Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 μg ml^?1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 g ml^–1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.Imazalil remt differentieel de toename in drooggewicht van 10-uur-oude gekiemde sporen van wild-type en DMI-resistente isolaten vanPenicillium italicum in vloeistofcultures van moutextract. De EC₅₀ waarden voor groei van de verschillende isolaten lopen uiteen van 0,005 tot 0,27 g ml^–1. In afwezigheid van het fungicide is in alle isolaten ergosterol het belangrijkste sterol (meer dan 95% van het totaal). DMI-resistentie kan daarom niet in verband staan met deficiëntie van het C-14 demethyleringsenzym in de ergosterol biosynthese. Imazalilbehandeling van mycelium bij concentraties rond de EC₅₀ waarde voor groeiremming, resulteerde bij alle isolaten in een afname van het ergosterolgehalte en een gelijktijdige toename van het gehalte aan 24-methyleen-24,25-dihydrolanosterol. Er bestaat dus een nauwe correlatie tussen de imazalilconcentratie die noodzakelijk is om vergelijkbare veranderingen in sterolsamenstelling te induceren en de EC₅₀ waarde voor remming van myceliumgroei van de verschillende isolaten. De differentiële effecten van imazalil op de sterolsamenstelling van de verschillendeP. italicum isolaten kunnen worden veroorzaakt door verminderde accumulatie van het fungicide in het mycelium en door andere, nog niet geïdentificeerde resistentiemechanismen.     

Effect of Phenylpyrroles on Glycerol Accumulation and Protein Kinase Activity of Neurospora crassa

The effect of phenylpyrroles on glycerol synthesis in Neurospora crassa has been investigated and compared with the mode of action of vinclozolin (a dicarboximide). The results indicated that fenpiclonil, fludioxonil and vinclozolin at concentrations which inhibit growth by 50% induce accumulation of glycerol in the mycelium of N. crassa. Furthermore a protein kinase (PK-III) possibly involved in the regulation of the glycerol synthesis is inhibited by phenylpyrroles, whereas vinclozolin is without effect. This implies that the target sites of phenylpyrroles and dicarboximides in the osmosensing signal transmission pathway are different. Comparative experiments with enzymes from human and animal sources revealed that PK-III could be a protein kinase Cδ. It is suggested that inhibition of PK-III activity may result in an increased concentration of a non-phosphorylated regulatory protein which may activate a MAP-kinase cascade of reactions resulting in increased glycerol synthesis. © 1997 SCI.     

Interference of the phenylpyrrole fungicide fenpiclonil with membranes and membrane function

The phenylpyrrole fungicide fenpiclonil accumulates to high levels in mycelium of Fusarium sulphureum and artificial liposomes. The accumulation is a result of a physicochemical distribution of the fungicide over lipid-like material of the mycelium and the medium. Accumulation is reversible, as the fungicide can easily be removed from the mycelium by washing with water. Fenpiclonil is not metabolized by the fungus during incubation for 24 h. The fungicide affects neither membrane fluidity in artificial liposomes nor amino acid accumulation in bacterial vesicles. Thus, accumulation of the fungicide does not seem to affect the functioning of membranes. Fenpiclonil induces the accumulation of the membrane-potential probe tetra-phenylphosphonium bromide by F. sulphureum. Since accumulation of this probe probably reflects the cumulative potential over all cell membranes, the increased accumulation is more likely a result of changes of potentials over membranes of intracellular organelles rather than plasma membrane hyperpolarization. Hence, the previously described effects of fenpiclonil on amino acid and saccharide uptake cannot be explained by plasma membrane hyperpolarization.     

Inhibition of Methionine Biosynthesis in Botrytis cinerea by the Anilinopyrimidine Fungicide Pyrimethanil

When mycelium of Botrytis cinerea was treated with low concentrations of the anilinopyrimidine fungicide pyrimethanil the total amount of free amino acids increased. Qualitative variations were also induced: alanine, glutamine, lysine, glycine, histidine, asparagine, arginine, threonine and moreover, α-aminobutyrate and β-alanine were accumulated; cyst(e)ine, valine, leucine and citrulline were reduced. When mycelium of B. cinerea was incubated with Na₂[³⁵S]O₄, pyrimethanil at 1·5 μM induced a decrease of [³⁵S]methionine and simultaneously an increase of [³⁵S]cystathionine. These data indicate that the anilinopyrimidine fungicide pyrimethanil inhibits the biosynthesis of methionine and suggest that the primary target could be the cystathionine β-lyase. © 1997 SCI.     

Mode of action of the fungicide flusilazole in ustilago maydis

Flusilazole is a potent inhibitor of Ustilago maydis sporidial growth (I₅₀= 20 μg liter⁻¹). Incorporation of [¹⁴C]acetate into ergosterol of growing sporidia is inhibited 50% by 0.5 μg liter⁻¹of the fungicide. Inhibition of ergosterol biosynthesis is concomitant with the accumulation of the precursors eburicol, obtusifoliol and 14α-methylfecosterol. A novel cell-free assay has been developed to measure the 14α-demethylation of [³H]dihydrolanosterol. Flusilazole inhibits the cell-free demethylation with an I₅₀of 15 μg liter⁻¹. These data provide strong evidence that the mode of action of flusilazole is by inhibiting ergosterol biosynthesis through direct inhibition of the 14α-demethylation of ergosterol precursors.     

Effect of the d‐glucose analog,d‐allose,on the growth of Arabidopsis roots

Although d ‐glucose increased the root growth of Arabidopsis seedlings, d ‐allose (a d ‐glucose epimer at the third carbon atom) inhibited the root growth at concentrations >0.1 mmol L^?1 and the inhibition increased with increasing d ‐allose concentrations. Allitol (a reduction product of d ‐allose) did not show any significant effect on the growth. The addition of d ‐glucose into the growth medium of Arabidopsis reversed the d ‐allose‐induced growth inhibition, which suggests that the inhibition is not caused by the toxicity of the accumulation of d ‐allose and/or its metabolites in the seedlings. d ‐Allose is phosphorylated by hexokinase, using ATP and phosphate, to allose‐6‐phosphate, with no known capacity for further metabolism. The addition of phosphate into the growth medium did not affect the d ‐allose‐induced growth inhibition and d ‐allose did not reduce the ATP level in the roots. These results suggest that the inhibition is not due to phosphate starvation and ATP depletion. d ‐Mannoheptulose, a specific competitive inhibitor of hexokinase, defeated the d ‐allose‐induced growth inhibition. Hexokinase is known to have a sugar‐sensing function and possibly triggers a signal cascade, resulting in the change of several gene expressions. Therefore, the phosphorylation of d ‐allose by hexokinase might trigger a signal cascade, resulting in the inhibition of Arabidopsis root growth. This is probably a useful model system for studies of the hexokinase‐mediated sugar‐sensing function and for developing new types of weed‐control agents.     

Action of the fungicide tridemorph on the glucose,lactate and succinate dehydrogenase activities of some tridemorph-sensitive and -resistant bacteria

The systemic morpholine fungicide tridemorph, which is known to exert its antifungal action through inhibition of ergosterol biosynthesis, can also inhibit the growth of organisms which are incapable of sterol biosynthesis. It was found to inhibit strongly glucose and lactate dehydrogenase activities in cultures of four Gram( +) bacteria, Rhodococcus sp. AK 1, Bacillus cereus Frankland & Frankland, Bacillus subtilis (Ehrenberg) Cohn, Nocardia asteroides and a Gram(?) bacterium, Rhizobium leguminosarum. Growth of these bacteria was inhibited by tridemorph at concentrations between 7 and 60 mg litre^?1. In contrast, similar dehydrogenase activities in other Gram(?) organisms, Escherichia coli Cast. & Chalm. and Azotobacter vinelandii, which showed no growth inhibition at 200 mg litre ^?1 tridemorph, were either not inhibited or inhibited only slightly. Similarly, succinate dehydrogenase activity in Rhodococcus sp. AK 1 was more strongly inhibited by tridemorph than that in E. coli. In cell-free extracts of Rhodococcus sp. AK1 and E. coli, lactate dehydrogenase activity was also inhibited by tridemorph to a much greater extent in the sensitive strain (63%) than in the resistant ones (8%).     

An energy-dependent efflux mechanism for fenarimol in a wild-type strain and fenarimol-resistant mutants of Aspergillus nidulans

Uptake of [¹⁴C]fenarimol (30 μM) by mycelium of wild-type Aspergillus nidulans was characterized by a rapid initial accumulation during the first 10 min of incubation with the fungicide and a subsequent gradual release with time. Uptake appeared to be the result of influx and efflux. Influx of fenarimol could not be inhibited by low temperature, anaerobiosis, starvation of mycelium, or incubation with several respiratory inhibitors and is, therefore, a passive process. Under identical test conditions efflux activity was severely inhibited and should, therefore, be regarded as an energy-dependent mechanism. After prolonged incubation (90 min) an equilibrium between influx and efflux was established, resulting in an energy-dependent permeability barrier, since uptake could instantaneously be enhanced by addition of oligomycin or N,N′-dicyclohexylcarbodiimide. It also indicates that efflux activity is inducible; this hypothesis is supported by the observation that pretreatment of mycelium with unlabeled fungicide prevented subsequent uptake of [¹⁴C]fenarimol. Uptake by fenarimol-resistant mutants J146, M193, and R264 of A. nidulans, all possessing the imaB gene for resistance, was relatively low and almost constant in time. In this case, uptake appeared to be considerably enhanced by low temperature, anaerobiosis, starvation of mycelium, and incubation with respiratory inhibitors. Low uptake by these mutants is ascribed to a higher energy-dependent efflux activity for fenarimol compared with the wild-type strain. Upon inhibition of the barrier activity, net uptake resulted from remaining passive influx, which in that case may be as high as in the wild-type strain. The results suggest that both wild-type and fenarimol-resistant mutants possess an energy-dependent efflux mechanism with different efficiencies to excrete fenarimol and probably other chemicals to which cross-resistance or collateral sensitivity is present.     

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.     

紫外光诱导灰葡萄孢产生抗多菌灵菌株的研究

测得多菌灵对Botrytis cinerea野生菌株B_2的最低抑制生长浓度(MIC)值为0.5ppm。然而,经紫外光照射定向诱导,从B_2获得了突变频率为I/0.9—4.5×10~4的抗多菌灵突变菌株。这种抗性菌株在含多菌灵1000ppm的马铃薯蔗糖琼脂培养基上,菌丝生长很少受到抑制,在多菌灵2000ppm情况下仍能生长,难以测定MIC值。诱变抗性菌株(B-UV1)在无药培养基上连续培养8个月,抗性程度仍未下降,但生长速率降低。 B-UV1菌株对苯来持、托布津杀菌剂表现高度的交互抗性,但对克菌丹等4种测试药剂的敏感性,与野生菌株相似。接种草莓时,B-UV1菌株引起的发病率为野生菌株的77.8％,而且病情较轻。     

Mode of action of the systemic fungicides furalaxyl,metalaxyl and ofurace

The systemic fungicides furalaxyl, metalaxyl and ofurace,

1 Ofurace is the proposed BSI and ISO common name for α-(2-chloro-N-2,6-xylylacetamido)-γ-butyrolactone.

used to control diseases caused by phycomycetes, were extremely active in vitro against Pythium ultimum, Phytophthora nicotianae and Phytophthora palmivora. Sporangia production was reduced more than mycelial growth but germination of sporangia and zoospores was relatively unaffected. Less than 1% of the metalaxyl or furalaxyl, present in media at the ED₅₀ for hyphal growth, was firmly absorbed by Phyt. palmivora mycelium; uptake was against a concentration gradient and was characterised by a rapid accumulation followed by a more gradual release. Respiration and wall synthesis were not inhibited, whilst membrane permeability was unaffected. Lipid patterns were altered but these changes were probably of secondary importance. The fungicides inhibited protein and nucleic acid synthesis; RNA production was particularly affected. There was some evidence of a reduction in nuclear division. The primary effect of furalaxyl and metalaxyl probably involves impaired biosynthesis of RNA so that mitosis is inhibited; ofurace may act in the same way.     

Impact of tricyclazole and azoxystrobin on growth,sporulation and secondary infection of the rice blast fungus,Magnaporthe oryzae

BACKGROUND: Rice blast, caused by Magnaporthe oryzae B. Couch sp. nov., is one of the most destructive rice diseases worldwide, causing substantial yield losses every year. In Italy, its management is based mainly on the use of two fungicides, azoxystrobin and tricyclazole, that restrain the disease progress. The aim of this study was to investigate and compare the inhibitory effects of the two fungicides on the growth, sporulation and secondary infection of M. oryzae. RESULTS: Magnaporthe oryzae mycelium growth was inhibited at low concentrations of azoxystrobin and relatively high concentrations of tricyclazole, while sporulation was more sensitive to both fungicides and was affected at similarly low doses. Furthermore, infection efficiency of conidia obtained from mycelia exposed to tricyclazole was affected to a higher extent than for conidia produced on azoxystrobin‐amended media, even though germination of such conidia was reduced after azoxystrobin treatment. CONCLUSIONS: This study presents for the first time detailed azoxystrobin and tricyclazole growth–response curves for M. oryzae mycelium growth and sporulation. Furthermore, high efficacy of tricyclazole towards inhibition of sporulation and secondary infection indicates an additional possible mode of action of this fungicide that is different from inhibition of melanin biosynthesis. Copyright © 2012 Society of Chemical Industry     

Inhibition of Botrytis cinerea growth and suppression of botrytis bunch rot in grapes using chitosan

Chitosan inhibited growth of Botrytis cinerea in liquid culture and suppressed grey mould on detached grapevine leaves and bunch rot in commercial winegrapes. Germination of B. cinerea was completely inhibited in malt extract broth containing chitosan at concentrations greater than 0·125 g L^?1. However, treated conidia were able to infect detached Chardonnay leaves and pathogenicity was not affected, even after incubation for 24 h in chitosan at 10 g L^?1. When added after conidial germination, chitosan inhibited B. cinerea growth and induced morphological changes suggestive of possible curative activity. The effective concentration of chitosan that reduced mycelial growth by 50% (EC₅₀) was 0·06 g L^?1. As a foliar treatment, chitosan protected detached Chardonnay leaves against B. cinerea and reduced lesion diameter by up to 85% compared with untreated controls. Peroxidase and phenylalanine ammonia‐lyase activities were also induced in treated leaves. In vineyard studies, Chardonnay winegrapes exhibited 7·4% botrytis bunch rot severity at harvest in 2007 after treatment with an integrated programme that included chitosan sprays from bunch closure until 2 weeks preharvest, compared with 15·5% in untreated controls and 5·9% with fungicide treatment. In the following season, botrytis bunch rot severity was 44% in untreated Chardonnay at harvest and the integrated programme (21%) was less effective than fungicides (13·8%). However, in Sauvignon blanc winegrapes, the integrated and the fungicide programme each reduced botrytis bunch rot severity to 4% and were significantly different from the untreated control (11·5%). This study provides evidence that suppression of botrytis in winegrapes by chitosan involves direct and indirect modes of action.     

寒地水稻秆腐菌核病菌的生物学研究

研究结果表明,水稻秆腐菌核病菌可利用多种碳源和氮源,碳源以可溶性淀粉最好,氮源以酒石酸铵最好,混合碳源(蔗糖+鼠李糖)对病菌菌丝生长有促进作用,比单独蔗糖和鼠李糖生长的好;病菌能在多种天然培养基上生长并形成菌核;菌丝在碳氮比为60∶1的培养基上生长最好;生长物质VB₆+肌醇、VB₆+谷氨酰胺等11个组合对菌丝生长有促进作用;微量元素中亚铁、锰和硼对菌丝生长有抑制作用;液体培养中以Czapek培养基生长最好,但不利于菌核的形成;菌核在15～35℃均可萌发,最适温度为30℃;菌核在0.05%葡萄糖溶液、蒸馏水、水琼脂上均可萌发,以0.05%葡萄糖溶液萌发最好。     

地肤子提取物对苹果树腐烂病菌的抑制作用及对其菌体结构和功能的影响

为开发新型安全高效的植物源苹果树腐烂病防治剂,采用菌丝生长速率法测定中草药地肤子乙醇提取物及各层析流分L1～L9对苹果树腐烂病菌Valsa mali的抑制作用,同时通过室内常规分析方法观测流分L7对该菌菌丝形态结构、细胞膜通透性及物质吸收和代谢的影响。结果表明,地肤子乙醇粗提物浓度为2 mg/mL时对苹果树腐烂病菌具有较强的离体抑制活性,处理后96 h的抑菌率达到92.16%。分别用石油醚、氯仿、正丁醇萃取醇提物,石油醚萃取物对苹果树腐烂病菌的抑制效果最为明显,其EC_(50)为0.07 mg/mL;在石油醚萃取物通过硅胶柱层析分离得到的9种流分中,流分L7对苹果树腐烂病菌的抑制作用显著,离体抑菌率高达96.73%;且在流分L7处理下,病菌菌丝体出现肿胀、膨大或畸形等现象,细胞膜通透性增大,可溶性蛋白、还原糖以及丙酮酸的含量随流分L7浓度的升高而持续降低,当浓度为100μg/mL时,菌丝体可溶性蛋白、还原糖及丙酮酸的含量分别降低70.78%、71.74%和78.68%。表明在离体培养条件下地肤子乙醇提取物能明显抑制苹果树腐烂病菌的生长,并对菌丝体细胞膜结构和功能稳定性产生显著影响,具有进一步开发为新型植物源苹果树腐烂病防治剂的潜力。     

四种拟除虫菊酯类杀虫剂对枸杞蚜虫的毒力及对三磷酸腺苷酶和谷胱甘肽S-转移酶活性的影响

为寻找防治枸杞蚜虫的适用药剂,采用玻璃管药膜法,测定了4种拟除虫菊酯类杀虫剂对枸杞蚜虫的毒力及对其三磷酸腺苷酶(ATPase)和谷胱甘肽S-转移酶(GSTs)活性的影响。结果表明:枸杞蚜虫对联苯菊酯最敏感,LC50值为4.34 mg/L;氯菊酯、高效氯氰菊酯和甲氰菊酯的LC50值分别为17.08、40.50和184.84 mg/L。4种杀虫剂对枸杞蚜虫两种ATPase活性均有抑制作用,药剂浓度为1×10-4mol/L时,4种药剂对Na+-K+-ATPase活性的抑制率均高于对Ca2+-M g2+-ATPase的抑制率,其中对Na+-K+-ATPase活性的抑制率从高到低依次为:联苯菊酯高效氯氰菊酯氯菊酯甲氰菊酯,而对Ca2+-M g2+-ATPase的抑制率则是联苯菊酯最高(46.41%),高效氯氰菊酯最低(33.04%)。4种药剂对枸杞蚜虫GSTs活性的影响差异较大:联苯菊酯在低浓度时对GSTs具有诱导作用,高浓度时则表现为一定的抑制作用;不同浓度高效氯氰菊酯和氯菊酯对GSTs活性均表现为抑制作用,抑制率最高达85.02%;而甲氰菊酯处理后GSTs的活性则升高了193.07%~249.96%。     

禾谷镰孢菌对氟唑菌酰羟胺敏感性基线的建立及药剂田间防效

为明确琥珀酸脱氢酶抑制剂类新型吡啶酰胺杀菌剂氟唑菌酰羟胺在中国小麦赤霉病防治中的应用潜力,分别采用菌丝生长速率法和孢子萌发法,测定了氟唑菌酰羟胺对湖北省6个地区106株禾谷镰孢菌的室内毒力、田间防效及其与多菌灵和氰烯菌酯的交互抗性。结果显示:氟唑菌酰羟胺对106株禾谷镰孢菌菌丝生长的EC₅₀值为 (0.018 0 ± 0.209 0) mg/L,平均值为 (0.072 8 ± 0.025 9) mg/L;对分生孢子萌发的EC₅₀值为 (0.052 7 ± 0.473 2) mg/L,平均值为 (0.176 0± 0.059 6) mg/mL;且其EC₅₀值频率分布均呈单峰曲线,因此可分别将菌丝生长和孢子萌发的平均EC₅₀值作为禾谷镰孢菌对氟唑菌酰羟胺的敏感性基线。初步的交互抗性测定结果表明,抗多菌灵或氰烯菌酯的菌株对氟唑菌酰羟胺均未表现出抗性。田间试验显示,氟唑菌酰羟胺有效剂量200 g/hm2处理的防效 (超过90.0%) 显著高于对照药剂氰烯菌酯600 g/hm2的防效 (78.0%),与空白对照相比增产效果在127%~135%之间。经氟唑菌酰羟胺处理后,小麦籽粒中由禾谷镰孢菌产生的毒素脱氧雪腐镰刀菌烯醇 (DON) 的含量比空白对照降低了55.09%。研究表明,氟唑菌酰羟胺对禾谷镰孢菌呈现出较高的室内活性且田间防效优越,同时还能降低小麦籽粒中DON毒素的含量及提高小麦产量,因此可作为生产中防治小麦赤霉病的替代或后备药剂,同时也可考虑用作为禾谷镰孢菌对多菌灵抗性治理的替代药剂。     

Relationship between chemical structure and biological activity of triazole fungicides against Botrytis cinerea

The inhibitory activity of commercial and experimental triazole fungicides on the target enzyme, sterol 14α-demethylase (P450_14DM), was studied in a cell-free sterol synthesis assay of Botrytis cinerea Pers. ex Fr. In order to assess structure-activity relationships, the inhibitory activities of the compounds on radial growth of the fungus were tested as well. The EC₅₀ values (concentrations of fungicide inhibiting radial growth of B. cinerea on PDA by 50%) of all triazoles tested ranged between 10^?8 and 10^?5 m. IC₅₀ values (concentrations of fungicide inhibiting incorporation of [2-¹⁴C]mevalonate into C4-desmethyl sterols by 50%) generally ranged between 10^?9 and 10^?7 M and correlated with inhibition of radial mycelial growth. However, differences in IC₅₀ values did not reflect quantitatively the observed differences in EC₅₀ values, since the ratio between EC₅₀ and IC₅₀ increased with decreasing fungitoxicity. For a limited number of compounds the correlation between intrinsic inhibitory activity and fungitoxicity was low. Both in-vitro tests were used to investigate structure-activity relationships for stereoisomers of cyproconazole, SSF-109 and tebucona-zole. Fungitoxicity and the potency to inhibit cell-free C4-desmethyl sterol synthesis correlated for all stereoisomers tested. Mixtures of isomers of tebucona-zole or cyproconazole were slightly less active than the most potent isomer. The high activity of several commercial triazoles in both experiments implies that poor field performance of triazole fungicides against B. cinerea is due neither to insensitivity of the P450_14DM nor to low in-vitro sensitivity of the fungus.     

Mechanism of action and fate of the fungicide chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) in biological systems: I. Reactions with cells and subcellular components of Saccharomyces pastorianus

The mechanism and sequence of reaction of chlorothalonil in cells of Saccharomyces pastorianus was investigated by the use of either ¹⁴C-labeled fungicide or Na₂³⁵SO₄-labeled cells. The initial uptake of fungicide resulted in rapid formation of substituted chlorothalonil-reduced glutathione (GSH) derivatives. Chlorothalonil reacted with proteins during derivative formation but decreased cell viability did not occur until all the GSH was reacted and inhibition of specific NAD thiol-dependent glycolytic and respiratory enzymes occurred. It is postulated that enzyme activity and cell viability are controlled either directly or indirectly by the concentration of GSH. The loss of GSH through derivative formation with chlorothalonil and the lack of regeneration of the reduced thiol could be an important feature in the sequence of toxicity of the fungicide. The general mechanism of action of chlorothalonil resembles the trichloromethyl sulfenyl fungicides in that treated cells accumulate large concentrations of fungicide; reactions involve both low and high molecular-weight thiols, with the formation of glutathione-fungicide derivatives; and toxicity resides ultimately with the inhibition of thiol-dependent enzymes.