植物病原真菌对甾醇脱甲基抑制剂类杀菌剂抗性分子机制研究进展

甾醇脱甲基抑制剂 (DMI) 可通过抑制病原真菌的14α-去甲基化酶(CYP51)而干扰或阻断细胞膜麦角甾醇的生物合成,造成有毒甾醇积累,从而影响细胞膜的结构及功能,进而发挥抗菌作用。随着DMI类杀菌剂的广泛应用,病原菌对其的抗性问题日益严重。本文从抗药性分子机制出发,总结出病原菌对DMI类杀菌剂产生抗性的主要原因为:CYP51氨基酸突变引起其与杀菌剂间的亲和力下降;启动子区域基因片段的插入引起CYP51基因过表达;转录因子激活突变或启动子区域基因片段插入导致外排蛋白基因过表达。本文基于杀菌剂的作用方式及病原菌抗性机制研究展开综述,可为杀菌化合物的结构修饰与优化、新靶点改进和研发以及病原真菌的抗药性治理提供参考。     

植物病原菌抗药性遗传研究

 植物病原菌对杀菌剂的抗药性是由遗传基因控制的,抗药基因位于细胞质遗传因子或细胞核染色体基因上,细菌对许多药剂如铜制剂、链霉素等的抗药性和真菌对少数药剂如甲氧丙烯酸酯类药剂的抗性属于前一种情况,而真菌对大多数药剂的抗性则属于后一种情况。核基因控制的抗药性又可分为主效基因(major-gene)抗性和微效多基因(poly-gene)抗性,分别使病菌对药剂的抗性表现质量性状和数量性状。病原菌对苯并咪唑类药剂、春日霉素、羧基酰胺类药剂、苯酰胺类药剂、芳烃类药剂、二甲酰亚胺类药剂等的抗性通常为主效基因控制;使病菌表现微效多基因抗性的杀菌剂主要有多果定、羟基嘧啶类药剂、甾醇合成抑制剂(SBIs)等     

玉米黑粉菌CYP51基因的克隆与表达

细胞色素P450甾醇14-a-脱甲基酶(P45014DM或CYP51)是真菌细胞膜麦角甾醇生物合成过程中的一个关键酶,它的缺乏将导致膜结构的破坏和功能消失,最终导致真菌死亡。从玉米黑粉菌中克隆得到一缺失102个核苷酸的玉米黑粉菌CYP51基因,并构建重组表达载体pE30YH1-14DM。玉米黑粉菌CYP51的克隆为研究CYP51结构和功能提供了实验基础,同时也为研究其它植物病原真菌CYP51进而为设计开发新型的具有更强结合作用和更高选择性14 a-脱甲基化酶抑制剂类杀菌剂提供了实验依据。     

植物病原真菌对杀菌剂抗性的研究进展

科学施用杀菌剂是植物病害综合治理的重要措施之一, 然而由于杀菌剂的长期使用, 病菌抗药性问题逐渐加重, 严重影响药剂的防治效果和使用寿命。近年来, 随着分子生物学技术的快速发展, 人们对杀菌剂抗性机制有了更深入的理解, 并开发出了病菌抗药基因型快速检测的方法。本文总结了植物病原真菌对苯并咪唑类杀菌剂(BZD)、肌球蛋白合成抑制剂、甾醇脱甲基抑制剂(DMI)、QoI类抑制剂、琥珀酸脱氢酶抑制剂（SDHI）和二甲酰亚胺类杀菌剂(DC)的抗药性现状与抗性机制。在此基础上, 介绍了聚合酶链反应(PCR)、限制性片段长度多态性(RFLP)、等位基因特异性PCR和环介导等温扩增(LAMP)技术在杀菌剂抗性快速检测方面的研究进展。此外, 对抗药性治理对策进行了讨论和展望。     

植物病原菌抗药性及其抗性治理策略

随着现代高活性的选择性杀菌剂的研发和广泛使用,病原菌的抗药性问题日趋严重,这已成为植物病害化学保护领域最受关注的问题之一。本文阐释了抗药性相关术语的定义,概述了病原菌的抗药性现状,并从自然选择和诱导突变两种学说的角度分析了抗药性产生的原因。进一步分析了抗药性群体流行与病原菌自身特点、杀菌剂类型和作用机制等影响因子密切相关,综述了抗药性风险评估、抗药性机制、抗药性进化以及抗药性常规和分子检测方法等内容。最后,提出了抗药性治理的目标和策略,即根据抗药病原群体形成的主要影响因素,针对性地设计抗药性治理短期和长期策略,特别是需进一步加强对新药剂和新防治对象开展抗药性风险评估、制定抗药性管理策略、建立再评价机制等。综上,明确植物病原菌抗药性发生发展特点并制定科学合理的抗性治理策略,对进一步开展植物病害的科学防控具有重要的参考价值。     

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。     

5种甾醇生物合成抑制剂类(SBIs)抗真菌药物对9种植物病原真菌的活性筛选

为了寻找高效、低毒以及环境友好型的农药先导化合物,通过菌丝生长速率法测定了5种甾醇生物合成抑制剂类(SBIs)抗真菌药物(益康唑、氟康唑、伏立康唑、酮康唑和咪康唑)对7种植物病原菌的抑制效果,选择其中活性较高的药物进行了防治小麦白粉病和水稻纹枯病的盆栽试验及防治小麦条锈病和水稻纹枯病的田间药效试验。室内毒力测定结果表明:伏立康唑对供试的7种植物病原真菌的杀菌活性最高,其EC50值均低于0.349 mg/L,咪康唑对小麦赤霉病菌、梨黑斑病菌、西瓜枯萎病菌和香樟炭疽病菌,益康唑对梨黑斑病菌和西瓜枯萎病菌,以及酮康唑对水稻稻瘟病菌均表现出较强的杀菌活性,且均高于对照药剂苯醚甲环唑。盆栽试验结果显示:在药剂质量浓度为37.5 mg/L时,伏立康唑和氟康唑对小麦白粉病的防治效果分别为98.26%和89.11%,明显高于商品化杀菌剂三唑醇;在质量浓度为150 mg/L时,益康唑对水稻纹枯病的防治效果最好,为86.14%。田间试验结果表明:在有效剂量为240 g/hm^2时,氟康唑对小麦条锈病的防效为98.42%,益康唑对水稻纹枯病的防效为75.21%。研究结果表明,临床上的抗真菌药物氟康唑、伏立康唑和益康唑对植物病原真菌也具有很高的活性,可望作为农用杀菌剂的先导化合物进一步研究。     

真菌源蛋白类激发子及其转基因植物研究进展

真菌源蛋白类激发子是广泛存在于植物病原真菌的信号传导分子,不仅在植物与病原菌互作中起重要作用,而且还具有广谱诱导植物抗性和促进植物生长的功能。本文就真菌源蛋白类激发子的种类、功能及蛋白激发子基因转化植物等研究进行了综述。     

植物挥发性有机化合物在农业病害防控中的潜能与应用

植物病害对全球粮食安全造成严重威胁，而病原物对杀菌剂日益严重的抗药性问题和杀菌剂施用导致的环境暴露风险极大限制了传统杀菌剂的开发。植物释放大量挥发性有机化合物 (volatile organic compounds, VOCs) 到大气中作为植物与周围环境交流互动的信号分子。植物VOCs可以保护自身免受食草动物的侵害，或吸引传粉者和种子传播者，它们还能够直接抑制病原菌的生长或者激活植物的防御系统。本文综述了植物VOCs在生物合成、收集分析、诱导释放、对病原微生物的活性和诱导植物免疫反应等方面的研究进展；总结了典型绿叶挥发物反-2-己烯醛的抑菌和抗性诱导机理；归纳和展望了植物VOCs在田间应用的局限性和今后的研究方向，可为该类化合物在可持续病害防控中的应用提供帮助。     

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。     

The 14alpha-Demethylasse(CYP51A1) Gene is Overexpressed in Venturia inaequalis Strains Resistant to Myclobutanil

ABSTRACT We identified the cytochrome P450 sterol 14alpha-demethylase (CYP51A1) gene from Venturia inaequalis and optional insertions located upstream from CYP51A1 and evaluated their potential role in conferring resistance to the sterol demethylation-inhibitor (DMI) fungicide my-clobutanil. The CYP51A1 gene was completely sequenced from one my-clobutanil sensitive (S) and two myclobutanil-resistant (R) strains. No nucleotide variation was found when the three sequences were aligned. Allele-specific polymerase chain reaction (PCR) analysis indicated that a previously described single base pair mutation that correlated with resistance to DMI fungicides in strains of other filamentous fungi was absent in 19 S and 32 R strains of V. inaequalis from Michigan and elsewhere. The sequencing results and PCR analyses suggest that resistance in these strains was not due to a mutation in the sterol demethylase target site for DMI fungicides. Expression of CYP51A1 was determined for strains from an orchard that had never been sprayed with DMI fungicides (baseline orchard), and the data provided a reference for evaluating the expression of strains collected from a research orchard and from three commercial Michigan apple orchards with a long history of DMI use and a high frequency of R strains. Overexpression of CYP51A1 was significantly higher in 9 of 11 R strains from the research orchard than in S strains from the baseline orchard. The high expression was correlated with the presence of a 553-bp insertion located upstream of CYP51A1. Overexpression of the CYP51A1 gene was also detected in eight of eight, five of nine, and nine of nine R strains from three commercial orchards, but the insertion was not detected in the majority of these strains. The results suggest that overexpression of the target-site CYP51A1 gene is an important mechanism of resistance in some field resistant strains of V. inaequalis, but other mechanisms of resistance also appear to exist.     

Activity of fungicides and modulators of membrane drug transporters in field strains of Botrytis cinerea displaying multidrug resistance

In Botrytis cinerea, multidrug resistant (MDR) strains collected in French and German vineyards were tested in vitro, at the germ-tube elongation stage, towards a wide range of fungicides. Whatever the MDR phenotype, resistance was recorded to anilinopyrimidines, diethofencarb, iprodione, fludioxonil, tolnaftate and several respiratory inhibitors (e.g., penthiopyrad, pyraclostrobin). In MDR1 strains, overproducing the ABC transporter BcatrB, resistance extended to carbendazim and the uncouplers fluazinam and malonoben. In MDR2 strains, overproducing the MFS transporter BcmfsM2, resistance extended to cycloheximide, fenhexamid and sterol 14α-demethylation inhibitors (DMIs). MDR3 strains combined the overexpression of both transporters and exhibited the widest spectrum of cross resistance and the highest resistance levels. The four transport modulators, amitriptyline, chlorpromazine, diethylstilbestrol, and verapamil, known to affect some ABC transporters, were tested in B. cinerea. In our experimental conditions, the activity of several fungicides was only enhanced by verapamil. Interestingly, synergism was only recorded in MDR2 and/or MDR3 isolates treated with tolnaftate, fenhexamid, fludioxonil or pyrimethanil, suggesting that verapamil may inhibit the MFS transporter BcmfsM2. This is the first report indicating that a known modulator of ABC transporters could also block MFS transporters.     

Secretion of Natural and Synthetic Toxic Compounds from Filamentous Fungi by Membrane Transporters of the ATP-binding Cassette and Major Facilitator Superfamily

This review provides an overview of members of the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) of transporters identified in filamentous fungi. The most common function of these membrane proteins is to provide protection against natural toxic compounds present in the environment of fungi, such as antibiotics produced by other microorganisms. In plant pathogenic fungi, these transporters can also be an important determinant of virulence on host plants by providing protection against plant defence compounds or mediating the secretion of host-specific toxins. Furthermore, they play a critical role in determining base-line sensitivity to fungicides and other antimycotic agents. Overexpression of some of these transporters can lead to the development of resistance to chemically-unrelated compounds, a phenomenon described as multidrug resistance (MDR). This has been observed in a variety of organisms and can impose a serious threat to the effective control of pathogenic fungi.     

真菌甲硫氨酸生物合成途径研究进展

 甲硫氨酸是蛋白质的重要组分,同时还可通过生成S-腺苷甲硫氨酸（SAM）调控细胞内多种生理过程。本文根据酿酒酵母、粗糙脉孢菌和构巢曲霉3种模式真菌中甲硫氨酸生物合成的最新研究进展,勾画出真菌的甲硫氨酸合成途径,综述了该途径中关键酶的生物学功能,探讨了该途径的调控机制,为病原真菌甲硫氨酸合成的研究提供理论基础,同时为新杀菌剂的研发提供新思路。     

Analysis of the CYP51 gene and encoded protein in propiconazole‐resistant isolates of Mycosphaerella fijiensis

BACKGROUND: Mycosphaerella fijiensis Morelet causes black sigatoka, the most important disease in bananas and plantains. Disease control is mainly through the application of systemic fungicides, including sterol demethylation inhibitors (DMIs). Their intensive use has favoured the appearance of resistant strains. However, no studies have been published on the possible resistance mechanisms. RESULTS: In this work, the CYP51 gene was isolated and sequenced in 11 M. fijiensis strains that had shown different degrees of in vitro sensitivity to propiconazole, one of the most widely used DMI fungicides. Six mutations that could be related to the loss in sensitivity to this fungicide were found: Y136F, A313G, Y461D, Y463D, Y463H and Y463N. The mutations were analysed using a homology model of the protein that was constructed from the crystallographic structure of Mycobacterium tuberculosis (Zoff.) Lehmann & Neumann. Additionally, gene expression was determined in 13 M. fijiensis strains through quantitative analysis of products obtained by RT‐PCR. CONCLUSION: Several changes in the sequence of the gene encoding sterol 14α‐demethylase were found that have been described in other fungi as being correlated with resistance to azole fungicides. No correlation was found between gene expression and propiconazole resistance. Copyright © 2009 Society of Chemical Industry