病原菌对Strobilurin类杀菌剂抗药性机理的研究进展

结合strobilurin类杀菌剂作用机制的特点,通过室内和田间研究的结果总结了病原菌对该类杀菌剂产生抗药性的主导机制。Strobilurin类杀菌剂主要结合于病原菌细胞色素bc1复合物中细胞色素b部分的Qo位点,而细胞色素b由突变率很高的线粒体基因组编码,所以病原菌很容易因该基因的突变而导致抗药性发生。至今已至少有14种寄主植物上的25种病原菌在田间表现出了对strobilurin类杀菌剂的抗药性。抗药性产生的主要机理是病原菌细胞色素b的氨基酸残基发生了单点取代。田间抗性菌株的主要取代位点是G143A、F129L和G143R。一些非靶标生物细胞色素b的143位为丙氨酸,这可能是strobilurin类杀菌剂对其表现安全性的原因之一。由于锈病病菌Puccinia spp.中内含子剪切位点恰好出现在编码G143的GGT之后,G143A的突变会导致突变菌株的死亡,因而它们在田间不会表现出抗药性。此外,Qo抑制剂阻断电子传递后会导致胞内活性氧(ROS)的增加,而ROS可以诱导细胞交替氧化酶(AOX)的表达,进而启动线粒体的旁路氧化途径,使病原菌表现出抗药性。因此,病原菌氨基酸残基的单点取代和交替氧化途径是导致病原菌对strobilurin杀菌剂产生抗药性的主导机制。     

新型杀菌化合物靶标识别及其靶向候选药剂设计概述

新型杀菌剂的创制在农业可持续发展和克服抗药性中具有重要作用。由于杀菌剂作用机制或靶标是新杀菌剂分子设计合成和创制开发的重要基础,因此作用机制或靶标研究的滞后会阻碍新先导化合物的发现或新品种的创制。鉴于此,本文对目前杀菌剂作用靶标的识别及靶向杀菌剂的分子设计进行了概述。首先阐述了基于转录组学、蛋白质组学的研究和药物亲和层析技术在靶标识别中的应用;其次概述了多学科交叉的方法在分子靶标验证中的应用;最后以strobilurin A为先导的Qo位点抑制剂、以肉桂酸为先导的卵菌抑制剂、以萎锈灵为先导的琥珀酸脱氢酶和植物免疫激活剂等不同类型的靶向杀菌剂为例进行了举例说明。     

复合物Ⅱ抑制剂的作用机制和研究进展

复合物II是线粒体电子传递链上重要功能复合物,已经成为农用杀菌剂方面的关键靶标。该类杀菌剂在植物病原真菌保护中发挥着重要的作用。本文在综述复合物II结构和泛醌结合位点作用机制的基础上,总结了复合物II抑制剂的类型和结构特征,介绍了最新的研究进展,旨在为开发新的复合物II抑制剂提供思路。     

琥珀酸脱氢酶抑制剂类杀菌剂抗性研究进展

作用于琥珀酸脱氢酶复合体的新型杀菌剂-琥珀酸脱氢酶抑制剂 (succinate dehydrogenase inhibitors, SDHIs) 已逐步成为继Qo位点呼吸抑制剂类 (QoIs) 和麦角甾醇生物合成抑制剂类(EBIs)杀菌剂之后的世界第3大类杀菌剂。近年来,SDHIs杀菌剂的市场占有份额逐年增加,新品种不断涌现,在植物病害化学防治中发挥着重要作用。然而,由于该类杀菌剂作用位点单一,抗药性已成为制约该类杀菌剂创制发展与科学应用的重要科学问题。本综述归纳了琥珀酸脱氢酶抑制剂类杀菌剂的开发、品种、抗性发生发展、抗性分子机制与应用现状,并结合作者研究团队的最新研究成果对其靶标生物学及应用技术研究进行了总结,以期为更高活性的SDHIs杀菌剂创制和应用提供参考。     

致病疫霉诱导的马铃薯酵母双杂交文库构建及无毒蛋白PiAVR3b寄主靶标筛选

致病疫霉是引起番茄、马铃薯晚疫病的植物病原卵菌,其分泌的效应蛋白在抑制寄主免疫方面发挥重要功能。鉴定效应蛋白的寄主靶标对研究植物与病原菌互作具有重要意义。本研究构建了高效马铃薯酵母双杂交cDNA文库,并通过筛选致病疫霉无毒蛋白PiAVR3b的互作蛋白来评价构建文库质量。分别提取接种致病疫霉后24 h和48 h的马铃薯栽培种‘合作88’叶片RNA,等量混合后构建cDNA文库。次级文库容量为1.6×10~7 cfu/mL,重组率为100%,插入片段平均在1 000 bp以上。以PiAVR3b为诱饵,通过酵母双杂交筛选初步获得10个候选靶标蛋白。经点对点验证,进一步确定了4个与PiAVR3b互作的靶标蛋白,分别是马铃薯MYB-like蛋白、线粒体外膜孔蛋白、碳分解代谢抑制蛋白、肽基脯氨酰异构酶。本研究构建的酵母双杂交文库为致病疫霉效应蛋白靶标筛选及植物与病原菌互作研究奠定了基础。     

植物病原菌对解偶联剂的抗性机制研究进展

以氟啶胺为代表的解偶联剂具有低毒、广谱和高效的特点，对病原真菌、卵菌和细菌均表现出良好的抑菌活性。然而随着杀菌剂频繁而大量的使用，有害生物发展出越来越严重的抗药性。开展病原菌对杀菌剂的抗性机制研究，能够有效预防或治理病原菌抗药性。病原菌对杀菌剂的抗性机制解析方法通常以杀菌剂的靶标蛋白为线索展开，但由于氟啶胺这类杀菌剂在病原菌体内可能不是通过与靶标蛋白结合而产生的抑菌作用，使得通过寻找抗性突变体中发生变化的氨基酸位点，进而进行抗性机制解析的方法难以奏效。本综述以氟啶胺和我国自主创制的杀菌剂双苯菌胺为研究对象，对其作用机制及病原菌对其抗性机制的研究进展进行归纳总结，旨在为这类杀菌剂的田间科学使用提供参考，同时可为病原菌多药抗性机制的解析提供借鉴，丰富杀菌剂抗性研究体系，并能够在实践中为病原菌的抗性治理提供依据。     

戊唑醇的作用特点及其应用概况

甾醇类化合物是生物维持生命及生长发育不可缺少的物质。在真菌和细菌中 ,主要是合成麦角甾醇 ,它是构成细胞膜的重要成分 ,对细胞膜的渗透性起重要作用。麦角甾醇的生物合成已成为新型杀菌剂的重要靶标之一 ,其抑制剂由于活性高、不易产生抗药性等特点 ,而在应用初期受到普遍重视。从化学结构来看 ,目前已开发出具有实用价值的品种主要是三唑类、咪唑类、吡啶类、吗啉类和哌嗪类等化合物。戊唑醇 (ｔｅｂｕｃｏｎａｚｏｌｅ)是一种羟乙基三唑衍生物 ,属三唑类内吸杀菌剂。毒性分类属低毒杀菌剂。该化合物由德国拜耳公司于１９８６年最先开…     

6种杀菌剂对梨火疫病菌的室内毒力测定和混配试验

梨火疫病是梨、苹果等蔷薇科仁果类植物上的一种毁灭性细菌病害。为筛选新的有效防治药剂,本研究采用比浊法测定了6种杀菌剂对梨火疫病菌的毒力,并测定了这6种杀菌剂混配成的7类35种混剂的毒力,将3种具有增效作用的混剂及4种组成混剂的单剂在盆栽杜梨苗上进行防效测定。结果表明,6种杀菌剂对梨火疫病菌均有毒力,毒力最强的是丙硫唑,EC50为0.02 mg/L;35种混剂中有9种具有增效作用,其中增效作用最强的是春雷霉素∶喹啉铜=1∶9,CTC值为184。3种混剂在盆栽苗上防效显著高于4种单剂,效果最好的是40%春雷·噻唑锌悬浮剂。本研究结果对创制新的梨火疫病防治复配药剂提供了借鉴。     

杀菌剂毒力及其生物测定

杀菌剂毒力是化合物具有开发和应用价值的固有生物学性状，不仅是农药登记部门予以注册登记的前置条件，而且也是反映杀菌剂生物活性及其安全性和有效性的关键技术参数。本文明确了杀菌剂毒力术语的定义，强调了杀菌剂可能的未知作用方式及通过与病原菌、寄主和环境因子相互作用的病害及流行防控效力在杀菌剂毒力评价中的重要性。深入分析了化合物与受体蛋白相互作用的结构特异性和精确性及干扰靶标蛋白功能的毒理学机制，揭示了杀菌剂毒力的选择性原理。综述了杀菌剂毒力生物测定及其结果分析方法，分析了可能影响毒力测定结果评价的常见因素。本文所述内容可为从事农药创制、加工、应用、毒理与抗性及分子互作科研工作者提供参考。     

植物病原真菌对甾醇生物合成抑制剂类(SBIs)杀菌剂的抗药性研究进展

甾醇生物合成抑制剂类(SBIs)杀菌剂通过抑制植物病原真菌甾醇生物合成途径中不同环节的酶,干扰或阻断病原菌麦角甾醇生物合成而发挥抗真菌作用。综述了植物病原真菌对SBIs杀菌剂的抗药性发生现状、遗传机制、生理生化机制、分子机制及治理策略等方面的最新研究进展。室内及田间有关SBIs杀菌剂抗药性的研究结果表明,植物病原菌对该类杀菌剂的抗药性可能是由1种或多种机制共同作用的结果。ABC和MFS运输蛋白基因及CYP51蛋白基因是植物病原真菌对SBIs杀菌剂产生抗药性的主要分子机制。其中ABC运输蛋白基因能够通过翻转酶将药剂从膜内层转移至外层而排出细胞体外;MFS运输蛋白基因的超表达和本底表达则是导致病原菌产生抗药性的关键因素;而CYP51蛋白基因与药剂作用时易在病原菌体内发生基因点突变或基因超表达,造成编码蛋白与药剂亲和力下降,导致病原菌产生抗药性。随着分子生物学的迅速发展,可从基因水平上寻找出与抗药性直接相关的基因、蛋白及调控途径等信息,同时与其他学科结合,合理设计新的、多作用位点的高效甾醇生物合成抑制剂,从而延长该类杀菌剂的使用寿命。     

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.     

Impact of alternative respiration and target-site mutations on responses of germinating conidia of Magnaporthe grisea to Qo-inhibiting fungicides

Qo-inhibiting fungicides act as respiration inhibitors by binding to the Qo center of cytochrome b. Sensitivities of fungi to Qo inhibitors can be influenced by the induction of alternative respiration or by mutational changes of the cytochrome b target site. Previous studies on both mechanisms in Magnaporthe grisea (Hebert) Barr were focused on the mycelial stage of the pathogen. The present study describes the expression and impact of both resistance mechanisms during the stage of conidia germination. In the absence of a host, alternative respiration provided a >500-fold rescue from azoxystrobin during the germination of conidia derived from four wild-type isolates of M. grisea. This rescue potential during conidia gemination was substantially more pronounced than for mycelial growth. However, the pronounced effectiveness of alternative respiration during conidia germination was not apparent when barley leaves were protected with azoxystrobin prior to inoculation with conidia. In a comparison of a wild-type strain and an alternative respiration-deficient mutant, azoxystrobin efficacies in suppressing symptom development differed by a factor of two, with full disease control achieved for both genotypes at 1 microg ml(-1) azoxystrobin. In contrast, conidia derived from two QoI-resistant target site mutants were highly resistant to azoxystrobin and trifloxystrobin and fully capable of infecting leaf surfaces protected with 10 microg ml(-1) of azoxystrobin. Both target-site mutants had emerged spontaneously in the presence of high azoxystrobin doses when residual mycelial growth was supported by alternative respiration. The effective silencing of alternative respiration in protective applications of Qo-inhibiting fungicides might constitute a strategy of slowing the emergence of highly resistant target site mutants.     

Re-examination of inhibitor resistance conferred by Qo-site mutations in cytochrome b using yeast as a model system

Cytochrome b from yeast (Saccharomyces cerevisiae Meyer ex Hansen) provides a convenient model system for the study of Qo-site inhibitor (QoI) resistance mutations from a variety of organisms. QoI resistance mutations from fungal plant pathogens (G143A and F129L), malaria agent Plasmodium sp (Y279C/S), and Pneumocystis carinii (L275F), an opportunistic pathogenic fungus of man, were introduced into yeast cytochrome b and their effect on the binding of a variety of natural (myxothiazol and stigmatellin) and synthetic (atovaquone, azoxystrobin and pyraclostrobin) inhibitors to the bc1 complex monitored. L275S (from a myxothiazol-resistant yeast) was also re-examined. Stigmatellin binding was relatively unaffected by the introduction of these mutations. Significant increases in resistance were observed for the strobilurin-class inhibitors myxothiazol, azoxystrobin and pyraclostrobin, with the largest increase in resistance conferred by G143A. In contrast, atovaquone binding was most effected by Y279C/S and L275S. Notably, F129L, G143A and L275S had a minor effect on bc1 activity, and so are unlikely to confer significant fitness penalties in vivo. These data are discussed in the light of the atomic structures for myxothiazol- and azoxystrobin-inhibited bovine bc1 which have recently become available. We propose that QoI resistance due to G143A arises from steric hindrance between the inhibitor and cytochrome b, whereas the mechanism of resistance for the other mutations is due to an increase in binding energy between the protein and inhibitor molecule. Site-directed mutagenesis was also used to model selected regions of the mammalian Qo site in yeast cytochrome b in order to further understand the differential efficacy of these QoI in the mammalian and pathogen bc1 complexes.     

Sensitivity of mitochondrial respiration to different inhibitors in Venturia inaequalis

The sensitivity of Venturia inaequalis field isolates to inhibitors of the cytochrome bc1 complex at the Qo site (QoIs) was characterised at the molecular, biochemical and physiological level, and compared to other respiration inhibitors. Comparison of a sensitive and a QoI-resistant isolate revealed very high resistance factors both in mycelium growth and spore germination assays. Cross-resistance was observed among QoIs such as trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. In the mycelium growth assay, antimycin A, an inhibitor of the cytochrome bc1 complex at the Qi site, was less active against the QoI-resistant than against the sensitive isolate. The mixture of QoIs with salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase, exerted synergistic effects in the spore germination but not in the mycelium growth assay. Thus, the cytochrome and the alternative respiration pathways are assumed to play different roles, depending on the developmental stage of the fungus. Induction of alternative oxidase (AOX) by trifloxystrobin was observed in mycelium cells at the molecular level for the sensitive but not the resistant isolate. Following QoI treatment, respiration parameters such as oxygen consumption, ATP level, membrane potential and succinate dehydrogenase activity were only slightly reduced in Qo-resistant mycelium cells, and remained at much higher levels than in sensitive cells. In contrast, no difference was observed between sensitive and resistant isolates when NADH consumption was measured. Comparison of the cytochrome b (cyt b) gene of the sensitive and resistant isolates did not reveal any point mutations as is known to occur in resistant isolates of other plant pathogens. It is assumed that QoI resistance in V inaequalis may be based on a compensation of the energy deficiency following QoI application upstream of the NADH dehydrogenase of the respiratory chain.     

The strobilurin fungicides

Strobilurins are one of the most important classes of agricultural fungicide. Their invention was inspired by a group of fungicidally active natural products. The outstanding benefits they deliver are currently being utilised in a wide range of crops throughout the world. First launched in 1996, the strobilurins now include the world's biggest selling fungicide, azoxystrobin. By 2002 there will be six strobilurin active ingredients commercially available for agricultural use. This review describes in detail the properties of these active ingredients--their synthesis, biochemical mode of action, biokinetics, fungicidal activity, yield and quality benefits, resistance risk and human and environmental safety. It also describes the clear technical differences that exist between these active ingredients, particularly in the areas of fungicidal activity and biokinetics.     

Cloning and expression analysis of the ATP-binding cassette transporter gene MFABC1 and the alternative oxidase gene MfAOX1 from Monilinia fructicola

Brown rot, caused by Moniliniafructicola (G Wint) Honey, is a serious disease of peach in all commercial peach production areas in the USA, including South Carolina where it has been primarily controlled by pre-harvest application of 14-alpha demethylation (DMI) fungicides for more than 15 years. Recently, the Qo fungicide azoxystrobin was registered for brown rot control and is currently being investigated for its potential as a DMI fungicide rotation partner because of its different mode of action. In an effort to investigate molecular mechanisms of DMI and Qo fungicide resistance in M fructicola, the ABC transporter gene MfABC1 and the alternative oxidase gene MfAOX1 were cloned to study their potential role in conferring fungicide resistance. The MfABC1 gene was 4380 bp in length and contained one intron of 71 bp. The gene revealed high amino acid homologies with atrB from Aspergillus nidulans (Eidam) Winter, an ABC transporter conferring resistance to many fungicides, including DMI fungicides. MfABC1 gene expression was induced after myclobutanil and propiconazole treatment in isolates with low sensitivity to the same fungicides, and in an isolate with high sensitivity to propiconazole. The results suggest that the MfABC1 gene may be a DMI fungicide resistance determinant in M fructicola. The alternative oxidase gene MfAOX1 from M fructicola was cloned and gene expression was analyzed. The MfAOX1 gene was 1077 bp in length and contained two introns of 54 and 67 bp. The amino acid sequence was 63.8, 63.8 and 57.7% identical to alternative oxidases from Venturia inaequalis (Cooke) Winter, Aspergillus niger van Teighem and A nidulans, respectively. MfAOX1 expression in some but not all M fructicola isolates was induced in mycelia treated with azoxystrobin. Azoxystrobin at 2 microg ml(-1) significantly induced MfAOX1 expression in isolates with low MfAOX1 constitutive expression levels.     

植物病原菌对杀菌剂抗性风险评估

植物病原菌对杀菌剂的抗性风险由基本风险和治理风险组成。杀菌剂使用之前或之初可根据 人工诱变、药剂选择或驯化实验、田间野生敏感菌株敏感性变异、抗药菌株的生物及遗传特 征、杀菌剂作用方式等进行基本抗药风险预测；杀菌剂使用数年之后可根据人工诱变、药剂 选择或驯化、田间药效与抗药性发生、抗药菌株的生物及遗传特征、杀菌剂作用方式与使用 对策等已有资料进行抗药风险评估。目前已有4种方法用于抗药风险评估。由杀菌剂与病害 共同决定的基本抗药风险可分成低、中和高度。基本抗性风险高的药剂合理使用可延缓田间 抗药性发生，中度基本抗性风险药剂不合理使用也可引发田间抗药性发生和药效明显降低。     

Following the dynamics of strobilurin resistance in Blumeria graminis f.sp. tritici using quantitative allele-specific real-time PCR measurements with the fluorescent dye SYBR Green I

Strobilurin-resistant isolates of Blumeria ( Erysiphe ) graminis f.sp. tritici , the cause of wheat powdery mildew, were more than 10-fold less sensitive to azoxystrobin than sensitive isolates. In all resistant isolates, a mutation resulting in the replacement of a glycine by an alanine residue at codon 143 (G143A) in the mitochondrial cytochrome b gene was found. Allele-specific primers were designed to detect this point mutation in infected wheat leaves. Using quantitative fluorescent allele-specific real-time polymerase chain reaction (PCR) measurements, strobilurin-resistant A143 alleles could be detected amongst strobilurin-sensitive G143 alleles at a frequency of at least 1 in 10 000, depending on the amount of target and nontarget DNA. Most isolates tested were dominant homoplasmic for either the A143 or G143 allele, although mixed populations of alleles could be detected in some isolates. In some of these isolates, strobilurin resistance was not always stable when they were maintained for many generations in the absence of selection. The allele-specific real-time PCR assay was also used to follow the dynamics of A143 alleles in field populations of B . graminis f.sp. tritici before and after application of fungicides. As expected, the A143 allele frequency only increased under selection pressure from a strobilurin fungicide. After three sprays of azoxystrobin, a pronounced selection for the strobilurin-resistant allele, with an increase in average frequency from 2·2 to 58%, was measured. The use of quantitative real-time PCR diagnostics for early detection of fungicide resistance genes at low frequency, coupled with risk evaluation, will be invaluable for further resistance risk assessment and validation of antiresistance strategies.