Chlorophyll fluorescence as a marker for herbicide mechanisms of action

Photosynthesis is the single most important source of O₂ and organic chemical energy necessary to support all non-autotrophic life forms. Plants compartmentalize this elaborate biochemical process within chloroplasts in order to safely harness the power of solar energy and convert it into usable chemical units. Stresses (biotic or abiotic) that challenge the integrity of the plant cell are likely to affect photosynthesis and alter chlorophyll fluorescence. A simple three-step assay was developed to test selected herbicides representative of the known herbicide mechanisms of action and a number of natural phytotoxins to determine their effect on photosynthesis as measured by chlorophyll fluorescence. The most active compounds were those interacting directly with photosynthesis (inhibitors of photosystem I and II), those inhibiting carotenoid synthesis, and those with mechanisms of action generating reactive oxygen species and lipid peroxidation (uncouplers and inhibitors of protoporphyrinogen oxidase). Other active compounds targeted lipids (very-long-chain fatty acid synthase and removal of cuticular waxes). Therefore, induced chlorophyll fluorescence is a good biomarker to help identify certain herbicide modes of action and their dependence on light for bioactivity.     

Natural products that have been used commercially as crop protection agents

Many compounds derived from living organisms have found a use in crop protection. These compounds have formed the basis of chemical synthesis programmes to derive new chemical products; they have been used to identify new biochemical modes of action that can be exploited by industry-led discovery programmes; some have been used as starting materials for semi-synthetic derivatives; and many have been used or continue to be used directly as crop protection agents. This review examines only those compounds derived from living organisms that are currently used as pesticides. Plant growth regulators and semiochemicals have been excluded from the review, as have living organisms that exert their effects by the production of biologically active secondary metabolites.     

Physiological effects of the hydrophilic phytotoxins produced by <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>, the causal agent of black sigatoka in banana plants

Although Mycosphaerella fijiensis, the causal agent of black sigatoka disease of banana, has been known to produce numerous lipophilic host-selective (HSTs) and nonhost selective phytotoxins (non-HSTs), only recently we have reported that the pathogen also produces hydrophilic phytotoxins. Here we examined the effect of light on the toxicity of the hydrophilic phytotoxins and estimated the electrolyte leakage and H₂O₂ and superoxide generation in detached banana leaves to study their mode of action at the cellular level. Nonhost plant species were also tested to determine whether the toxins are HSTs or non-HSTs. Our results suggest that the hydrophilic phytotoxins are non-HSTs, that their phytotoxicity is not light dependent, and that they may act at the plasma membrane by altering permeability through oxidative damage, by inducing ROS production as part of their mechanism of action.     

Isophorone‐induced light‐independent lipid peroxidation and loss of cell membrane integrity

Isophorone (3,5,5‐trimethylcyclohex‐2‐en‐1‐one) is a plant‐derived volatile compound with strong phytotoxic activity. Here, we aimed to elucidate the mechanism of action of isophorone, and to this end, the effects of isophorone on shoot fresh weight, chlorophyll content, electrolyte leakage, and lipid peroxidation of Lactuca sativa L. and photosynthetic electron transport activity in chloroplast isolated from Spinacia oleracea L. were investigated. Isophorone induced light‐independent decreases in shoot fresh weight and light‐dependent chlorosis. In addition, increased electrolyte leakage and lipid peroxidation occurred under light conditions. However, the inhibitory activity on photosynthetic electron transport was unexpectedly low, and electrolyte leakage and lipid peroxidation were induced even under dark conditions. These results suggest that the inhibition of photosynthetic electron transport is not the main mechanism of action of isophorone and that the phytotoxic effects are mainly due to light‐independent oxidative damage and subsequent loss of cell membrane integrity.     

Microscopic, Biochemical and Physiological Assessment of the Effect of Methylene Bisthiocyanate on the Sapstain Fungus Ophiostoma floccosum

In vitro effects of methylene bisthiocyanate (MBT) on hyphal morphology and ultra-structure of Ophiostoma floccosum were examined using differential interference contrast, epifluorescence and transmission electron microscopy (TEM). To understand the mode of action of MBT, experiments were undertaken to measure potassium ion (K⁺) leakage from cells, oxygen consumption, glucose and ATP levels. Differential interference contrast microscopy indicated that MBT caused rapid changes in O. floccosum hyphae resulting in extensive vaculoation and accumulation of granular materials within the cytoplasm. Epifluorescence microscopy provided evidence that MBT treatment causes a loss in the permeability properties of the plasma membrane. TEM showed retraction of the plasma membrane from the cell wall, aggregation of cytoplasmic contents, vesiculation of membranous components, a dramatic increase in vacuolation, and eventually a complete loss in the integrity of organelles. There was a rapid efflux of intracellular K⁺ ions from cells, a substantial loss in K⁺ ions occurring within the first 5 min of MBT treatment. The rate of K⁺ leakage was MBT concentration treatment-time dependent. The study also showed that the effect of lower concentrations of MBT (0.01 and 0.1 mM) on respiratory activity was negligible. However, at the same concentrations, glucose consumption and ATP production were affected. Taken together, these observations suggest that the target site of MBT in O. floccosum alters membrane properties and uncouples oxidative phosphorylation from the respiratory chain.     

Why have no new herbicide modes of action appeared in recent years?

Herbicides with new modes of action are badly needed to manage the evolution of resistance of weeds to existing herbicides. Yet no major new mode of action has been introduced to the market place for about 20 years. There are probably several reasons for this. New potential products may have remained dormant owing to concerns that glyphosate-resistant (GR) crops have reduced the market for a new herbicide. The capture of a large fraction of the herbicide market by glyphosate with GR crops led to significantly diminished herbicide discovery efforts. Some of the reduced herbicide discovery research was also due to company consolidations and the availability of more generic herbicides. Another problem might be that the best herbicide molecular target sites may have already been discovered. However, target sites that are not utilized, for which there are inhibitors that are highly effective at killing plants, suggests that this is not true. Results of modern methods of target site discovery (e.g. gene knockout methods) are mostly not public, but there is no evidence of good herbicides with new target sites coming from these approaches. In summary, there are several reasons for a long dry period for new herbicide target sites; however, the relative magnitude of each is unclear. The economic stimulus to the herbicide industry caused by the evolution of herbicide-resistant weeds, especially GR weeds, may result in one or more new modes of action becoming available in the not too distant future.     

Effect of fusaric acid on the leaf physiology of cucumber seedlings

The damaging effects of fusaric acid (FA), a fungal toxin produced by Fusarium oxysporum, on cucumber seedlings were investigated in a greenhouse experiment. The accumulation of red ink was introduced as damage determination, the vines and mesophyll cells of the plants treated with high concentration of FA were acutely stained to a deep red colour, and the quantity of red ink in the shoots and roots was significantly increased. The leaf plasma membrane H⁺-ATPase was significantly inhibited after treatment with FA. Moreover, transmission electron microscopy and electrolyte leakage experiments revealed severe FA-induced injury to leaf cell membranes. The membrane injury and wilt in the leaves of FA-treated plants disturbed the water status, and the leaf water potential was significantly decreased. The present results suggested that FA inhibits the leaf plasma membrane H⁺-ATPase and reduce the cell membrane integrity of cucumber seedlings, thus leading to leaf wilting and a reduction of the leaf water potential.     

AAL-toxin,a potent natural herbicide which disrupts sphingolipid metabolism of plants

AAL-toxin, a product of Alternaria alternata (Fr.) Keissl., is effective as a herbicide at low concentrations against a range of broadleaf plants (e.g. jimsonweed, prickly sida and black nightshade). However, monocotyledonous crops such as maize and wheat, as well as some varieties of tomato, are tolerant to it. The IC50 values for cellular electrolyte leakage and chlorophyll loss in duckweed (Lemna pausicostata L.) after 72 h treatment were 20–40 nM. Similar results were obtained with a susceptible tomato variety. AAL-toxin caused rapid cellular leakage of electrolytes, followed by cellular collapse; the first symptom at the ultrastructural level is disruption of the plasma membrane. The effects of the toxin are not light-dependent and appear to be associated with dysfunction of the plasma membrane. Fumonisins and sphingoid bases such as phytosphingosine cause similar effects, although these compounds are less potent (fumonisins, about 10-fold; sphingoid bases, about 100-fold). Recent studies suggest that in duckweed and in susceptible tomato varieties, AAL-toxin- and fumonisin B1 -induced disruption of sphingolipid metabolism is an early event in the cascade of events leading to phytotoxic injury and cell death.     

新型杀菌剂氟醚菌酰胺对辣椒疫霉的作用机制初探

为研究氟醚菌酰胺(N-(3-氯-5-(三氟甲基)吡啶-2-甲基)-2,3,5,6-四氟-4-甲氧基苯甲酰胺)对辣椒疫霉的作用机制,采用生物测定法系统测定了氟醚菌酰胺对辣椒疫霉菌丝生长、游动孢子释放和菌丝生长量的影响,并进一步对药剂处理后的辣椒疫霉菌丝的超微结构进行了观察;同时测定了氟醚菌酰胺对病原菌糖酵解、三羧酸循环和磷酸戊糖3种呼吸代谢途径的影响,以及其对辣椒疫霉菌丝体细胞膜通透性、可溶性蛋白和DNA含量的影响。结果表明:氟醚菌酰胺对辣椒疫霉菌丝生长、游动孢子释放和菌丝生长量的EC50值分别为7.14、16.34和5.12μg/mL;其可使辣椒疫霉菌丝分支增多变短,且出现细胞壁增厚和细胞变形现象;ATP的存在能降低氟醚菌酰胺对辣椒疫霉菌丝的抑制作用,说明氟醚菌酰胺对辣椒疫霉能量产生过程有一定的抑制作用;其对三羧酸循环途径的抑制作用最为明显。电导率法测定结果表明,氟醚菌酰胺处理均能提高辣椒疫霉的细胞膜通透性,用100μg/mL的氟醚菌酰胺处理400 min后,菌株的相对渗率达78.23%。但氟醚菌酰胺对辣椒疫霉生物大分子的影响较小。研究结果初步表明,氟醚菌酰胺有多个作用位点,但主要是通过抑制辣椒疫霉能量产生和细胞膜通透性而起到抑菌作用。     

Susceptibility of a broad-leaved weed, Acanthospermum hispidum, to the grass herbicide fluazifop-butyl

Broad-leaved plants are generally resistant to aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) herbicides. In laboratory experiments, however, we confirmed that Acanthospermum hispidum , a Compositae weed, was susceptible to one of the AOPP herbicides, fluazifop-butyl, but tolerant to other AOPP herbicides (quizalofop-ethyl and fenoxaprop-ethyl) and a CHD herbicide (sethoxydim). The symptoms induced by fluazifop-butyl in A. hispidum (wilting and necrosis) were distinctly different from those induced in oat (chlorosis). The period required to cause seedling death of A. hispidum (48–72 h) was shorter than that of oat ( ca 15 days). The ( R )-enantiomer of fluazifop-butyl was more active on this weed. In oat, lipid biosynthesis and acetyl-CoA carboxylase (ACCase) activity were inhibited, and electrolyte leakage from the shoots was increased by fluazifop-butyl and sethoxydim. In the case of A. hispidum , the membrane permeability increased and the lipid biosynthesis was inhibited only by fluazifop-butyl. These results indicate that A. hispidum is particularly sensitive to fluazifop-butyl, and its mechanism of action in the plant may be different from its mechanism of action in oat.     

Rationale for a natural products approach to herbicide discovery

Weeds continue to evolve resistance to all the known modes of herbicidal action, but no herbicide with a new target site has been commercialized in nearly 20 years. The so-called 'new chemistries' are simply molecules belonging to new chemical classes that have the same mechanisms of action as older herbicides (e.g. the protoporphyrinogen-oxidase-inhibiting pyrimidinedione saflufenacil or the very-long-chain fatty acid elongase targeting sulfonylisoxazoline herbicide pyroxasulfone). Therefore, the number of tools to manage weeds, and in particular those that can control herbicide-resistant weeds, is diminishing rapidly. There is an imminent need for truly innovative classes of herbicides that explore chemical spaces and interact with target sites not previously exploited by older active ingredients. This review proposes a rationale for a natural-products-centered approach to herbicide discovery that capitalizes on the structural diversity and ingenuity afforded by these biologically active compounds. The natural process of extended-throughput screening (high number of compounds tested on many potential target sites over long periods of times) that has shaped the evolution of natural products tends to generate molecules tailored to interact with specific target sites. As this review shows, there is generally little overlap between the mode of action of natural and synthetic phytotoxins, and more emphasis should be placed on applying methods that have proved beneficial to the pharmaceutical industry to solve problems in the agrochemical industry.     

Contact activity of difenzoquat differs from that of paraquat

Difenzoquat herbicide is used for post-emergence control of wild oat (Avena fatua L) in small grain crops. Its mechanism of action is not known, but appears to have both paraquat-like contact activity and systemic growth-inhibition activity. Experiments were conducted in vitro to compare the contact activity of difenzoquat and paraquat, to examine the contact activity of difenzoquat in difenzoquat-resistant (accession LCS) and -susceptible (accession SB 18) A fatua biotypes and -tolerant 'Ernest' and -susceptible 'Verde' hard red spring wheat (Triticum aestivum L) cultivars, and to investigate the role of differential DNA synthesis as a mechanism for difenzoquat resistance. Difenzoquat at 1 mM induced significant electrolyte leakage from resistant and susceptible A fatua leaf tissue in both light and dark, but paraquat at 1 mM induced electrolyte leakage only in light and not in dark. Difenzoquat at 1 mM under light induced more electrolyte leakage from resistant and susceptible A fatua than from tolerant 'Ernest' and susceptible 'Verde' wheat. Paraquat under light conditions induced more electrolyte leakage than difenzoquat, but no significant differences among A fatua accessions and wheat cultivars were detected. Difenzoquat under light did not decrease chlorophyll levels for either A fatua accessions or wheat cultivars, whereas paraquat decreased chlorophyll content of all four plant types. Inhibition of DNA synthesis was not significantly different between susceptible and resistant A fatua, but was significantly greater in susceptible 'Verde' than tolerant 'Ernest' wheat. These data suggest that difenzoquat and paraquat, have different modes of contact activity.     

黑曲霉xj粗提物的拮抗机制及其抗氧化活性

为探究黑曲霉Aspergillus niger xj 菌株粗提物SE对魔芋白绢病病原菌——齐整小核菌Sclerotium rolfsii R-67的拮抗机制及其抗氧化活性,采用菌丝生长速率法,研究了SE对R-67的抑制作用,进而通过测定处理后菌株的电导率、核酸蛋白泄露、三羧酸循环关键酶[琥珀酸脱氢酶 (SDH) 及苹果酸脱氢酶 (MDH)]比活力、胞内活性氧 (ROS) 水平及核酸含量,对SE的拮抗机制进行了研究,并通过1,1-二苯基-2-三硝基苯肼 (DPPH) 法及2,2-联氮基-双-(3-乙基苯并噻唑啉-6-磺酸) 二铵盐 (ABTS) 法测定了其抗氧化活性。通过气相色谱-质谱联用 (GC-MS)方法,从粗提物SE中共检测到12种化合物,其中主要成分为5-羟甲基糠醛(质量分数 97.96%)。生物试验结果表明:SE能够抑制R-67菌丝的生长,其IC₅₀值为0.46 mg/mL;SE处理可导致R-67菌液的电导率升高及核酸蛋白含量增加,抑制其三羧酸循环中关键酶SDH与MDH 的活力,增加菌体细胞内ROS含量,使细胞核4',6-二脒基-2-苯基吲哚 (DAPI) 染色的荧光强度减弱,据此初步推测其作用机制是通过影响R-67菌体细胞膜完整性、损伤细胞结构、抑制三羧酸循环及核酸合成等途径而发挥抑菌作用。研究表明,黑曲霉xj粗提物SE在魔芋白绢病的生物防治中具有良好的应用前景,此外SE对DPPH及ABTS自由基有一定的清除能力,有望应用于抗氧化功能性产品中。     

Acaricide mode of action

The last few years have seen the introduction of an unprecedented number of new classes of acaricides with novel or under-exploited modes of action, discovered by traditional screening. Acaricide research has uncovered several unrelated compounds that possess improved properties. Pyridaben, acequinocyl, diafenthiuron, etoxazole, spirodiclofen and bifenazate, in particular, are acaricides that are safe to beneficials, have low mammalian toxicity and short environmental persistence. Many of the new acaricides appear to affect mitochondrial respiration, and previously unknown targets affecting mite growth and development have been identified, offering new opportunities for mite control.     

Requirement of Host Signaling Mechanisms for the Action of Ptr ToxA in Wheat

Ptr ToxA, the host-selective toxin produced by Pyrenophora tritici-repentis, is genetically associated with the development of tan spot disease of wheat. The toxin was shown previously to cause a programmed cell death in the host that requires de novo mRNA and protein synthesis. In the present study, inhibitors of plant signaling mechanisms protected wheat leaves from toxin action, as determined by electrolyte leakage bioassays, when applied to leaves with toxin. Okadaic acid, calyculin A and phenylarsine oxide, all inhibitors of protein phosphatase activity, reduced toxin-induced electrolyte leakage by more than 90%. Inorganic calcium channel blockers (LaCl₃ and CoCl₂ reduced toxin-induced electrolyte leakage by 78–95%, depending on inhibitor and time of measurement. By comparison, about 50% protection was achieved by the application of the protein kinase inhibitors staurosporine and K-252A. Nonetheless, the reduction in toxin-induced electrolyte leakage by protein kinase inhibitors was reproduced in multiple trials and was statistically significant. The data indicate that host signaling mechanisms, including calcium fluxes and a protein phosphorylation cascade, are required for the Ptr ToxA-induced cell death in wheat. Our current model holds that the signaling events occur between toxin perception by the cell and the toxin-directed gene expression in the host associated with cell death. As an alternative, the toxin-induced mRNA synthesis required for cell death may be for protein phosphatase and/or protein kinase genes. Additional work is required to resolve these possibilities.     

Effect of δ-aminolevulinic acid on the herbicidal efficacy of foliar-applied MTB-951, a mycoherbicide to control Echinochloa crus-galli L.

The conidia of a plant pathogen ( Drechslera monoceras [Drechsler] Subram. et Jain [=  Exserohilum monoceras [Drechsler] Leonard et Suggs]), MTB-951, which was isolated from native Echinochloa species in Japan, were used as the herbicidal active ingredient. The effects of δ-aminolevulinic acid (ALA) on the efficacy of foliar-applied MTB-951 on Echinochloa crus-galli L. were examined. First, foliar-applied MTB-951 exhibited almost no efficacy when no dew period was provided. However, the efficacy increased depending on the length of the dew period. MTB-951 and ALA exhibited an additive effect on E. crus-galli under 3 day dew conditions, although they exhibited a significant synergistic effect under no-dew conditions. When the leaves of E. crus-galli were immersed in deionized water containing the conidia of MTB-951, cellular electrolyte leakage from the leaves was observed. The ALA increased the electrolyte leakage by MTB-951 synergistically. Also, ALA increased the number of lesions on the leaves of E. crus-galli caused by MTB-951 under light conditions but failed to increase the number under dark conditions. These results suggest that ALA promotes infection with MTB-951 for E. crus-galli through its photodynamic action.     

Do we have the tools to manage resistance in the future?

Pesticide resistance is a major factor affecting world food and fibre production, but that has been contained so far by the availability of diverse modes of action. Overcoming resistance by switching to a new mode of action is a concept easily grasped by growers but threatened by losses through resistance and new registration requirements. Opportunities for innovation and development of a diversity of novel modes of action exist through harnessing recent advances, fundamental to all eukaryotes and largely funded for medical rather than agricultural objectives, in understanding cell biology and development. The cystoskeleton, cell wall synthesis, signal transduction and RNAi are discussed as examples where new targets are now exposed. However, new modes of action will be delivered not only by sprayer or seed treatment but also through transgenic crops, although these still need to be transferred from experiment to practice. Improvements in modelling protein structures and target-site changes, supplemented by rapid diagnostics to detect resistance early, will improve resistance risk management and integrate chemical, biopesticide, transgenic and conventional breeding around the concept of diversity in modes of action. However, before agronomy can translate this into practical antiresistance strategies, there is a need to direct more resources to the biochemistry and cell biology of pests, diseases and weeds to translate these new discoveries into key tools needed to manage resistance in the future.     

中生菌素的抗生作用

 本文研究了中生菌素对革兰氏阳性菌、革兰氏阴性菌、丝状真菌的抗生作用。结果表明,中生菌素是一种抗菌谱比较广的农用抗生素。其对菌体细胞膜无明显的影响。100 ppm时引起部分菌体原生质凝聚。同位素标记前体物掺入实验表明,中生菌素15.6 ppm对白菜软腐病菌DNA、RNA的合成影响很小,但强烈抑制菌体蛋白质的合成。用化学测定法分析了中生菌素对枯草杆菌细胞内大分子物质含量的影响,得到了和前体物掺入法相似的结果。说明了中生菌素的作用机制是抑制菌体蛋白质的合成。     

小麦抗条锈反应中活性氧及细胞质钙离子的作用

 为探讨活性氧和细胞质钙离子在小麦抗条锈病反应中的作用,以小麦品种洛夫林13与具有不同致病力的单孢锈菌CY29、CY25的互作体系为平台,对锈菌侵染后小麦叶片中活性氧(ROS)积累、保护酶系(SOD、CAT和APX)活性变化动态、细胞质膜的透性改变及细胞质钙离子浓度变化做了研究。结果表明,不亲和锈菌CY25侵染可引起小麦叶片内2次ROS的爆发,第1次出现在接种后前期(接种后第2天),强度较小,第2次出现在接种后期(接种后第5天),强度较大;亲和锈菌CY29侵染只引起1次ROS爆发,出现在接种后期(接种后第5和第6天之间),但强度极大。过敏性坏死反应HR只出现在不亲和互作小麦叶片上前期ROS爆发之后,表明前期ROS爆发与HR的产生有关。伴随着后期小麦叶片中强度极高的ROS的爆发,叶片细胞原生质膜遭到了破坏,细胞内物质外渗,细胞不久便死亡,表明高强度的ROS爆发会导致细胞死亡。根据不同互作体系ROS爆发时期SOD、CAT和APX等保护酶的活性变化分析,不亲和互作体系前期强度较小的ROS爆发主要成分是H₂O₂,后期强度较高的ROS爆发主要成分是O₂^-_·和H₂O₂;亲和互作体系强度极高的ROS爆发主要成分是O₂^-_·和H₂O₂。由此说明H₂O₂是引起小麦抗病反应HR发生的因素,而O₂^-_·则是引起细胞死亡的因素。细胞质钙离子浓度变化研究表明,HR的发生与细胞质钙离子浓度增加相关。细胞质钙离子浓度的降低推迟了HR的发生,这说明小麦叶片细胞内细胞质钙离子浓度的增加是HR的必要条件,同时也说明Ca²⁺是植物HR的胞内第二信使参与植物抗病防卫反应。