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.     

A comparison of the potency of some fungicides as inhibitors of sterol 14-demethylation

An enzymatic assay system has been developed to measure the relative potency of fungicides such as triadimefon, triarimol, triforine, and buthiobate as inhibitors of sterol 14-demethylation. The enzyme preparation used is the 8000g supernatant derived from a homogenate of an aerobically adapted, anaerobically grown, high sterol strain of Saccharomyces cerevisiae. After incubation of the enzyme with [2-¹⁴C]mevalonic acid and the fungicide the ratio, radioactivity in 4,4-dimethyl sterols/radioactivity in 4-demethyl sterols is determined. The higher this ratio is, the more efficient is the fungicide as an inhibitor of fungal sterol 14-demethylation. The ratio has been determined for a number of commercial fungicides and two series of triazole compounds. A similar assay system based on the 10,000g supernatant from a rat liver homogenate was also tested but gave an inaccurate assessment of the relative potency of fungicides as inhibitors of fungal sterol 14-demethylation.     

微生物降解三唑类杀菌剂研究进展

三唑类杀菌剂因具有高效、低毒的特性已在中国得到广泛使用,但同时也因其残留期较长、易污染土壤而备受关注。微生物降解被认为是修复被污染土壤的有效措施。文章从降解三唑类杀菌剂的微生物种类、降解机理及影响微生物降解的因素等方面进行了综述,同时指出,目前有关微生物降解三唑类杀菌剂的研究多数仅限于降解菌的筛选及影响降解的因素分析等方面,对于三唑类杀菌剂的微生物代谢途径、降解过程中起关键作用的酶和基因,以及对手性三唑类杀菌剂的对映体选择性降解机制等机理方面的研究仍较少,相关研究有待进一步加强。     

Update on mechanisms of azole resistance in Mycosphaerella graminicola and implications for future control

This review summarises recent investigations into the molecular mechanisms responsible for the decline in sensitivity to azole (imidazole and triazole) fungicides in European populations of the Septoria leaf blotch pathogen, Mycosphaerella graminicola. The complex recent evolution of the azole target sterol 14α‐demethylase (MgCYP51) enzyme in response to selection by the sequential introduction of progressively more effective azoles is described, and the contribution of individual MgCYP51 amino acid alterations and their combinations to azole resistance phenotypes and intrinsic enzyme activity is discussed. In addition, the recent identification of mechanisms independent of changes in MgCYP51 structure correlated with novel azole cross‐resistant phenotypes suggests that the further evolution of M. graminicola under continued selection by azole fungicides could involve multiple mechanisms. The prospects for azole fungicides in controlling European M. graminicola populations in the future are discussed in the context of these new findings. Copyright © 2012 Society of Chemical Industry     

戊唑醇和三唑醇种衣剂对小麦幼苗生长发育的影响

三唑醇、戊唑醇用于小麦种子处理,当包衣浓度分别高于０．３ｇ ａｉ／ｋｇ种子和０．６ｇ ａｉ／ｋｇ种子时,对小麦的发芽及出苗都有较为严重的影响;对小麦的生长发育有明显的影响主要表现为株高降低,根长、胚芽鞘高度、第１片真叶长减小,而分根数增加,第１片真叶宽增大;除包衣用量大于１．２ｇ ａｉ／ｋｇ种子百株鲜重明显降低外,其余与空白对照无明显差异。相同用量下,戊唑醇对小麦幼苗生长发育的抑制作用要比三唑醇的     

含两个手性中心的三唑类杀菌剂色谱分离研究进展

三唑类杀菌剂是目前世界上广泛应用的杀菌剂,而绝大多数三唑类杀菌剂属于手性农药,具有1个或2个手性中心,存在2个或4个对映异构体。本文在近年有关三唑类杀菌剂研究成果的基础上,综述了采用高效液相色谱、超临界流体色谱、毛细管电泳、气相色谱和合相色谱法等方法对含两个手性中心的11种三唑类杀菌剂 (氟环唑、多效唑、三唑醇、联苯三唑醇、丙环唑、苯醚甲环唑、叶菌唑、苄氯三唑醇、环唑醇、糠菌唑和乙环唑) 进行分离测定的研究进展,可为多个手性中心三唑类化合物的分离、分析和深入研究提供参考。     

不同生育阶段黄瓜菌核病菌对几种三唑类杀菌剂的敏感性

采用菌丝生长速率法测定了6种三唑类杀菌剂和4种其他杀菌剂抑制黄瓜菌核病菌Sclerotinia sclerotiorum(Lib.)de Bary菌丝生长的毒力,同时测定了其对菌核形成及萌发的抑制作用。结果表明,甲基硫菌灵抑制菌丝生长的毒力最低,EC50值为65.75 μg/mL;己唑醇和戊唑醇的抑制活性较高, EC50值分别为0.09和0.16 μg/mL,毒力分别是甲基硫菌灵的763.7和423.4倍;其次是丙环唑、氟硅唑、腈菌唑,毒力是甲基硫菌灵的137.3~286.6倍;再次为三唑酮,毒力仅为甲 基硫菌灵的20.6倍;腐霉利、菌毒清抑制菌丝生长的毒力较低,EC50值均大于10 μg/mL。在EC50值 浓度下,所有供试药剂对菌核形成及其数量均没有影响,但三唑类杀菌剂对菌核单重影响较大,各处理所形成的菌核单重降低率均在70%以上;而当药剂浓度在10 μg/mL以下时,己唑醇等三唑类杀菌剂对菌核萌发均没有影响。     

Characterization of CbCyp51 from field isolates of Cercospora beticola

The hemibiotrophic fungus Cercospora beticola causes leaf spot of sugar beet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides. However, reduced sensitivity to DMIs has been reported recently in the Red River Valley sugar beet-growing region of North Dakota and Minnesota. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14α-demethylase in C. beticola. CbCyp51 is a 1,632-bp intron-free gene with obvious homology to other fungal Cyp51 genes and is present as a single copy in the C. beticola genome. Five nucleotide haplotypes were identified which encoded three amino acid sequences. Protein variant 1 composed 79% of the sequenced isolates, followed by protein variant 2 that composed 18% of the sequences and a single isolate representative of protein variant 3. Because resistance to DMIs can be related to polymorphism in promoter or coding sequences, sequence diversity was assessed by sequencing >2,440 nucleotides encompassing CbCyp51 coding and flanking regions from isolates with varying EC(50) values (effective concentration to reduce growth by 50%) to DMI fungicides. However, no mutations or haplotypes were associated with DMI resistance or sensitivity. No evidence for alternative splicing or differential methylation of CbCyp51 was found that might explain reduced sensitivity to DMIs. However, CbCyp51 was overexpressed in isolates with high EC(50) values compared with isolates with low EC(50) values. After exposure to tetraconazole, isolates with high EC(50) values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 μg ml(-1).     

Strobilurin fungicides induce changes in photosynthetic gas exchange that do not improve water use efficiency of plants grown under conditions of water stress

The effects of five strobilurin (beta-methoxyacrylate) fungicides and one triazole fungicide on the physiological parameters of well-watered or water-stressed wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and soya (Glycine max Merr.) plants were compared. Water use efficiency (WUE) (the ratio of rate of transpiration, E, to net rate of photosynthesis, A(n)) of well-watered wheat plants was improved slightly by strobilurin fungicides, but was reduced in water-stressed plants, so there is limited scope for using strobilurins to improve the water status of crops grown under conditions of drought. The different strobilurin fungicides had similar effects on plant physiology but differed in persistence and potency. When applied to whole plants using a spray gun, they reduced the conductance of water through the epidermis (stomatal and cuticular transpiration), g(sw), of leaves. Concomitantly, leaves of treated plants had a lower rate of transpiration, E, a lower intercellular carbon dioxide concentration, c(i), and a lower net rate of photosynthesis, A(n), compared with leaves of control plants or plants treated with the triazole. The mechanism for the photosynthetic effects is not known, but it is hypothesised that they are caused either by strobilurin fungicides acting directly on ATP production in guard cell mitochondria or by stomata responding to strobilurin-induced changes in mesophyll photosynthesis. The latter may be important since, for leaves of soya plants, the chlorophyll fluorescence parameter F(v)/F(m) (an indication of the potential quantum efficiency of PSII photochemistry) was reduced by strobilurin fungicides. It is likely that the response of stomata to strobilurin fungicides is complex, and further research is required to elucidate the different biochemical pathways involved.     

杀菌剂对水稻旱育秧立枯病的控制作用及其对秧苗的生理效应

为了筛选防治水稻旱育秧立枯病的有效药剂,通过秧盘试验和室内致病菌离体毒力测定,结合考察药剂对秧苗生长的影响,对保护性杀菌剂代森锰锌和福美双以及内吸治疗性杀菌剂稻瘟灵、 FDA1 霉灵等进行了系统评价。结果表明, FDA1 霉灵、甲基硫菌灵和稻瘟灵对致病菌离体毒力虽然不高,但由于 FDA1 霉灵和甲基硫菌灵对水稻秧苗生长均有促进作用,稻瘟灵能显著提高秧苗抗低温胁迫能力,这3种药剂对水稻立枯病的防治效果较好,防效达65%~81%;而离体毒力虽然高,但对秧苗生长不利的三唑酮、丙环唑和烯唑醇等三唑类药剂效果稍差,防效为51%~75%。根据试验结果提出稻瘟灵是防治该类病害的一种较理想的单用或混用药剂;针对水稻立枯病的发生受环境影响大、病原种类多等特点,指出在研究防治药剂时应采用活体筛选为主、离体筛选为辅的筛选方法,而且提倡不同杀菌谱品种的混用。     

Fungicide modes of action and resistance in downy mildews

Among oomycetes, Plasmopara viticola on grape and Phytophthora infestans on potato are agronomically the most important pathogens requiring control measures to avoid crop losses. Several chemical classes of fungicides are available with different properties in systemicity, specificity, duration of activity and risk of resistance. The major site-specific fungicides are the Quinone outside inhibitors (QoIs; e.g. azoxystrobin), phenylamides (e.g. mefenoxam), carboxylic acid amides (CAAs; e.g. dimethomorph, mandipropamid) and cyano-acetamide oximes (cymoxanil). In addition, multi-site fungicides such as mancozeb, folpet, chlorothalonil and copper formulations are important for disease control especially in mixtures or in alternation with site-specific fungicides. QoIs inhibit mitochondrial respiration, phenylamides the polymerization of r-RNA, whereas the mode of action of the other two site-specific classes is unknown but not multi-site. The use of site-specific fungicides has in many cases selected for resistant pathogen populations. QoIs are known to follow maternal, largely monogenic inheritance of resistance; they bear a high resistance risk for many but not all oomycetes. For phenylamides, inheritance of resistance is based on nuclear, probably monogenic mechanisms involving one or two semi-dominant genes; resistance risk is high for all oomycetes. The molecular mechanism of resistance to QoIs is mostly based on the G143A mutation in the cytochrome b gene; for phenylamides it is largely unknown. Resistance risk for CAA fungicides is considered as low to moderate depending on the pathogen species. Resistance to CAAs is controlled by two nuclear, recessive genes; the molecular mechanism is unknown. For QoIs and CAAs, resistance in field populations of P. viticola may gradually decline when applications are stopped.     

Effects of triazole inhibitors of sterol biosynthesis on the free and esterified sterol composition of celery cell suspension cultures

Treatment of celery cell suspension cultures with paclobutrazol and three other triazoles resulted in decreased growth and an accumulation of 14α-methylsterols in both the free sterol and the steryl ester pools, thus indicating that the triazoles were inhibiting the action of the plant obtusifoliol 14α-demethylase system. Obtusifoliol, 14α-methylcampesta-8,24(24¹)-dien-3β-ol and 14α-methylcampest-8-en-3β-ol were the main 14α-methylsterols to increase in the free sterol pool. In the presence of the triazoles the steryl esters became virtually depleted of sitosterol, campesterol, stigmasterol and isofucosterol, which were replaced by 14α-methylsterols with obtusifoliol becoming the dominant esterified sterol. Treatment of the celery culture with a fourth triazole which did not have a noticeable effect on growth and caused negligible accumulation of 14α-methylsterols in the free sterol pool nevertheless produced some enrichment of the steryl esters in obtusifoliol. The results indicate that, following triazole treatment, a rapid esterification of the accumulating 14α-methylsterol intermediates occurs before they start to build up in significant amount in the free sterol pool.     

Baseline sensitivity of Monilia yunnanensis to the DMI fungicides tebuconazole and triadimefon

Monilia yunnanensis was recently identified as a new species causing brown rot of peach in China. Sterol 14α-demethylase inhibitors (DMIs) continue to be important in the management of brown rot of Monilinia spp. worldwide. Tebuconazole and triadimefon are two kinds of DMI fungicides that may be used for brown rot control in China. To establish the baseline sensitivity of M. yunnanensis to these two DMI fungicides, 203 M. yunnanensis single spore isolates were collected. Measurements of sensitivity to the two fungicides were based on inhibition of mycelial growth. For both fungicides, the sensitivity distribution was a unimodal curve, with an EC₅₀ range (the effective concentration to inhibit mycelial growth by 50 %) of 0.0001–0.0644 μg/ml for tebuconazole and 0.2311–1.7477 μg/ml for triadimefon. The M. yunnanensis isolates were obtained from orchards where DMI fungicides have not been used for peach brown rot control, thus the fungicide sensitivity distribution established in this study can be considered as the baseline for monitoring the resistance development in M. yunnanensis once the DMI fungicides are used to control peach brown rot.     

Molecular basis for the antimycotic and antibacterial activity of N-substituted imidazoles and triazoles: The inhibition of isoprenoid biosynthesis

The antimycotic N-substituted imidazoles and triazoles, such as imazalil, ketoconazole and itraconazole, interfere selectively at low concentrations (≥0.01nm) with the 14α-demethylase system (which is dependent on cytochrome P-450) of fungal cells, for example, Candida albicans and Penicillium italicum. This results in a decreased availability of ergosterol and the accumulation of 14α-methyl-sterols such as lanosterol. Cholesterol synthesis in a subcellular fraction of rat liver, in intact fibroblasts, and in vivo in rat liver, was much less sensitive, for example, to ketoconazole. The imidazole derivatives imazalil, miconazole, ketoconazole and parconazole, and the triazole derivatives propiconazole, terconazole and itraconazole affect the cytochrome P-450 species of microsomal fractions from Saccharomyces cerevisiae and rat liver. Cytochrome P-450 of rat-liver microsomes was much less sensitive to these azole derivatives, in parallel with the lower sensitivity of cholesterol synthesis. Using unilamellar vesicles composed of phosphatidylcholine, phosphatidyl-ethanolamine and diphosphatidylcholine, multilamellar vesicles of dipalmitoylphos-phatidylcholine, and intact S. cerevisiae, it was shown that the substitution of ergosterol by lanosterol leads to functional changes in the membranes. It is speculated that the selective interaction of the azole derivatives with the yeast microsomal cytochrome P-450 leads to the accumulation of 14a-methyl-sterols and results in changes in the permeability of the membranes and leakages. The observed inhibition of growth may have its origin in these changes. Miconazole, ketoconazole and deacylated ketoconazole (R-39519) also affect the growth of Staphylococcus aureus, miconazole being 12.5 and 14 times, respectively, more active than R-39519 and ketoconazole. The greater antibacterial activity of miconazole coincides with its greater inhibition of the biosynthesis of C-55 isoprenoid alcohol and vitamin K. The phosphorylated derivative of C-55 isoprenoid alcohol has functional importance in the biosynthesis of bacterial cell wall and membrane polymers, and the menaquinone vitamin K plays a role in the electron transport of Gram-positive bacteria. The reduced synthesis of these vital compounds may contribute to the antibacterial activity of miconazole.     

Reactivity of a critical sulfhydryl group of the acetylcholinesterase from aphids (Myzus persicae)

The acetylcholinesterase of aphids contains a cysteine residue that reacts covalently with thiol reagents, blocking catalysis of substrate hydrolysis. The SH residue is not at the active site. The enzyme inactivated by 5,5-dithio-bis-2-nitrobenzoic acid could not be reactivated by an excess of sulfhydryl, because the native enzyme is also inactivated by such a treatment. A third peculiarity of this enzyme is its instability at higher pHs. The main difference between the acetyl-cholinesterase from aphids and that of other sources may be a relatively loose tertiary structure of the former that mainly depends on a S-S bridge. The possibility of “specific aphidical thiol reagents” due to reaction with this unusual cholinesterase is discussed. A slight adaptation of the Ellman method enables its application for the routine assay of the aphid enzyme.     

Effects of triazole fungicides on sterol biosynthesis during spore germination of Botrytis cinerea,Venturia inaequalis and Puccinia graminis f. sp. tritici

With three plant pathogens,Botrytis cinerea, Venturia inaequalis and Puccinia graminis f. sp.tritici, the time course of sterol biosynthesis during spore germination was examined by labeling experiments along with the question whether this pathway could be inhibited by triazole fungicides. Conidia ofB. cinerea andV. inaequalis are able to synthesize sterols immediately after the beginning of the germination process when the germ tubes have not yet emerged. On the contrary uredospores ofP. graminis start sterol biosynthesis after 6 to 8 h germination time almost at the end of the germ tube phase, indicating that sterol reserves of the spores are likely to be used for the germ tube growth.The sterol C-14 demethylation appeared to be the rate limiting step within the sterol biosynthetic pathway: the half life of 24-methylenedihydrolanosterol was less than 1 h forB. cinerea. It was more than 1 h forV. inaequalis and 3 h forP. graminis. Independent of these differences in the time course of sterol biosynthesis and in the C-14 demethylation rate, the synthesis of sterols in germinating spores was strongly inhibited by triazole fungicides in all three pathogens examined. In contrast toP. graminis, this inhibition could be demonstrated withB. cinerea andV. inaequalis even in ungerminated conidia, indicating that the fungicides were rapidly taken up and reached their target within 1 or 2 h. These results are discussed along with the question whether spore germination can be used as a bioassay for the estimation of sensitivities of triazole fungicides.     

中国小麦纹枯病化学防治研究进展

随着秸秆还田等耕作栽培措施的推广,中国小麦纹枯病发生日趋严重,对小麦的高产、稳产造成了很大威胁。由于缺乏免疫及高抗病性小麦品种,生产中对纹枯病一直采用播期拌种及春季喷雾相结合的化学防治方法。文章总结了当前中国小麦纹枯病的发生现状及主要病原;评述了三唑类药剂对纹枯病菌的毒力及对纹枯病的防治效果,介绍了生产中小麦纹枯病菌对三唑类药剂的抗药性现状及机理,分析了三唑类药剂对小麦的安全性;同时阐述了井冈霉素、甲基立枯磷及其他种类药剂在小麦纹枯病综合防治中的应用;指出小麦纹枯病化学防治的发展方向应是将生防菌剂同化学药剂相结合,实现生物防治与化学防治的协同应用。     

Genetic control of the response of Erysiphe graminis f.sp. hordei to ethirimol and triadimenol

A study was conducted on the genetic control of the response of Erysiphe graminis f.sp. hordei , the causal agent of barley powdery mildew, to two fungicides: the hydroxypyrimidine ethirimol, and the triazole, sterol demethylation inhibitor triadimenol. In tests of responses to both fungicides, sets of progeny of various crosses were classified by principal components analysis into discrete resistant and sensitive classes. A single allele controlled the response to ethirimol of the resistant isolate DH14 in crosses with the sensitive isolates CC52 and CC138. The ethirimol-resistance alleles of DH14 and another resistant isolate. CC107, are at the same locus or are closely linked. Alleles at single loci controlled resistance and sensitivity to triadimenol in crosses of DH14 (sensitive) with CC107 (moderately resistant) and CC138 (highly resistant). There was no evidence for polygenic control of response to either fungicide. The ethirimol response locus and the two putative triadimenol response loci are designated Eth1 and Tdl1 and Tdl2 , respectively. There was no evidence for linkage of Eth1 and Tdl2 in the cross CC138 × DH14, in which responses to both fungicides segregated.     

New tools for studying epidemiology and resistance of grape powdery mildew to DMI fungicides

Using a Random Amplified Polymorphic DNA (RAPD) assay, we investigated the genetic polymorphism existing among 62 European isolates of the grape powdery mildew fungus (Uncinula necator [Schw.] Burr.). Isolates overwintering as mycelium in buds were genetically distinct from isolates overwintering as ascospores, suggesting the existence of two genetically isolated powdery mildew populations, and consequently of two independent sources of inoculum in the vineyard. Isolates resistant to fungicides inhibiting sterol 14α-demethylation (DMIs) were found in both populations, suggesting that resistance to DMIs may arise independently in the two powdery mildew populations. A PCR assay targeting the gene encoding U. necator 14α-demethylase has been developed which will permit an early, specific detection of U. necator infections, and may be useful for spraying programmes. ©1997 SCI     

Control of take-all of wheat in the field with benzimidazole and triazole fungicides applied at seeding

Control of take-all ( Gaeumannomyces graminis var. tritici ) of wheat, with fungicides applied at seeding, was investigated at three naturally infested field sites. Fungicides were applied with seed either as granules or as pellets at rates ranging from 1-5 to 140 kg a.i./ha. Treatment with benzimidazole and triazole fungicides resulted in lower disease severity on seminal roots, up to 100% fewer deadheads and more than 200% greater yields.