杀菌剂抗性分子检测技术的研究进展

靶标病菌基因突变是许多内吸性杀菌剂出现抗性的根本原因,检测与抗药性相关的靶标病菌基因突变对阐明抗药性发生的分子生物学机制及进行早期诊断具有重要意义。目前已成功用于检测靶标病菌抗药性菌株的分子检测技术有6种:等位基因特异性扩增、限制性片段长度多态性、等位基因特异性寡核苷酸杂交、单链构象多态性、实时定量PCR、变性高效液相色谱分析。这些技术能够快速、灵敏地检测田间早期出现的抗药性或抗药性种群的发展动态,在病害的可持续管理系统中科学使用杀菌剂方面发挥着重要作用。     

浙江省西 (甜) 瓜蔓枯病菌对甲基硫菌灵和啶酰菌胺的抗性检测及抗性机制

从浙江省5地采集了112株西 (甜) 瓜蔓枯病菌Stagonosporppsis citrulli,采用区分剂量法检测其对苯并咪唑类杀菌剂甲基硫菌灵 (以下简称Ben) 和琥珀酸脱氢酶抑制剂类 (SDHIs) 杀菌剂啶酰菌胺 (简称Bos) 的抗性。结果显示:112株西 (甜) 瓜蔓枯病菌对Ben和Bos的抗药性频率分别为100%和28.6%,其中对甲基硫菌灵产生高水平抗性 (BenHR) 的菌株达100%,对啶酰菌胺产生低水平抗性 (BosLR) 和高水平抗性 (BosHR) 的菌株分别为18.8%和9.8%。抗药性分子机制研究表明:BenHR菌株中β-tubulin的第198位氨基酸由Glu (E) 突变成了Ala (A);BosHR菌株中Sdh B的第277位氨基酸由His (H) 突变成了Tyr (Y),但BosLR的抗性机制还需进一步研究。研究结果表明,浙江省西 (甜) 瓜蔓枯病菌对苯并咪唑类杀菌剂甲基硫菌灵的抗性已十分严重,尽管大多数菌株对啶酰菌胺仍表现敏感,但在一些地区已存在高水平抗性菌株,应在加强抗药性监测的同时,注意SDHIs类杀菌剂的科学使用。     

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。     

琥珀酸脱氢酶抑制剂类杀菌剂吡噻菌胺的研究进展

吡噻菌胺作为新型的琥珀酸脱氢酶抑制剂类手性杀菌剂,自研发以来便在越来越多的国家登记使用。相较于传统的琥珀酸脱氢酶抑制剂（Succinate Dehydrogenase Inhibitors,SDHI）类杀菌剂,吡噻菌胺对多种病菌都表现出了较好的防治效果,弥补传统SDHI类杀菌剂抗药性日益增强的同时,具有更广阔的应用前景和市场。本文旨在通过对吡噻菌胺的合成工艺、生物活性和生态毒性、环境行为和农药抗性研究进行综述,为其农业合理应用、科学有效管理与生态风险规避提供科学参考。     

梨黑斑病菌抗药性检测及其对啶酰菌胺的敏感性基线

黑斑病是梨的主要病害之一,近年来不少地区反映多菌灵等传统常用杀菌剂对其防治效果已出现下降。作者从浙江、江苏和安徽3省分离了252株梨黑斑病菌Alternaria kikuchiana,采用菌丝生长速率法检测了其抗药性发生情况。结果发现:所检测的黑斑病菌群体(n=252)对苯并咪唑类杀菌剂多菌灵的抗性频率为57.1%,且全部为高水平抗性(HR);对二甲酰亚胺类杀菌剂异菌脲的抗性频率为46.8%,全部为低水平抗性(LR);对甾醇脱甲基抑制剂类杀菌剂苯醚甲环唑的抗性为低水平(LR)及中等水平(MR),抗性频率均为28.6%;表明梨黑斑病菌对常用杀菌剂已产生较为严重的抗性。供试252株梨黑斑病菌对琥珀酸脱氢酶抑制剂啶酰菌胺的EC50值分布在0.12~3.85μg/m L之间,平均EC50值为(1.21±0.12)μg/m L,且其分布呈近似正态的单峰曲线。研究表明,啶酰菌胺可作为潜在的梨黑斑病防治替代药剂,其平均EC50值(1.21±0.12)μg/m L可作为梨黑斑病菌对啶酰菌胺的敏感性基线。     

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     

大麦云纹斑病菌对杀菌剂的抗性检测及同工酶谱类型

1993～1994年大麦云纹斑病菌(Rhynchosporium secalis)对杀菌剂多菌灵、三唑醇的抗药性监测表明,该菌对两种杀菌剂的敏感程度发生了变化,并且首次在田间发现了大麦云纹斑病菌的多抗菌株。这类菌株对多菌灵、乙霉威及三唑醇都具有抗药性,且抗性程度很高。与1990～1991年得到的资料相比,随着杀菌剂选择压力的增加,大田群体中三唑醇高抗菌株比例逐年增加。对该菌同工酶分析结果表明,α—酯酶有7种表现型,磷酸葡萄糖变位酶有2种表现型,过氧化氢酶有3种表现型,无论对去甲基抑制剂类(DMI)表现抗性或敏感菌株,无论调节酶还是非调节酶,其酶谱表现型的变异基本相似。这表明大麦云纹斑病菌对DMI类抗药性突变存在于多个菌系的基因型背景群体中,而不是只起源于单一的广泛分布的菌系。     

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。     

二化螟对双酰胺类杀虫剂抗性的分子机制研究进展

新型双酰胺类杀虫剂已广泛用于保障水稻生产,而二化螟作为危害水稻生产的钻蛀性害虫,已经对该类杀虫剂产生了抗性,明确该类杀虫剂抗性分子机制,可为二化螟抗性快速检测和绿色防控提供技术支撑。本文在总结二化螟对双酰胺类杀虫剂抗性现状的基础上,重点综述了近年来有关其抗性分子机制研究的进展,主要包括解毒酶和转运蛋白基因过表达介导的代谢抗性,以及鱼尼丁受体基因突变介导的靶标抗性;指出了该研究领域在抗性分子检测、抗性新基因鉴定、抗性基因调控网络和多重抗性机制等方面存在的问题,并展望了其发展方向,认为：利用高通量测序技术检测害虫种群抗药性;利用多组学技术鉴定新抗性基因及调控网络,以探明多重抗性机制;将反向遗传学工具放射性配基结合及电生理技术深入验证抗性基因功能;需开发靶向抗性基因的dsRNA转基因作物、纳米农药及选择性新型化学杀虫剂,以达到杀虫剂减施增效的目的。     

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。     

Target and non-target site mechanisms of fungicide resistance and their implications for the management of crop pathogens

Fungicides are indispensable for high-quality crops, but the rapid emergence and evolution of fungicide resistance have become the most important issues in modern agriculture. Hence, the sustainability and profitability of agricultural production have been challenged due to the limited number of fungicide chemical classes. Resistance to site-specific fungicides has principally been linked to target and non-target site mechanisms. These mechanisms change the structure or expression level, affecting fungicide efficacy and resulting in different and varying resistance levels. This review provides background information about fungicide resistance mechanisms and their implications for developing anti-resistance strategies in plant pathogens. Here, our purpose was to review changes at the target and non-target sites of quinone outside inhibitor (QoI) fungicides, methyl-benzimidazole carbamate (MBC) fungicides, demethylation inhibitor (DMI) fungicides, and succinate dehydrogenase inhibitor (SDHI) fungicides and to evaluate if they may also be associated with a fitness cost on crop pathogen populations. The current knowledge suggests that understanding fungicide resistance mechanisms can facilitate resistance monitoring and assist in developing anti-resistance strategies and new fungicide molecules to help solve this issue. © 2023 Society of Chemical Industry.     

桃褐腐病菌抗药性分子机理研究进展

桃褐腐病是严重危害桃生产的重要病害之一。在实际生产中,该病害的控制主要以化学防治为主。由于杀菌剂的大量使用,桃褐腐病菌已对几类常用的杀菌剂产生了不同程度的抗药性。本文就桃褐腐病菌(Moniliniaspp.)对生产上使用的几大类杀菌剂的抗药性情况及抗药性分子机制研究进展进行简单概述和分析,以供国内同行借鉴与参考。     

Do some IPM concepts contribute to the development of fungicide resistance? Lessons learned from the apple scab pathosystem in the United States

One goal of integrated pest management (IPM) as it is currently practiced is an overall reduction in fungicide use in the management of plant disease. Repeated and long‐term success of the early broad‐spectrum fungicides led to optimism about the capabilities of fungicides, but to an underestimation of the risk of fungicide resistance within agriculture. In 1913, Paul Ehrlich recognized that it was best to ‘hit hard and hit early’ to prevent microbes from evolving resistance to treatment. This tenet conflicts with the fungicide reduction strategies that have been widely promoted over the past 40 years as integral to IPM. The authors hypothesize that the approaches used to implement IPM have contributed to fungicide resistance problems and may still be driving that process in apple scab management and in IPM requests for proposals. This paper also proposes that IPM as it is currently practiced for plant diseases of perennial systems has been based on the wrong model, and that conceptual shifts in thinking are needed to address the problem of fungicide resistance. © 2013 Society of Chemical Industry     

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。     

An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides

BACKGROUND: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self‐splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. RESULTS: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5′–3′) 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above‐mentioned 1166 bp intron, a self‐splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. CONCLUSION: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron. Copyright © 2010 Society of Chemical Industry     

French vineyards provide information that opens ways for effective resistance management of Botrytis cinerea (grey mould)

Resistance to fungicides is an evolutionary process resulting from the selection of advantageous genotypes in naturally diverse populations. Seven fungicide modes of action are authorised to control grey mould caused by Botrytis cinerea on grapevine in France, and five of them have encountered specific resistance, with variable frequencies in populations and possible consequences for field fungicide efficacy. Moreover, multidrug resistance is caused by fungicide efflux and allows a weak resistance towards six unrelated modes of action. Here, a review is given of the fungicide resistance status of B. cinerea in France, particularly in the vineyards of Champagne, which are the most affected. Recently developed resistance and recent findings concerning the associated resistance mechanisms are focused upon in particular. Finally, antiresistance strategies are presented, and examples of managed resistance are discussed in a more general manner with the aim of extending this knowledge to other crops and countries undergoing similar resistance problems. © 2013 Society of Chemical Industry     

Genetic correlations in resistance to morpholine and piperidine fungicides inPyrenophora teres populations

Highly significant genetic variation (P<0.001) in resistance to the morpholine fungicides fenpropimorph, tridemorph and dodemorph and the piperidine fungicide, fenpropidin was found in different populations ofPyrenophore teres in North America and Europe which had not been previously exposed to these fungicides. Resistance phenotypes were continuously distributed for each fungicide in each population. Cross resistance relationships were determined by estimating genetic correlation coefficients in resistance to all pairwise combinations of fungicides. The majority of the correlation coefficients were highly positive for all fungicide combinations in all populations; eight of 36 (22%) coefficients were not significantly different from 1 (P>0.05). This result is consistent with the hypothesis that many of the same genes, or genes in gametic disequilibrium, control resistance to more than one fungicide in most populations ofP. teres and that these fungicides comprise a single cross resistance group. Three of 36 (8%) correlation coefficients were not significantly different from 0 (P>0.05) indicating that, in these populations, independent genes controlled resistance to these fungicides. The results of this study indicate that although most of the same genes control resistance to morpholine and piperidine fungicides inP. teres, differences in frequencies of these genes among populations can result in different cross resistance relationships from one population to another.     

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。     

Molecular and biochemical characterization of boscalid resistance in laboratory mutants of Sclerotinia sclerotiorum

The plant‐pathogenic fungus Sclerotinia sclerotiorum has a broad host range and a worldwide distribution. Boscalid, an inhibitor of succinate dehydrogenase in the electron transport chain of fungi, is highly effective in controlling sclerotinia stem rot caused by S. sclerotiorum. The current study characterized the S. sclerotiorum boscalid‐resistant (BR) mutants obtained by fungicide induction. Among the bioactive fungicides against S. sclerotiorum, cross‐resistance was not detected between boscalid and dimethachlon, fluazinam or carbendazim; positive cross‐resistance was detected between boscalid and carboxin; and negative cross‐resistance was detected between boscalid and kresoxim‐methyl. Compared to their parental isolates, BR mutants had slower radial growth, no ability to produce sclerotia, lower virulence and oxalic acid content but higher mycelial respiration and succinate dehydrogenase (SDH) activity. Moreover, BR mutants had decreased sensitivity to salicylhydroxamic acid (SHAM) but not to oxidative stress. All the results indicated that the risk of resistance to boscalid in S. sclerotiorum is low to moderate. DNA sequence analysis showed that all of the BR mutants had the same point mutation A11V (GCA to GTA) in the iron sulphur protein subunit (SDHB). Interestingly, expression of the cytochrome b (cytb) gene was reduced to different degrees in the BR mutants, and this might be correlated with the negative cross‐resistance between boscalid and kresoxim‐methyl. Such information is vital in the design of resistance management strategies.