Allelopathic compounds isolated from plants that exhibit inhibitory potential on seed germination and growth of weeds
Since the first implementation of synthetic herbicides in crop protection systems, weeds have continuously developed resistance. As a main reason of such evolution, long-lasting exploitation of herbicides with one target site in plants is considered. This has been the case with the first widely-used triazine herbicides, photosynthesis inhibitors, which have effectively eliminated a wide range of weeds. Unfortunately, inappropriate adjustment of herbicides to weed species occupying fields, application of herbicides at the incorrect developmental stage and in unsuitable weather conditions have contributed to the accumulation of active compounds in the soil, accumulation of weed species and acceleration evolution of resistant biotypes . To date, there have been 211 species and 393 biotypes of herbicide resistant weeds identified . Most of them are resistant to B, C1 and A groups of herbicides, inhibitors of: acetolactate synthase (ALS), photosystem II and acetyl CoA carboxylase, respectively. Ten species pose the biggest threat for crops due to causing yield losses, including the most important herbicide-resistant species which are characterized by multiple resistances: rigid ryegrass (
Evolution of weeds resistant to herbicides demands new solutions to cope with the problem since economic losses generated by weeds can be higher than those caused by other pests. Due to the fact that abandoning chemical weed control is, with current agricultural practices, rather impossible, it is necessary to create new classes of herbicides with new mechanisms of action and target sites not previously exploited. Presently used synthetic herbicides are not approved for use in organic agriculture. Moreover, using crop protection chemicals also need public acceptance. . The number of synthetic chemicals with new target sites are decreasing dramatically. Eco-friendly trends in weed management force scientists to reach for innovative sources and tools. Natural compounds pose a great field for the discovery of new environmentally safe herbicides, so called “bioherbicides”, which are based on compounds produced by living organisms. According to the CAS (Chemical Abstracts Service) registry, among the 24 million organic compounds, a large group of secondary plant metabolites is represented. Some of these compounds take part in allelopathic interactions.
2. Allelopathic interactions and allelopathic compounds
Allelopathy is considered a multi-dimensional phenomenon occurring constantly in natural and anthropogenic ecosystems . It is defined as the interaction between plants and microorganisms by a variety of compounds usually referred to as allelopathins, allelochemicals, or allelopathic compounds. This review is focused mainly on compounds taking part in complex allelopathic interactions between higher plants. However, determination of quality, quantity, direct or indirect effects of allelopathins on plant or microorganism communities in the natural environment is very difficult owing to the multi-dimensional character of those interactions. The development of analytical techniques allowing better specification of direct effects of allelopathins, have moved the exploration (or the research on) of this phenomenon from fields into laboratories. The term “allelopathy” refers rather to interactions occurring in the natural environment . For studies with plant extracts, allelopathins isolated from plant tissue, collected from exudates or even synthetic compounds identical to natural ones, it was established the term ‘‘phytotoxicity’’ to distinguish allelopathy (as a phenomenon occurring in natural environment) from studies conducted in laboratory.
Allelopathins are products of the secondary metabolism and are non-nutritional primary metabolites [6,7]. These compounds belong to numerous chemical groups including: triketones, terpenes, benzoquinones, coumarins, flavonoids, terpenoids, strigolactones, phenolic acids, tannins lignin, fatty acids and nonprotein aminoacids. A wide range of these biochemicals are synthesized during the shikimate pathway  or, in the case of essential oils, from the soprenoid pathway. Allelochemicals can be classified into 10 categories  according to their different structures and properties:
water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes, and ketones;
long-chain fatty acids and polyacetylenes;
quinines (benzoquinone, anthraquinone and complex quinines);
cinnamic acid and its derivatives;
steroids and terpenoids (sesquiterpene lactones, diterpenes, and triterpenoids).
Allelochemicals are released into the environment by plant organs such as roots, rhizomes, leaves, stems, bark, flowers, fruits and seeds (Figure 1a). The huge number of allelopathic interactions is typically negative in character, with positive relations being rare. Allelopathic compounds affect germination and growth of neighboring plants by disruption of various physiological processes including photosynthesis, respiration, water and hormonal balance. The underlying cause of their action is mainly inhibition of enzyme activity. Ability of an allelochemical to inhibit or delay plant growth and/or seed germination is usually defined as its “allelopathic (or phytotoxic) potential”. An excellent example of allelopathic interaction is seen in soil exhaustion due to the accumulation of allelopathins that can be prevented by using fertilizers and rotating crops. Plants producing allelopathins are considered as “donor” organisms while the plants which allelopathins are directed to are referred to as “target” plants or “acceptors”. The after-effects and strength of allelopathic interactions are diverse due to modifications of the allelopathins taking place in soil (Fig 1b). Most of the allelochemicals penetrate the soil as already plant-active compounds, e.g. phenolic acids, cyanamide, momilactones, heliannuols etc. Some have to be modified into the active form by microorganisms or by specific environmental conditions (pH, moisture, temperature, light, oxygen etc.), e.g. juglone, benzoxazolin-2-one (BOA), 2-amino-3-H-phenoxazin-3-one (APO).
3. Advantages and disadvantages of allelopathins as bioherbicides
Mode of action of some allelochemicals is similar to synthetic herbicides. These features have allowed them to be considered for possible use in weed management as bioherbicides. However, the field of knowledge is poorly studied but it is a very attractive area to explore.
Allelochemicals are highly attractive as new classes of herbicides due to a variety of advantages. However, in the perspective of bioherbicides based on allelopathins, effects caused by these compounds on target plants are also classified as “phytotoxic”.
Most of allelopathins are totally or partially water-soluble which makes them easier to apply without additional surfactants [3, 10]. Their chemical structure is more environmentally friendly than synthetic ones. They possess higher oxygen- and nitrogen-rich molecules with relatively few so called ‘heavy atoms’, a halogen substitute, and are characterized by the absence of ‘unnatural’ rings. These properties decrease a chemical’s environmental half-life, prevent accumulation of the compound in soil and eventual influence on non-target organisms. On the other hand, these properties are an allelochemical’s Achille’s heel due to less than satisfactory duration of activity. Structure complexity generates more stereocenters making them more reactive and unstable. Therefore, rapid degradation of one of the chemical groups can significantly decrease bioactivity of the whole compound.
The diversity of allelopathins makes them promising tools possessing specific properties in discovering novel, specific target sites in acceptor plants. Even if they inhibit photosynthesis or respiration, they may also bind to proteins at different sites than synthetic herbicides [11, 12]. This provides the opportunity to eliminate weeds that are already resistant to commercialized herbicides with the same mode of action. Allelochemicals are also characterized by multi-site action in plants without high specificity which is achieved in the case of synthetic herbicides. Therefore, this feature excludes the application of an allelopathic compound as a selective herbicide or totally prohibits its usage in weed management. On the other hand, effects of allelopathins in acceptor plants are highly dose-dependent . This allows the opportunity to search out compounds exhibiting selectivity. Generally, monocotyledonous plants are more resistant to allelochemicals than dicotyledonous ones. Therefore, usage of a compound as a potential herbicide is possible but rather restricted to cultivation of exact crops with a defined weed composition.
The route of discovery is much more complicated with allelopathins. In contrast to synthetic herbicides where synthesis, bioassay, evaluation and quantitative structure-active relationship follow Quantitative Structure-Activity Relationship (QSAR), allelochemicals have to be first isolated from plant extracts . The amount of recovered compounds is usually low in comparison to chemical synthesis. After extraction, purification and selection of the most attractive compound and determination of its mode of action in plants is done. At the end of the process, similar to synthetic herbicides, allelpathins are subjected to QSAR. The long discovery process is usually offset by a shorter, less expensive track of registration . It is worth noting that before an allelochemical can become an herbicide, the following conditions have to be performed: phytotoxic activity at the range between 10-5 and 10-7 M, identified chemical structure, known mode of action in plants, time of residence in soil, possible influence on microbial ecology and non-target plants, possible toxic properties on human health and profitability of production on a commercial scale .
A high number of limitations does not exclude allelochemicals as possible herbicides. In particular, they can be alternatives in weed management strategy. Widely developed bioinformatics and cheminformatics support development of new herbicides [3, 15, 16]. Identified chemical structure of a particular allelochemical is a starting point to design a product with the compound-like properties using computer programs. Thanks to cheminformatics we are able to predict the potential structure of analogues and make several modifications, which make it more or less active, with higher environmental stability, as it was done for leptospermone. We may also predict the target site of compound action in plants due to comparison studies. Similar structure of a compound to a commercialized herbicide or other natural compound whose mode of action is well-known may allow us to predict the target site.
4. Allelopathic plant extracts as bioherbicides
Plant protection is effective but rather costly and problematic due to environmental pollution. Exploration of the allelopathic potential of some species allows the introduction of alternative techniques for weed management, e.g. extracts from allelopathic plants can be applied as foliar sprays. Apart from decreasing the costs of herbicide application, this method also improves crop production.
The best known examples of natural bioherbicides are phytotoxic water extracts from herbage of sorghum (
Effects of sorgaab on weeds is time- and dose-depend but is typically used at 5% or 10% (w/v) concentration as double spray 20/30 and 40/60 days after sowing (DAS) or after seedling transplantation (AT) [17-19]. The best results to account for net profits have been elicited with a double spray of 10% extract in cotton (
Sunfaag has been widely used in wheat. The extract has been usually applied three times at 7-day intervals starting between 3-4 weeks post-emergence. This system of application has reduced biomass of the two most commonly occurring weeds, lambsquarters (
Aqueous extracts of sorghum and sunflower are effective on weed growth but unfortunately might not be profitable enough in crop production; however, crop allelopathy can be manipulated for achieving sustainable weed management. Combination of phytotoxic crop water extracts with lower rates of herbicides may provide reduced weed control levels with reduced herbicide usage. The interesting review of allelopathic crop plants in weed management strategy is presented in reference . Two field studies were conducted utilizing water extracts of sorghum, sunflower and rapeseed (
Selectivity of plant extracts on weeds without any negative implications on crop productivity is probably due to differences in the physiological stage of plants and following plant competition. Sunfaag has been applied when wheat seedlings were 3-4 weeks old while lambsquarters and toothed dock 1-week old at the stage of three to four leaf [20, 21].
High allelopathic potential conditioned by glucosinolates and isothiocyanates is present in Brassica sp. [27, 28]. Isothiocyanates have been strong suppressants of germination of spiny sowthistle (
Legumes crops may also be applied as a source of allelochemicals useful in weed suppression. Mulch of dead pea plants could be used to control growth of weeds. Pea cover crop has regulated germination and growth of lady’s thumb (
Application of plant extracts as pre-emergence or as early post emergence herbicides resulted in reduction of doses of synthetic herbicide due to their synergistic or additive action. However, not all phytotoxic extracts are effective enough to inhibit weed growth or germination when applied as spray even when plants show high allelopathic potential as mulch, intercropping system or in rotation. This may be the result of masking the activity of one compound by another in water solution or other factors such as impossibility of extract penetration through the cuticle . A new opportunity to enhance effectiveness of usage of bioherbicides based on natural extracts is associated with extraction of individual allelochemicals and/or its comparison with synthetic herbicides. The extraction of sesquiterpene lactone, dehydrozaluzanin C (DHZ) produced among Compositae family serves as an example . Comparison studies of isolated DHZ (1 mM) and the commercial herbicide Logran® showed high inhibitory activity of DHZ on dicotyledonous plants while the synthetic herbicide showed no activity . Also pure 2-benzoxazolinone (BOA) isolated from several graminaceous crops such as rye (
5. Plant allelopathins as sources of bioherbicides
Plant phytotoxic extracts, after evaluation, can be successfully used in integrated weed management. However, as was aforementioned, not all systems of its application under field conditions are suitable and profitable enough. To circumvent masking effects of one allelopathin by another in plant extract, research is now focused on isolation and application of a single, specific compound for the purpose of weed elimination. The list of allelochemicals isolated from various plants that may act as inhibitors of weed seed germination and/or weed growth are summarized in Table 1. A purified allelopathic compound may act on target plants with much higher or much lower strength. Even in situations when an allelopathin is active at unprofitably high doses but has a favorable environmental profile, it still may be a source to explore due to several reasons such as biodegradability. Modifications of chemical structure can make a compound more active on target plants while preserving desire properties.
Herein, examples of purified allelopathins with possible roles as herbicides are described. Some herbicides based on modified allelopathins already launched on the market are also included.
|Glucosinolates, Isothiocyanates||mustard (|
garden radish (
|spiny sowthistle (|
|Sorgoleone||sorghum (||littleseed canarygrass (|
|Momilactone||rice (||barnyardgrass, (|
|Artemisinin||annual wormwood (||redroot pigweed, pitted morning-glory (|
|Leptospermone||bottle brush (||barnyard grass, hairy crabgrass, yellow foxtail (|
|Essential oils||eucalyptus ||barnyard grass, |
|Sarmentine||pepper ||barnyard grass, redroot pigweed, crabgrass, Sprangletop (|
The inhibitory effect of sorghum on various plant species has been known for many years. Accumulation of sorghum phytotoxins in soil affects crop growth and imposes the need for a crop rotation system. Besides crops, weeds are also vulnerable to its allelopathic influence [16, 36]. Sorghum toxicity is mainly determined by both hydrophilic phenols in herbage, as well as hydrophobic sorgoleone and its analogs exuded by the root hairs [37, 38]. Therefore, sorghum herbage reach can be successfully used against weeds as a foliar spray as it is discussed in detail in the previous chapter.
However, allelochemical sorgoleone has enormous potential as an herbicide due to its high activity against various weed species. Studies conducted under laboratory conditions have shown that low doses of sorgoleone (100 μM) inhibit growth of the following weeds by 80%, black nightshade (
Sorgoleone released into the soil may act as a pre-emergence herbicide. Its persistence in the soil during or after sorghum cultivation inhibits germination and growth of small-seeded weeds, e.g. hairy crabgrass and green bristlegrass (
Inhibition of H+-ATPase in plant roots makes sorgoleone an effective growth inhibitor and potential post-emergence herbicide . Decreased activity of that enzyme affects ion uptake and water balance by decreasing water uptake and affecting plant growth. Redroot pigweed, Jimson weed (
Sorgoleone may be taken up by roots but cannot be translocated acropetally by xylem due to high lipophilic properties. Therefore, its application as a post-emergence herbicide may be limited. However, as a spray (0.6 kg ha-1), it has inhibited growth by 12% of green foxtail (
Due to the structural similarity of sorgoleone to plastoquinon, it acts as a photosystem II (PSII) inhibitor [11, 43]. It binds to the niche of the D1 protein in PSII, gathers electrons and does not allow reoxidation of plastoquinon A by the secondary electron acceptor, plastoquinone B. Competition studies under sorgoleone
Extracts and residues of rice, the well-known cereal plant, also have allelopathic potential. Among isolated secondary metabolites, phenolic acids, hydroxamic acids, fatty acids, terpenes and indoles were identified . The key role in rice allelopathy plays momilactone A and B isolated from root exudates. High allelopathic rice varieties release up to 2-3 μg of momilactone B per day . These compounds inhibited the growth of typical weeds in rice, e.g. barnyard grass and awnless barnyard grass (
Artemisinin is a sesquiterpenoid lactone of annual wormwood (
Other controversies around the phytotoxic potential of artemisinin arose when the dichloromethane extracts of annual wormwood leaves containing artemisinin showed a stronger phytotoxic effect on redroot pigweed seed germination and seedling growth than pure artemisinin . Moreover, aqueous extract with disposed artemisinin had equal inhibitory effects on both physiological processes as allelopathin alone. This experiment suggests a marginal role of artemisinin in plant extract and joint action of other allelochemicals. Although, most studies analyzing allelopathic weed–crop interferences using annual wormwood were conducted under laboratory and greenhouse conditions .
Toxic studies on duckweed (
Leptospermone (1-hydroxy-2-isovaloryl-4,4,6,6-tetramethyl cyclohexen-3,5-dione) is a natural triketone produced by the roots of the bottlebrush (
However, leptospermone has lately been found as the main herbicidal component of manuka oil (
5.5. Essential oils
Lately, there has been a growing interest for using essential oils as allelopathins with bioherbicide potential. Some of them have already been commercialized and successfully launched in organic agriculture. They disrupt the cuticle and contribute to desiccation or burn down young tissues. Examples of this are the commercially available bioherbicide with the trade name of GreenMatch EX which consists of lemongrass (
Sarmentine was first isolated from long pepper (
As an herbicide, sarmentine and its derivatives may be both obtained from fruits of long pepper and successfully chemically synthesized . Despite the fact that the compound is active under laboratory conditions, its chemical and biological instability under field conditions may limit its application as an herbicide. However, it has been shown that crucial for sarmentine herbicidal activity is the presence of an amine bond with a secondary amine. Replacement of the acid moiety with structurally similar fatty acids has not changed its phytotoxic potential. Moreover, natural herbicides based on sarmentine may also contain other derivatives with similar modes of action on plants but higher environmental stability . Sarmentine may be successfully applied in combination with synthetic herbicides, e.g. aryloxyphenoxypropionic, benzoic acid, dicarboximide, organophosphorus, triazine, sulfonamide herbicides and with many others. This gives an opportunity to further the structural modification that makes the compound more stable without any disadvantages on bioherbicide action in plants. It is worth noting that sarmentine has already been patented as an herbicide but not commercialized yet .
6. Biotechnology in bioherbicide investigation
A lot of effort has been done to explore the nature of allelopathic interactions. Studies on allelopathic compounds greatly increased thanks to chemical and biochemical techniques, which improved identification and knowledge about its mode of action. Since then, the crucial role of secondary metabolites synthesized and released by plants became better understood. It has been clearly demonstrated that allelopathins may take part in very complex inter- and intra-specific ecological interactions including soil microorganisms. However, despite the extensive research carried out under laboratory conditions, the higher level of such interactions at the ecosystem level has not been sufficiently explored. Structure, chemical properties, and mode of action in plants of multitude allelochemicals are already known but, unfortunately, only a part-per thousand of them have been successfully introduced in agricultural practices. This is mainly due to limitations of compounds as plant protection agents but also due to extended field experiments. A very important aspect that allows the introduction of allelopathy to natural weed management is knowledge about biology of donor and target plants and the exact chemicals responsible for the interaction . All formerly described limitations of natural compounds as bioherbicides decreasing in case of plant extracts as herbicides due to simple and low cost of application. However, separation of one, specific compound that is the most interesting for us among hundreds synthesized by plants often required information about its synthesis
One of the problems is to obtain adequate amounts of the compound, when its chemical synthesis is impossible or collection of plants, unprofitable. Increased synthesis of an allelopathin gives triple profits. First of all, enhanced allelopathic potential of a plant makes it more competitive against weeds. Second of all, increased concentration of a compound makes plant extract more active. Thirdly, this allows collection of the compound at a sufficient amount and makes it more profitable. However, it is much easier to obtain active compounds from the crop species than wild living ones. Difficulties in introducing plants to cultivation are due to the low ability to grow outside their natural ecosystem .
Cells and organ cultures provide opportunities to circumvent these limitations. Abilities of undifferentiated and differentiated cells to produce allelochemicals may be commercialized in bioreactors using cell suspension cultures . Such attempts have been made on Artemisia suspension culture for artemisinin production; however, obtained amounts of that compound were insufficient. The addition of β-cyclodextrins to the growing medium has increased artemisinin synthesis up to 300% . Allelochemicals produced by roots may be obtained from hairy root cultures, both
The recombinant DNA technology can be useful to improve allelochemical production. Enhancing or suppression of gene expression, metabolic engineering and genetic transformation are promising new tools for allelochemical synthesis . This approach is based on elucidation of the metabolic pathway, enzyme activities and identification of genes encoding crucial enzymes, associated with metabolite (allelochemical) synthesis.
Allelopathy is a quantitative trait. A genetic analysis of quantitative trait loci (QTL) is a promising approach to identify genes underlying this trait. Only a few crops are under genetic screening for its allelopathic properties including: rice, wheat, barley and oat [83, 84]. The first QTL map associated with allelopathic properties was developed in rice. A segregating population derived from a cross of two cultivars varying with allelopathic potential against barnyardgrass. The map contained 140 DNA markers with four main-effects QTL located on chromosome 2, 3 and 8 . Proteomic studies on allelopathy of rice against barnyardgrass confirmed the crucial role of three enzymes: phenylalanine ammonia-lyse (PAL), thioredoxin and 3-hydroxy-3-methilglutarilcoenzyme A reductase 3 (HMGR) is highly involved in phenols biosynthesis . Such a genetic approach may allow the location of the gene in the genome and better understanding of its function in plant allelopathy and create the chance of applying marker assisted selection (MAS) to enhance allelopathic abilities.
Just like breeding programs allow improved crop production, they may also improve production of allelopathic compounds increasing allelopathic potential.
Scopoletin has been known as allelopathic root exudates of oats (
The situation becomes more complicated when more than one gene encoding special enzymes is required to increase synthesis of a plant compound. Such difficulties have been encountered for DIBOA, synthesized by various grass species . In maize, biosynthesis of this compound is determined by five genes (
Monoterpenes are a large family of compounds produced by a varied family of aromatic plants. Some of the monoterpenes also take part in allelopathic interactions, e.g. linalool, cineole camphene, pinene, limonene, etc. Currently, metabolic engineering allows improved production of specific compounds in heterologous systems . The most interesting are monoterpene synthases which catalyzed geranyl diphosphate (GPP) into output structure of numerous monoterpenes family, e.g. enhanced expression of limonene synthase in transgenic peppermint (
However, we have to bear in mind that biosynthesis of natural compounds can be limited to organs, tissues or even cells. Specific locations of compound synthesis, accumulation or secretion often make that compound toxic to other tissues within the same plant organism. Moreover, even successful transformation of a plant does not guarantee successful and sufficient production of a desirable compound. The gene of (S)-linalool synthase (
All presented techniques provide greater knowledge on allelopathy. However, better understanding of such complex interactions among this phenomenon bring us one step forward to development of new strategies in weed management and finding new herbicides and new herbicidal target sites.
The phenomena of allelopathy and phytotoxic interactions between plants are strongly expanding branches of biological science. Allelochemicals, as a group of substances also called biocommunicators, seem to be a fruitful challenge for combining traditional agricultural practices and new approaches in pest management strategies. Allelochemicals have already been used to defend crops against pathogens, insects or nematodes, parallel to some attempts to use them for weed control. Crop rotation, cover crops, dead and living mulches are being employed in agriculture. Both in natural and agricultural ecosystems allelopathic interactions are involved in practically every aspect of plant growth, as they can play the role of stimulants and suppressants. Complex plant-plant and plant-microbe interactions in ecosystems and currently developing studies on molecular, cytological and physiological levels bring us to a better understanding of processes occurring around us. The ancient knowledge of well-known toxic properties of water extracts of a variety of allelopathic plants give us a basis that could be used in the creation of a novel approach in weed control.
Some allelochemicals, mainly these that are mentioned in the text above, may act as a starting point for production of new bioherbicides with novel target sites, not previously exploited, as the understanding of their mode of action is still growing. Creation of bioherbicides based on allelochemicals generates the opportunity to exploit natural compounds in plant protection and shows the possibility to cope with evolved weed resistance to herbicides. Despite the fact that we have extensive knowledge about the chemical nature of natural compounds, we can synthesize its analogues, and we have basically explored its phytotoxic potential, we still have insufficient data. Until recently, most studies on phytotoxicity have been conducted under laboratory conditions due to the ability to eliminate other environmental factors such us temperature, soil texture and its chemical and physical properties. Such approach allows the recognition of only direct effects of allelochemical action. There is still a great need to transfer laboratory data into field conditions. Such experiments are not willing to be taken on due to troublesome field experiments dependent on environmental conditions and a few year repetitions. New tools of molecular genetics, proteomics and metabolomics profiling as well as modern and sophisticated methods of chemistry and biochemistry will lead to the creation of substances, maybe based on the structure of particular compounds occurring in nature, which could be used without any risks as selective and eco-friendly herbicides.