single amino acid change from glycine to alanine at position 143 in the resistant isolate. This change is known to occur also in the naturally tolerant basidiomycete Mycena galopoda. It is suggested that the field isolates of M fijiensis can acquire resistance to Qo inhibitors due to a target site alteration with a single base pair change. Resistant isolates do not seem to contain a mixture of mutated and non‐mutated DNA, indicating a complete selection of resistant mitochondria and a maternally donated mode of resistance. © 2000 Society of Chemical Industry     

Mitochondrial impacts of insecticidal formate esters in insecticide‐resistant and insecticide‐susceptible Drosophila melanogaster

BACKGROUND: Previous research on insecticidal formate esters in flies and mosquitoes has documented toxicity profiles, metabolism characteristics and neurological impacts. The research presented here investigated mitochondrial impacts of insecticidal formate esters and their hydrolyzed metabolite formic acid in the model dipteran insect Drosophila melanogaster Meig. These studies compared two Drosophila strains: an insecticide‐susceptible strain (Canton‐S) and a strain resistant by cytochrome P450 overexpression (Hikone‐R). RESULTS: In initial studies investigating inhibition of mitochondrial cytochrome c oxidase, two proven insecticidal materials (hydramethylnon and sodium cyanide) caused significant inhibition. However, for insecticidal formate esters and formic acid, no significant inhibition was identified in either fly strain. Mitochondrial impacts of formate esters were then investigated further by tracking toxicant‐induced cytochrome c release from mitochondria into the cytoplasm, a biomarker of apoptosis and neurological dysfunction. Formic acid and three positive control treatments (rotenone, antimycin A and sodium cyanide) induced cytochrome c release, verifying that formic acid is capable of causing mitochondrial disruption. However, when comparing formate ester hydrolysis and cytochrome c release between Drosophila strains, formic acid liberation was only weakly correlated with cytochrome c release in the susceptible Canton‐S strain (r² = 0.70). The resistant Hikone‐R strain showed no correlation (r² < 0.0001) between formate ester hydrolysis and cytochrome c release. CONCLUSION: The findings of this study provide confirmation of mitochondrial impacts by insecticidal formate esters and suggest links between mitochondrial disruption, respiratory inhibition, apoptosis and formate‐ester‐induced neurotoxicity. Copyright © 2009 Society of Chemical Industry     

Mitochondrial electron transport in a carboxin-resistant,antimycin A-sensitive mutant of Ustilago maydis

Succinoxidase activity of mitochondria from an antimycin A-sensitive (ants) mutant of Ustilago maydis is approximately five times less sensitive to the fungicide carboxin than that of mitochondria from wild-type cells. The antimycin A sensitivity is due to the absence of an alternative electron transport pathway in mitochondria obtained from mutant cells grown in control medium. The same mutant, however, develops high rates of alternative respiration if grown in the presence of chloramphenicol. Substrate and oxygen affinity as well as resistance to hydroxamates indicate that this respiration is mediated by only one mitochondrial electron transport pathway, similar to the inducible system described earlier in wild-type mitochondria. Induction appears to be regulated by the activity of the cytochrome pathway. The absence of the constitutive system from the ants mutant mitochondria which are resistant to carboxin, a selective inhibitor of the succinate dehydrogenase complex, supports the view that this system must be related to succinic dehydrogenase.     

Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK‐2A

BACKGROUND

Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated.

RESULTS

UK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides.

CONCLUSION

Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Inhibitory effect of bionic fungicide 2‐allylphenol on Botrytis cinerea (Pers. ex Fr.) in vitro

BACKGROUND: 2‐Allylphenol is a registered fungicide in China to control fungal diseases on tomato, strawberry and apple. It is synthetic and structurally resembles the active ingredient ginkgol isolated from Ginkgo biloba L. bark. 2‐Allylphenol has been used in China for 10 years. However, its biochemical mode of action remains unclear. An in vitro study was conducted on the biochemical mechanism of 2‐allyphenol inhibiting Botrytis cinerea (Pers. ex Fr.). RESULTS: The inhibition was approximately 3 times stronger when the fungus was grown on non‐fermentable source, glycerol, than that on a fermentable carbon source, glucose. Inhibition of B. cinerea and Magnaporthe oryzae (Hebert) Barr mycelial growth was markedly potentiated in the presence of salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial alternative oxidase. Furthermore, at 3 h after treatment with 2‐allylphenol, oxygen consumption had recovered, but respiration was resistant to potassium cyanide and sensitive to SHAM, indicating that 2‐allylphenol had the ability to induce cyanide‐resistant respiration. The mycelium inhibited in the presence of 2‐allylphenol grew vigorously after being transferred to a fungicide‐free medium, indicating that 2‐allylphenol is a fungistatic compound. Adenine nucleotide assay showed that 2‐allylphenol depleted ATP content and decreased the energy charge values, which confirmed that 2‐allylphenol is involved in the impairment of the ATP energy generation system. CONCLUSION: These results suggested that 2‐allylphenol induces cyanide‐resistant respiration and causes ATP decrease, and inhibits respiration by an unidentified mechanism. Copyright © 2009 Society of Chemical Industry     

Contribution to the knowledge of aerobic processes in Taenia crassiceps larvae (Zeder, 1800)

Respiration of homogenates and isolated mitochondria of T. crassiceps larvae was measured. Respiration (about 70%) could be inhibited by cyanide, indicating that an important part is played by classical respiratory chain. When succinate was used as substrate, relatively low respiratory rates were measured, mitochondria showing higher affinity to NADH. Above a half of respiratory rate remained unchanged even at very low oxygen concentrations. Fumarate exhibited inhibitory activity on respiration of T. crassiceps mitochondria. Respiration in which NADH was used as substrate was twice as much more sensitive to inhibition by fumarate than respiration stimulated by succinate.     

Effects of pyroquilon on the infection process of Monilinia laxa causing peach twig blight

The capacity of Monilinia laxa (Aderh. et Ruhl.) Honey to induce peach twig blight was lost in an albino mutant and in a wild-type strain of the pathogen treated with pyroquilon, a melanin biosynthesis inhibitor. Pyroquilon (10 ù ml^?1) inhibited melanin biosynthesis but did not inhibit mycelial growth of the pathogen or the albino mutant in vitro. These observations indicate that the compound interferes in some manner with the infection process of host cells.     

Inoculum density — host response relationships of spring wheat cultivars to infection by Septoria tritici

Eightinoculumdensities (ID), increasing in geometric progression from 0.1×10⁵ to 12.8×10⁵ conidia ml^–1 ofSeptoria tritici were sprayed onto the leaves of five cultivars ofTriticum aestivum, differing in resistance toS. tritici. Host response to infection byS. tritici, measured as either thelatencyperiod (LPD) orpercentage ofleafarea covered bylesions with pycnidia (PLAL), was significantly linearly correlated with log₁₀ID (r=0.84 to 0.96, with 6 degrees of freedom). As the ID increased from 0.2×10⁵ to 12.8×10⁵ conidia ml^–1, LPD decreased by 1 to 2 days for the susceptible and resistant cultivars and by 3 to 5 days for the moderately susceptible cultivars. Within the same ID range, PLAL increased from 73% to 90% for the susceptible cultivar and 2 to 12% for the resistant cultivar. The ID₅₀ value (the number of conidia required for 50% PLAL) calculated from the regression equations relating log₁₀ID to PLAL for each cultivar, ranged from 6.42×10³ conidia ml^–1 for the susceptible cultivar to 7.13×10¹³ conidia ml^–1 for the resistant cultivar. Estimates of the number of conidia ml^–1 required to initiate a lesion (obtained by substituting 1% PLAL into the regression equations relating log₁₀ ID to PLAL) ranged from 16 conidia ml^–1 for the susceptible cultivar to 1.0×10⁴ conidia ml^–1 for the resistant cultivar. Using the change of PLAL with ID, the restriction of lesion development for each cultivar could be partitioned into that due to host resistance and that due to interaction between lesions. Log₁₀LPD was significantly correlated negatively with PLAL. As a general rule when screening cultivars for resistance toS. tritici, it is advisable to inoculate them with several inoculum densities and to determine the latency period.Samenvatting Sporensuspensies vanSeptoria tritici die in dichtheid (ID) varieerden volgens een geometrische reeks van 0,1 tot 12,8×10⁵ sporen.ml^–1, werden gebruikt bij de inoculatie van kiemplanten van vijf cultivars vanTriticum aestivum die verschilden in resistentie tegenS. tritici.De reactie van de waardplanten werd gemeten aan de latentie periode (LPD) en aan het percentage bladoppervlak dat ingenomen werd door vlekken met pycniden (PLAL). Beide grootheden toonden een significante lineaire correlatie met log₁₀ID (r=0,84 tot 0,96 bij 6 vrijheidsgraden). Bij een toename van ID van 0,2×10⁵ tot 12,8×10⁵ sporen.ml^–1 nam de lengte van de latentie periode af met 1 tot 2 dagen bij de vatbare en de resistente cultivars, en met 3 tot 5 dagen bij de matig vatbare cultivars (Fig. 2).In hetzelfde traject van inoculumdichtheid nam de PLAL toe van 73 tot 90% bij vatbare cultivars, en van 2 tot 12% bij de resistente cultivars (Fig. 3). De ID₅₀-waarde, het aantal sporen dat nodig is om 50% aantasting te verkrijgen, werd berekend uit de regressievergelijkingen die voor iedere cultivar het verband aangeven tussen log₁₀ ID en het aantastingspercentage (PLAL). Deze waarde varieerde van 6,42×10³ sporen per ml voor vatbare tot 7,13×10³ sporen per ml voor de resistente cultivars (Tabel 1). Schattingen voor het aantal sporen dat nodig is om een vlek te doen ontstaan werden verkregen door de 1% aantastingswaarde te substitueren in de eerder genoemde regressievergelijkingen. Deze waarden varieerden van 16 sporen.ml^–1 voor de vatbare cultivar tot 10000 sporen.ml^–1 voor de resistente cultivar (Tabel 1). Via de toename van de aantastingsgraad (PLAL) per eenheid stijging in de inoculumdichtheid kon de remming op de vlekontwikkeling opgesplitst worden in een gedeelte dat toegeschreven werd aan de resistentie van de plant en een gedeelte dat te wijten was aan de interactie tussen de vlekken (Fig. 4 en 5; Tabel 2).Log₁₀ LPD was negatief gecorreleerd met PLAL (Fig. 6). Bij het toetsen van cultivars op resistentie tegenS. tritici zouden sporensuspensies van verschillende dichtheden gebruikt moeten worden en zou de latentie periode, evenals de aantastingsgraad bepaald moeten worden.     

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc ₁ complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc ₁ complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.     

Effect of propiconazole on growth and sterol biosynthesis by Sclerotium rolfsii

Germination of sclerotia ofSclerotium rolfsii on agar nutrient medium was delayed or slightly inhibited by concentrations of propiconazole between 0.4 and 4.0 μg ml^?1, but was strongly inhibited by 8 μg ml^?1 and completely inhibited by 16 μg ml^?1. On the other hand, growth of hyphae from the germinated sclerotia was strongly inhibited by propiconazole at 1 μg ml^?1 or greater. Hyphal growth from agar discs on agar medium was about 8 times less sensitive than hyphal growth from the sclerotia or from hyphal inoculum in liquid media. Propiconazole at 0.25 and 1.0 μg ml^?1 strongly inhibited ergosterol biosynthesis, but this was not associated with large accumulations of C-14 methyl sterols. The ratio of eburicol to ergosterol in hyphae grown in the presence of 0.25 μg ml^?1 propiconazole for 16, 30 or 45 h was 0.11, 0.13 and 0.04, respectively, for the three intervals while for hyphae grown in the presence of 1 μg ml^?1, the ratios were 0.29, 0.36 and 0.30, respectively, for the same intervals. In view of a ratio of 23.5 for¹⁴C-acetate incorporation into the two sterols during the initial 6 h growth period in the presence of propiconazole, it is believed that the lack of large accumulation of C-14 methyl sterols is due to the feedback inhibition by eburicol or to cell lysis when the content of ergosterol becomes too low in the actively growing cells.     

Laboratory studies on benzimidazole resistance in Septoria tritici1

A technique is described for testing isolates of Septoria tritici from winter wheat for resistance to benzimidazole fungicides. Secondary spores from 23 isolates were tested on Czapek Dox V-8 agar amended with benomyl at 1–10 μg ml. Twenty-one isolates were recovered from eight crops in England in 1984 and two (PBI isolates) were obtained in 1973. Thirteen isolates, including both PBI isolates, were sensitive to benomyl at 1 μg ml^?1 and nine were resistant at 10 μg ml^?1. The remaining isolate had a low proportion (1:3.7 x 10⁴) of resistant spores. The minimum inhibitory concentration for sensitive isolates was 0.2–0.4 μg ml ¹ benomyl and for resistant isolates was more than 1000 μg ml ¹. Benomyl-resistant isolates were cross-resistant to carbendazim, thiabendazole and thiophanate-methyl, but not to 12 other fungicides with different modes of action. The implications of these findings are discussed.     

Metabolic differentiation of bloodstream forms of Trypanosoma brucei brucei into procyclic forms in hemin-depleted medium and in the presence of respiratory inhibitors

Bloodforms of Trypanosoma brucei brucei (STIB 247) differentiated in vitro into procyclic forms as described in the accompanying paper (Markos et al. 1989). The importance of the respiratory chain for the process was tested by the inhibition of its development (omission of hemin from the medium) or function (respiratory inhibitors). In the absence of hemin, all enzyme markers of the procyclic state, except for hemoproteins, developed to 50-70 per cent of control values. The presence of hemin is therefore not essential for the onset of differentiation, although the process cannot be completed under hemin limitation. Addition of 1 mumole.dm-3 KCN, 10 mumole.dm-3 antimycin A, or 100 mumole.dm-3 salicyl hydroxamate (SHAM) did not block the differentiation, although it proceeded at a slower rate. The development of the inner mitochondrial membrane markers--succinate: cytochrome c reductase, and NADH: cytochrome c reductase--was strongly inhibited by KCN or antimycin. None of these inhibitors had a significant effect on the activity of procyclic state marker--glycosomal malate dehydrogenase.     

Effects of herbicidal carbamates on mitochondria and chloroplasts

At concentrations near 2 × 10^?4M, barban, chlorpropham, and phenmedipham are inhibitors of the electron transfer in potato and mung bean mitochondria. The inhibition seems to be localized in the flavoprotein region. It affects preferentially the exogenous NADH dehydrogenation, in potato mitochondria (I₅₀, 10^?4M). Succinate dehydrogenation is less inhibited. At noninhibiting concentrations, the studied carbamates cannot uncouple the oxidative phosphorylations. Photosynthesis is completely inhibited by 2.10^?7M phenmedipham, 5 × 10^?5M barban, and 2 × 10^?4M chlorpropham. The inhibition takes place at the PS II level. Moreover, barban and chlorpropham are uncouplers of the photophosphorylations for concentrations between 5 × 10^?5 and 5 × 10^?4M. The effects observed on mitochondrial respiration can also be found on respiration of Acer cultured cells. The effects on isolated chloroplast photosynthesis are also observed for slightly higher concentrations on cultured Chlorella and on pea and oat leaf fragments.     

Siderophore-mediated competition for iron and induced resistance in the suppression of fusarium wilt of carnation by fluorescent Pseudomonas spp

The mechanisms of suppression of fusarium wilt of carnation by two fluorescentPseudomonas strains were studied.Treatments of carnation roots withPseudomonas sp. WCS417r significantly reduced fusarium wilt caused byFusarium oxysporum f. sp.dianthi (Fod). Mutants of WCS417r defective in siderophore biosynthesis (sid^–) were less effective in disease suppression compared with their wild-type. Treatments of carnation roots withPseudomonas putida WCS358r tended to reduce fusarium wilt, whereas a sid^– mutant of WCS358 did not.Inhibition of conidial germination of Fod in vitro by purified siderophores (pseudobactins) of bothPseudomonas strains was based on competition for iron. The ferrated pseudobactins inhibited germination significantly less than the unferrated pseudobactins. Inhibition of mycelial growth of Fod by bothPseudomonas strains on agar plates was also based on competition for iron: with increasing iron content of the medium, inhibition of Fod by thePseudomonas strains decreased. The sid^– mutant of WCS358 did not inhibit Fod on agar plates, whereas the sid^– mutants of WCS417r still did. This suggests that inhibition of Fod by WCS358r in vitro was only based on siderophore-mediated competition for iron, whereas also a non-siderophore antifungal factor was involved in the inhibition of Fod by strain WCS417r.The ability of thePseudomonas strains to induce resistance against Fod in carnation grown in soil was studied by spatially separating the bacteria (on the roots) and the pathogen (in the stem). Both WCS417r and its sid^– mutant reduced disease incidence significantly in the moderately resistant carnation cultivar Pallas, WCS358r did not.It is concluded that the effective and consistent suppression of fusarium wilt of carnation by strain WCS417r involves multiple mechanisms: induced resistance, siderophore-mediated competition for iron and possibly antibiosis. The less effective suppression of fusarium wilt by WCS358r only depends on siderophore-mediated competition for iron.     

A molecular mechanism of resistance to streptomycin in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzicola</Emphasis>

Xanthomonas oryzae pv. oryzicola, the causal agent of rice leaf streak disease, was found to be sensitive to streptomycin (an aminocyclitol glycoside antibiotic), by inhibition of protein synthesis resulting from interference with translational proofreading. This study aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzicola to streptomycin. Seven streptomycin-resistant mutants were obtained by UV induction or streptomycin selection. These mutants can grow at 100 μg ml⁻¹ of streptomycin while the wild-type strain (RS105) cannot grow at 5 μg ml⁻¹. Sequencing indicated that the rpsL gene encoding ribosomal protein S12 has 375 bp encoding 125 amino acid residues. In all resistant strains, a mutation in which AAG was substituted for AGG (Lys→Arg) occurred either at codon 43 or 88. Two plasmids, pUFRRS and pUFRRX, were constructed by ligating the rpsL gene into the cosmid pUFR034. The plasmids pUFRRS and pUFRRX containing the Lys→Arg mutation of the rpsL gene conferred streptomycin resistance to the sensitive wild-type strain by electroporation. Both transformants, RS1 and RS2, could grow in the medium containing 50 μg ml⁻¹ of streptomycin. A mutation at codon 43 or 88 in rpsL can result in resistance of Xanthomonas oryzae pv. oryzicola to streptomycin.     

Mechanism of resistance to fenarimol in Aspergillus nidulans

Mycelial uptake of [¹⁴C]fenarimol (10 μg/ml) by 20 fenarimol-resistant mutants of Aspergillus nidulans was compared with uptake by wild-type strain 003. Uptake of the fungicide during the initial 10 min of incubation was significantly lower in all mutant strains than in the wild-type strain indicating that resistance is related with reduced uptake. Upon prolonged incubation a gradual decrease of accumulated radioactivity in the wild-type strain was observed. A few mutants displayed resistance to unrelated chemicals such as p-fluorophenylalanine or d-serine; this phenomenon appeared not to be due to a decreased uptake of the corresponding natural amino acids. Incorporation of [³H]adenine and [¹⁴C]leucine by mycelium of mutant M193 was hardly inhibited after 5 hr of incubation with the fungicide, whereas a distinct effect was found with the wild-type strain. At this time also fungitoxicity to the wild-type strain became apparent. Probably, this effect is indirectly caused by inhibition of ergosterol biosynthesis. Mycelium of mutant M193 incorporated [¹⁴C]acetate slightly less effectively than the wild-type strain. After 2 hr of incubation with this radiochemical leakage of [¹⁴C]acetate metabolites from mycelium of the mutant strain was observed. This indicates that resistance might be correlated with increased excretion of fungal metabolites, which in turn may be related with reduced fitness of fenarimol-resistant mutants.     

Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl

A new class of agricultural fungicides derived from strobilurins act as respiration inhibitors by binding to mitochondrial cytochrome b. The effects of the strobilurin, kresoxim-methyl, on conidia germination, mycelial growth and the protection of apple leaves from scab development were investigated for two isolates of Venturia inaequalis randomly selected from a culture collection. Inhibition of mycelial growth required relatively high doses of kresoxim-methyl (ED₅₀=1 μg ml^-1) for both isolates. In comparison, germination of conidia was highly sensitive for one of the isolates (ED₅₀=0·005 μg ml^-1), while the level of inhibition achieved for the second isolate was 60-fold less (ED₅₀=0·3 μg ml^-1). As deduced from identical sequences of cytochrome b cDNAs prepared from both isolates, the different responses of germinating conidia to kresoxim-methyl were not caused by differences in the sequence of cytochrome b as the target site for strobilurin action. Strong synergistic effects of salicylhydroxamic acid on kresoxim-methyl inhibitory potency suggested that the reduced kresoxim-methyl sensitivity observed for germinating conidia was caused by interference of the alternative respiratory pathway with inhibitor action. However, this mechanism of target site circumvention in germinating conidia had no adverse effects on the protection of apple leaves from scab infection by kresoxim-methyl. © 1998 Society of Chemical Industry     

Uptake,metabolism and effects of DDT,fenitrothion and chlorpyrifos on Tetrahymena pyriformis

The uptake and metabolism of DDT, fenitrothion and chlorpyrifos were studied in cultures of the ciliate protozoan Tetrahymena pyriformis. When cultures were treated with DDT in concentrations varying from 0.01 to 0.5 μg ml⁻¹, concentrations found in T. pyriformis were 3.8 to 335 μg g⁻¹ dry weight. The accumulation of fenitrothion ranged from 28.7 μg g⁻¹ in cultures treated with 1 μg ml⁻¹ to 2260 μg g⁻¹ in cultures treated with 10 μg ml⁻¹. Under similar experimental conditions chlorpyrifos was accumulated from 24.7 to 15400 μg g⁻¹. The patterns of uptake were dependent on the growth cycle, the ability of the organism to metabolise insecticide and the type of the insecticide used. Maximum accumulation of DDT, fenitrothion and chlorpyrifos occurred in 2, 4 and 6 h respectively. Tetrahymena metabolised DDT to DDD and DDE but failed to metabolise fenitrothion and chlorpyrifos. The effects on growth and morphology of T. pyriformis were studied over a period of 5 days. Higher concentrations (10, 50 and 100 μg ml⁻¹) of DDT inhibited only the growth of the organisms and did not change cell morphology. Fenitrothion was extremely toxic to the organisms and at 5 and 10 μg ml⁻¹ cells became more or less spherical and died after 48 h. However, concentrations of 0.5, 1 and 2.5 μg ml⁻¹ fenitrothion caused growth inhibition, but only at 2.5 μg ml⁻¹ was this permanent. Chlorpyrifos inhibited the growth of the organisms at 1, 5 and 10 μg ml⁻¹ but the morphology was affected only at 5 and 10 μg ml⁻¹.     

Effect of maleic hydrazide on parasitic fitness of Puccinia striiformis f. sp. tritici in wheat

As a growth inhibitor of the apical meristem in the plant stem, Maleic hydrazide plays an important role in modulation of plant growth. In this research, Puccinia striiformis f. sp. tritici and its host for reproduction was employed to characterize the effect of maleic hydrazide on parasitic fitness of the strain. Growth inhibition of the secondary leaves of wheat by maleic hydrazide was demonstrated. Results showed that root irrigation by maleic hydrazide at the seedling stage significantly increased the parasitic fitness of Puccinia striiformis f. sp. tritici, leading to the increase in sporulation amount, sporulation period, and germination rate of urediniospores. In addition, the ultrastructure of Puccinia striiformis f. sp. tritici urediniospores was not affected by maleic hydrazide treatment. Out data indicate that the optimal concentration and dose for the use of maleic hydrazide is 0.35 g?l^?1 and 1.5 ml/cm², respectively, facilitating the widespread application in wheat stripe rust studies.     

Inhibition of ergosterol biosynthesis in Ustilago maydis by the fungicide 1-[2-(2, 4-dichlorophenyl)-4-ethyl-1, 3-dioxolan-2-ylmethyl]-1H-1, 2, 4-triazole

Inhibition of sporidial multiplication in cultures of Ustilago maydis by 1-[2-(2, 4-dichlorophenyl)-4-ethyl-1, 3-dioxolan-2-ylmethyl]-1H-1, 2, 4-triazole^a (CGA-64251), at concentrations of 0.1, 1.0 and 5.0 μg ml^?1, increased from about 15% during the first 4 h, to 58–70% during the subsequent 4 to 12-h period. Sporidia became swollen and highly branched in the presence of the fungicide. Total lipid content as a percentage of the dry weight was not affected after exposure of the sporidia to the fungicide at 0.1 or 5 μg ml^?1 for 4 h, but synthesis of ergosterol and other demethyl-sterols was inhibited by 87–92%. Large quantities of methyl-sterol precursors of ergosterol and of free fatty acids accumulated in the treated sporidia. Fungitoxicity of CGA-64251 is attributed to inhibition of ergosterol biosynthesis at the stage of sterol C-14 demethylation.