Fungal phytotoxins: from basic studies to practical use (a review)

Recent materials are summarized, pertaining to classification of fungal phytotoxins, methods of their isolation, and assays for biological activity. Producers of phytotoxic substances have been characterized, and the chemical nature of phytotoxins has been subjected to analysis. The review gives consideration to the mechanisms of action of phytotoxins on susceptible plants and the mechanisms of plant resistance to such agents. Other matters discussed include prospects of utilizing basic knowledge of the nature and mechanisms of action of phytotoxins for developing means of plant protection against diseases and weeds and identifying or classifying fungi (chemosystematics).     

Physiological and cellular aspects of phytotoxicity tolerance in plants: the role of membrane transporters and implications for crop breeding for waterlogging tolerance

Waterlogging affects large areas of agricultural land, resulting in severe economic penalties because of massive losses in crop production. Traditionally, plant breeding for waterlogging tolerance has been based on the field assessment of a range of agronomic and morphological characteristics. This review argues for a need to move towards more physiologically based approaches by targeting specific cellular mechanisms underling key components of waterlogging tolerance in plants. Also, while the main focus of researchers was predominantly on plant anoxia tolerance, less attention was given to plant tolerance to phytotoxins under waterlogged conditions. This paper reviews the production of major elemental and organic phytotoxins in waterlogged soils and describes their adverse effects on plant performance. The critical role of plasma membrane transporters in plant tolerance to secondary metabolite toxicity is highlighted, and ionic mechanisms mediating the this tolerance are discussed. A causal link between the secondary metabolite-induced disturbances to cell ionic homeostasis and programmed cell death is discussed, and a new ethylene-independent pathway for aerenchyma formation is put forward. It is concluded that plant breeding for waterlogging tolerance may significantly benefit from targeting mechanisms of tolerance to phytotoxins.     

Flow cytometry as a method for assaying the biological activity of phytotoxins

A flow cytometric method has been developed for the qualitative and quantitative evaluation of the biological activities of phytotoxins from plant pathogenic fungi. The method utilized fresh wheat (Triticum aestivum L.) leaf protoplast preparations treated with purified phytotoxins, triticone A-B and triticone D. Subsequently, protoplasts were exposed to fluorescein diacetate, and analyzed by flow cytometry. Information acquired included fluorescence owing to esterase activity on fluorescein diacetate, and chlorophyll autofluorescence. Results indicate that triticone A-B has a rapid dose-dependent toxic effect on wheat protoplasts but triticone D has no toxic effect. This method can also yield information on the mechanism of action of phytotoxins that are relatively unstable or available only in small quantities.     

Phytotoxins as tools in studying plant disease resistance

Many plant pathogenic fungi and bacteria produce phytotoxins which are a vital part of their arsenal in causing plant disease. Some host-specific toxins have proved useful in enhancing our understanding of the biochemical mechanisms underlying plant disease resistance.     

The phytotoxicity ofFusarium metabolites: An update since 1989

The present article summarises the published phytotoxic effects of severalFusarium metabolites (mycotoxins, phytotoxins, antibiotics and pigments) since 1989. The phytotoxicity of many of the commonly isolated metabolites cannot be disputed, but their role in pathogenesis ofFusarium-induced plant diseases is uncertain. Plant species/varieties differ in their susceptibililty resistance to these toxinsin vitro, as well as toFusarium pathogens under field conditions. Such variations in plant response may reflect resistance mechanisms that operate at several levels, including an initial ability to prevent fungal invasion; prevention of fungal spread and toxin tolerance or degradation. Little is known about the mode of action of most of these metabolites on either animal or plant cells. Several novelFusarium metabolites have been isolated in the past few years. Many are toxic to animals and cell lines, but assessment of their phytotoxicity has largely been neglected. Since many plant pathogenic Fusaria produce a plethora of metabolites, the additive or synergistic actions of toxins in combination must be considered in plant pathology.     

Plant growth inhibitors isolated from sugarcane (Saccharum officinarum) straw

Several compounds related with plant defense and pharmacological activities have been isolated from sugarcane. Straw phytotoxins and their possible mechanisms of growth inhibition are largely unknown. A bioassay-guided fractionation of the phytotoxic constituents leachated from a sugarcane straw led to the isolation of trans-ferulic (trans-FA), cis-ferulic (cis-FA), vanillic (VA) and syringic (SA) acids. The straw leachates and their identified constituents significantly inhibited root growth of lettuce and four weeds. VA was more phytotoxic to root elongation than FA and SA. The identified phenolic compounds significantly increased leakage of root cell constituents, inhibited dehydrogenase activity and reduced chlorophyll content in lettuce. VA and FA inhibited mitotic index while SA increased cell division. Additive (VA-FA and FA-SA) and synergistic (VA-SA) interactions on root growth were observed at the response level of EC(25). Although the isolated compounds differed in their relative phytotoxic activities, the observed physiological responses suggest that they have a common mode of action. HPLC analysis indicated that sugarcane straw can potentially release 1.43 (ratio 2:1, trans:cis), 1.14 and 0.14mmolkg(-1) (straw dry weight) of FA, VA and SA, respectively. As phenolic acids are often found spatially concentrated in the top soil layers under plant straws, further studies are needed to establish the impact of these compounds in natural settings.     

Glycosyltransferases: managers of small molecules

Studies of the glycosyltransferases (GTs) of small molecules have greatly increased in recent years as new approaches have been used to identify their genes and characterize their catalytic activities. These enzymes recognize diverse acceptors, including plant metabolites, phytotoxins and xenobiotics. Glycosylation alters the hydrophilicity of the acceptors, their stability and chemical properties, their subcellular localisation and often their bioactivity. Considerable progress has been made in understanding the role of GTs in the plant and the utility of GTs as biocatalysts, the latter arising from their regio- and enantioselectivity and their ability to recognize substrates that are not limited to plant metabolites.     

Animal Toxicity of Phytopathogenic Fungi

Twelve genera of phytopathogenic fungi comprising 27 species previously reported to produce phytotoxins were tested concurrently for animal and plant toxicity. There appeared to be no direct relationship between plant and animal toxicity.     

Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents.     

Pseudomonas syringae phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases.

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents.     

Joint action of phenolic acid mixtures and its significance in allelopathy research

Although the ecological significance of mixtures of phytotoxins is recognized in research on chemical plant interference (allelopathy), few studies convincingly demonstrate the joint action of phytotoxin mixtures, key to understanding the ecological impact of these materials, using established models from other biological disciplines, e.g. toxicology and pharmacology. Addressing this need, the present study investigates the joint action of the phenolic acids, p -hydroxybenzoic, p -coumaric and ferulic acids on root growth inhibition of perennial ryegrass ( Lolium perenne L). The Additive Dose Model (ADM) isobole and estimated concentration of phenolic acid mixture were calculated on ED₂₀, ED₅₀ and ED₈₀ from the dose–response curves for the phenolic acids applied alone or in mixtures of fixed ratios. The binary combination of three selected phenolic acids is generally antagonistic relative to the ADM. No evidence for synergistic activities of phenolic acids in the mixture was noted. Since allelopathic activities in nature are largely due to the presence of several compounds in a mixture, the present study advances understanding of the joint action of binary combination of allelochemicals in a mixture.     

Rotting softly and stealthily

The soft rot erwiniae, which are plant pathogens on potato and other crops world-wide, synthesize and secrete large quantities of plant cell wall degrading enzymes that are responsible for the soft rot phenotype, earning them the epithet 'brute force' pathogens. They have been distinguished from classic 'stealth' pathogens, such as Pseudomonas syringae, which possesses an extensive battery of Type III secreted effector proteins and phytotoxins to manipulate and suppress host defences. However, recent studies, including whole-genome sequencing, are revealing many components of stealth pathogenesis within the soft rot erwiniae (SRE), suggesting that 'stealth' and 'brute force' should not be regarded as mutually exclusive modes of pathogenesis.     

Ecophysiological Approach in Allelopathy

Allelopathy is a process that can be present in many ecosystems, according to the literature. Nevertheless, the authors think that, due to the evolutionary constraints, especially from a co-evolution point of view, this process cannot be very extended except in some conditions. Allelopathy can be important when an invader plant affects to the autochthonous species, when soil microorganisms cannot cope with a new molecule and target plants have not co-evolved. It can be important in some conditions when there is a continuous release of allelochemicals into the environment or when there is a very limited metabolism of those substances. Most agricultural and forestry practices tend to increase the possibilities of such a process to be important. Allelopathy can be effective only when plants are in stress due to other mechanisms, for example, when there is a lack of water or competition for nutrients or light is strong. In those conditions, allelochemicals production has been shown to increase. Sometimes there are allelochemicals that are not normally produced if the plant is not under stress. Under stress, the target plant is also more susceptible to the effect of the released phytotoxins. A multidisciplinary ecophysiological approach is needed in studying allelopathy in conjunction with other mechanisms that affect plants. The study can range from the ecological level to the molecular one. New methods are requested to separate the effects of allelopathy from competition.     

Focus on Weed Control: Natural Compounds as Next-Generation Herbicides

Herbicides with new modes of action (MOAs) are badly needed due to the rapidly evolving resistance to commercial herbicides, but a new MOA has not been introduced in over 20 years. The greatest pest management challenge for organic agriculture is the lack of effective natural product herbicides. The structural diversity and evolved biological activity of natural phytotoxins offer opportunities for the development of both directly used natural compounds and synthetic herbicides with new target sites based on the structures of natural phytotoxins. Natural phytotoxins are also a source for the discovery of new herbicide target sites that can serve as the focus of traditional herbicide discovery efforts. There are many examples of strong natural phytotoxins with MOAs other than those used by commercial herbicides, which indicates that there are molecular targets of herbicides that can be added to the current repertoire of commercial herbicide MOAs.The evolutionary forces driving the survival of species include chemical interactions between organisms, which function in positive interactions such as mutualistic and symbiotic relationships and negative interactions such as competitive and parasitic relationships. These processes have led to the emergence of novel secondary metabolic pathways (often through gene duplication), producing a vast array of structurally diverse and biologically active molecules (Moore and Purugganan, 2005; Ober, 2005; Flagel and Wendel, 2009; Jiang et al., 2013). This evolutionary process is similar to a high-throughput screen. However, unlike conventional in vitro screens, which test many compounds on a single biochemical target over a very short period of time, this natural high-throughput process selects molecules based on their whole-organism activities, involving numerous chemical interactions between countless organisms and target sites over millions of years. To date, approximately 200,000 secondary metabolites have been identified (Tulp and Bohlin, 2005), with many more expected to be discovered. Few of these compounds have been examined for phytotoxicity, and the modes or mechanisms of action (MOAs) of even fewer known phytotoxins have been elucidated.The negative chemical interactions between organisms are often characterized using anthropomorphic language, such as chemical warfare, referring to the production of phytotoxins used by plant pathogens to invade their host plants (Maor and Shirasu, 2005), and the novel weapons hypothesis, which is associated with the chemical-based advantage of some invasive plant species over native plant populations (Callaway and Aschehoug, 2000; Callaway and Ridenour, 2004; Callaway and Maron, 2006; Cappuccino and Arnason, 2006; Callaway et al., 2008). While simplistic, this terminology illustrates how these toxin-based interactions exploit biochemical weaknesses between an organism and its host or enemy/competitor to enhance its own survival (Verhoeven et al., 2009). In fact, these interactions can even be multitrophic, such as when exotic plants enhance their invasiveness by promoting the growth of certain native soil pathogens noxious to native plants (Mangla et al., 2008; Barto et al., 2011).As humans evolved from a nomadic hunter-gatherer subsistence existence to an agricultural lifestyle, they learned to utilize certain biologically active secondary metabolites to manage agricultural pests. Indeed, the concept that nature is an excellent source of natural pesticides is captured in the following ancient Lithica poem (circa 400 B.C.): “All the pests that out of earth arise, the earth itself the antidote supplies” (Ibn et al., 1781). Less than a century later, Greek and Roman treatises described practices to control agricultural pests that include the use of essential oils. Similar documents are found in Chinese literature, such as a survey describing plant species used to control plant pests (Yang and Tang, 1988). The mid-20th century ushered in the use of synthetic pesticides, which have revolutionized agriculture. Like pharmaceuticals (Harvey, 1999, 2008; Newman and Cragg, 2012), many pesticides are based on natural compounds. However, natural products have not played a major role in herbicide discovery (Copping and Duke, 2007; Hüter, 2011).     

An overview of the effects of phytotoxins on Phragmites australis in relation to die-back

The accumulation in sediments of the low molecular weight, volatile, monocarboxylic acids and/or sulphide, generated by the decay of (i) the underground parts of the reed and/or (ii) organic deposits produced under eutrophic conditions, may play a crucial part in Phragmites die-back. In the field high levels of some of these phytotoxins have been detected at certain die-back sites and in sediments containing the rotting underground parts of the plant.Symptoms of die-back include a clumped habit, stunting and death of roots and shoots, bud death, premature senescence of shoots, weakened stems, impeded aeration of the underground parts of the plant due to callus development, blockages within the vascular systems, lignification and suberisation of laterals and apical regions of adventitious roots and lower levels of starch in rhizomes. Poor convective aeration of the rhizome system has also been associated with higher proportions of alanine, γ-amino butyric acid and serine in culm bases, indicating hypoxic metabolism in the underground system.In laboratory experiments plants developed almost all of the growth, morphological and anatomical symptoms of die-back when treated with various single phytotoxins, e.g. acetic, propionic, n- and iso-butyric and n-caproic acids and sulphide at concentrations reported for die-back sites and/or associated with the decaying underground parts of the plant. For each acid alone, a concentration of ca. 1 mM, was highly toxic at pH 4.5, but relatively non-toxic at pH 6.0. However, when a cocktail of the five acids was applied (where each acid was 1 mM) the mixture proved to be toxic even at pH 6.     

The Effect of Stereochemistry on the Biological Activity of Natural Phytotoxins,Fungicides, Insecticides and Herbicides

Phytotoxins are secondary microbial metabolites that play an essential role in the development of disease symptoms induced by fungi on host plants. Although phytotoxins can cause extensive—and in some cases devastating—damage to agricultural crops, they can also represent an important tool to develop natural herbicides when produced by fungi and plants to inhibit the growth and spread of weeds. An alternative strategy to biologically control parasitic plants is based on the use of plant and fungal metabolites, which stimulate seed germination in the absence of the host plant. Nontoxigenic fungi also produce bioactive metabolites with potential fungicide and insecticide activity, and could be applied for crop protection. All these metabolites represent important tools to develop eco‐friendly pesticides. This review deals with the relationships between the biological activity of some phytotoxins, seed germination stimulants, fungicides and insecticides, and their stereochemistry. Chirality 25:59–78, 2013. © 2012 Wiley Periodicals, Inc.     

Biotechnological detoxification: an unchanging source–sink balance strategy for crop improvement

The wide occurrence of natural phytotoxins renders many crops unfit for human consumption. To overcome this problem and produce detoxified crop varieties, we propose the use of biotechnological strategies that can enhance the harvest index without the need to increase crop biomass or alter whole plant architecture.     

Effects of Alternaria alternata f.sp. lycopersici toxins on pollen

Summary Effects of the phytotoxic compounds (AAL-toxins) isolated from cell-free culture filtrates of Alternaria alternata f.sp. lycopersici on in vitro pollen development were studied. AAL-toxins inhibited both germination and tube growth of pollen from several Lycopersicon genotypes. Pollen from susceptible genotypes, however, was more sensitive for AAL-toxins than pollen from resistant plants, while pollen of species not belonging to the host range of the fungus was not significantly affected by the tested toxin concentrations. AAL-toxins elicit symptoms in detached leaf bioassays indistinguishable from those observed on leaves of fungal infected tomato plants, and toxins play a major role in the pathogenesis. Apparently, pathogenesis-related processes and mechanisms involved in disease resistance are expressed in both vegetative and generative tissues. This overlap in gene expression between the sporophytic and gametophytic level of a plant may be advantageously utilized in plant breeding programmes. Pollen may be used to distinguish susceptible and resistant plants and to select for resistances and tolerances against phytotoxins and other selective agents.     

Modern aspects of studying phytohormones. Cytokinins

The review presents current data on mechanisms of cytokinin action in plants. By analogy with the first part (Ivanova et al., 1999), in which general principles of phytohormone action and cardinal trends of phytohormone investigations were examined, here the relevant information on mechanisms of action of auxins and gibberellins has been given, and taking cytokines as example an attempt has been done to summarize the literature data on the number of questions offered for analysing hormones of high animals (Gudwin, Merser, 1986). The review demonstrates that mechanisms of cytokine action at the cellular level are not known in many cases. One of the most significant factors in the action of phytohormones of this class on plants is their concentration, determined by their synthesis, transportation and further chemical conversions. This paper points to a poor knowledge of the relative role of these processes in regulation of cytokinin contents and their distribution among plant organs. Two possible ways of studying cytokinin action at the present day stage of investigations have been designated: 1) revealing the cytokinin expressed genes and establishing mechanisms of their action; 2) estimation of endogenous cytokinin alteration and the influence of this alteration on definite processes in the cell with the help of ipt-gene from t-DNA of Agrobacterium tumefaciens.