SPECIAL REPORT
ALLELOPATHIC CHEMICALS Allelopathy has been described as chemical warfare between plants. Although the war may be silent and the tactics subtle, the outcome is often decisive. There are winners and there are losers, and the winners often dominate the territory. Today, scientists are trying to exploit allelopathic agents (allelochemicals) for use in agriculture. Allelochemicals are attractive because they are natural products made by plants or microbes. One could either manipulate the producing organisms in the field or use them as "factories" for batch production of the chemicals. Many think that natural products are environmentally safe chemicals, but research shows that a few can be extremely toxic if ingested by animals. The Austrian botanist Hans Molisch, who coined the term allelopathy in 1937, indicated that he meant it to include toxicities exerted by microorganisms (bacteria, actinomycetes, fungi), as well as by higher plants. This use has been followed by almost all scientists since that time. The term allelopathy literally translates as "mutual harm." In reality, the emitting species generally gains some advantage, and the recipient is harmed. Humans have observed for centuries that certain plant species tend to become predominant in both cultivated and natural settings. Although plants differ in their capacity to use resources such as water, nutrients, or light, another mechanism that apparently contributes to their aggres34
April 4, 1983 C&EN
Natures herbicides in action Alan R. Putnam Michigan State University
Leaves, roots, and litter are allelochemical sources Volatilization from leaves
Leaching from leaves by rain, fog, or dew Leaching from plant litter
Decay of plant litter
*
Exudation from roots
Decay of sloughed tissue from roots
Allelopathic chemicals from plants may be released from living leaves as volatiles or leachates or from roots through exudation or sloughing off of dead tissues. They also may be leached from leaf litter on the soil surface
siveness is allelopathy resulting from the release of chemical inhibitors that have an adverse effect on neighboring recipient plants. These allelochemicals may influence the associated plant directly or may act in a more subtle manner by inhibiting symbionts (beneficial microorganism) of the plant. Allelopathy may occur by exudation of compounds from living roots, leaves, or fruits or by leaching them from decaying plant residues. In the latter instance, soil microbes (actinomycetes, fungi, bacteria, algae) may play a big role in synthesizing or releasing the toxic agents. Allelopathy represents the plantagainst-plant aspect of a larger field of chemical ecology, in which all organisms tend either to respond to or to regulate one another by producing chemical attractants, stimulators, or inhibitors. For example, insects or nematodes may be discouraged from attacking a specific plant species because of repellent chemicals it contains. Plants also may produce inhibitors (phytoalexins) in response to attack by pathogenic fungi or bacteria. Specific cases of allelopathic interaction have been observed in agricultural lands, forests, grasslands, deserts, and even aquatic systems. A variety of chemicals with herbicidal action have been isolated from plants, microbes, soils, air, and water. Most scientists believe that the surface has barely been scratched in characterizing allelochemicals of plant or microbial origin.
These native wild sunflowers produce allelopathic substances that interfere with the growth of such weeds as velvetleaf, pigweed, jimsonweed, and wild mustard
Much of the current excitement about allelopathy can be attributed to Elroy L. Rice at the University of Oklahoma, who has written extensively on the subject and has trained many scientists in this area of research. He has stimulated many agricultural scientists (including myself) to attempt manipulation of this phenomenon in the field or to consider it as a lead for designing synthetic herbicides. Some skeptics in the scientific community still believe that the actual existence of allelopathy has not been proved adequately, although now there are probably more than 1000 jscientific papers published on this subject. A legitimate criticism is that many investigators have failed to isolate and identify the toxic agents. However, numerous other pieces of evidence point to the existence of allelopathy. To not believe in it just be-
cause all the chemicals have not been isolated and identified is, to me, analogous to not believing in genetic inheritance before the structure of DNA was determined. Often, there is no reasonable alternative explanation. We have been able to convince most of our skeptical students by demonstrating in the greenhouse the vivid impact of chrysanthemum, sorghum, or walnut residues on the growth of susceptible plants. The real difficulty is to assess experimentally the relative importance of allelopathy in complex plant communities where a number of interference mechanisms may be working simultaneously. Chung-Shih Tang and Chin-Chung Young at the University of Hawaii recently developed a clever but relatively simple system that allows recovery of toxic root exudates from plants. The chemicals are adsorbed on a resin, through which the nutrient solution is circulated. The resin selectively adsorbs the toxins, while allowing essential nutrients to pass through. In a complex soil system, natural substances may be extremely short-lived and may exert their influences at microsites for short periods of time only when their concentrations are sufficient. In soil, perhaps as many as a dozen or more inhibitors may be present at one time and may be acting in an additive or synergistic manner. For example, when phenolic compounds have been implicated in allelopathy, complexes of many closely related compounds have been isolated, all of which may interact together. Procedures to characterize natural products such as allelopathic substances recently have evolved rapidly as more sophisticated instrumentation has been developed. No single method, of course, works for every tissue or chemical. Briefly, isolation of a compound involves extraction of either plants or soil, or collection in an appropriate solvent or adsorbant. Commonly used extraction solvents are cold water or alcohol in which either dried or live plant parts are soaked. After the material is extracted for a specified time, the extractant containing any dissolved natural products is filtered or centrifuged before bioassaying (testing the extract for toxicity on living plant tissues). Extraction with water attempts to simulate the natural release of compounds that might be caused by rain acting on the standing or fallen plant material. Other methods of extracting plant material use boiling water, autoclaving, or organic solvents. Hot water or autoclaving increases the diffusion of soluble chemicals April 4, 1983 C&EN
35
Special Report
Above left, sorghum is grown here to produce allelopathic chemicals that, when the plant dies, will be released to control weeds in the later rotational crop* Above, sorghum killed by winter freezing releases allelopathic compounds into the soil Left, snap beans are growing in sorghum residues whose released allelochemicals have inhibited weed growth
into an aqueous phase and eliminates microbial decay. Organic solvents permit the extraction of a wide array of organic substances, many of which are poisonous to plants. The danger of these harsh extraction methods, however, is that the toxic materials isolated may not be released under natural conditions. Nevertheless, if the objective is to find unique herbicides, more thorough extraction may be warranted. Several assay methods are used to detect the presence of plant inhibitors in root exudates. They include: • Using agar as a medium for plant growth and examining for the presence of biologically active chemicals left in the agar. • Growing donor and recipient plants together in sand and evaluating the effects early in plant development before competition can occur. • Growing donor plants in sand for specific times, leaching the sand, and evaluating the leachate's effect on recipient plants. • Growing donor and recipient plants in pots arranged in a so-called staircase system, in which the pots of donors and recipients are alternated so that the leaching solution (usually nutrient solution) flows from the donor to the recipient and back to a reservoir, from which it is recirculated for varying periods each day. 36
April 4, 1983 C&EN
In studies of the release of organic substances from decomposing organic matter, either live or dried plant material is placed in or on soil for a specific period of time before bioassaying with recipient plants. Some studies have consisted first of isolating specific fungi involved in decomposing the plant material and then evaluating the by-products of fungal metabolism on plant growth. As Rice has pointed out, in studies with plant litter in soil, it is difficult to determine whether the toxic agent comes from the plant or from a microorganism or whether it is a result of an additive or synergistic effect or both. The methodology for bioassaying volatile allelopathic products has received considerable attention from Cornelius H. Muller and associates at the University of California, Santa Barbara. They have germinated indicator seeds on or between filter paper sheets held on a cellulose sponge that is placed in a larger container adjacent to beakers containing the donor plants.The only contact between material from the donor plant and the seed is through the air. This technique gives results similar to those observed under natural conditions. Chemical separations are accomplished on the basis of such properties as polarity, molecular size, charge, or adsorptive characteristics. Various chromatographic methods are used, including column chromatography, thin-layer chromatography, gas-liquid chromatography, and, more recently, high-pressure liquid chromatography. HPLC has proved particularly useful in separating water-soluble compounds from crude plant extracts. At one time, the major effort in compound identification involved chemical tests to detect specific functional groups. Now, characterization usually is accomplished by a series of spectroscopic analyses. Initially, ultraviolet spectroscopy was reasonably effective, along
with x-ray diffraction analysis. More recently, infrared spectroscopy, XH and 13C nuclear magnetic resonance spectroscopy, and mass spectrometry have helped immensely in determining the structures of complex natural products. Tandem mass spectrometry, a recent development that allows analysis of mixtures of compounds, likely will be particularly useful for characterizing compounds in plant extracts (C&EN, Nov. 30,1981, page 40). Scientists have long been interested in the question of how chemicals kill plants. The answers could assist them in designing other products or in predicting how safe the chemicals may be to other organisms. Research on the mode of action of inhibitors has caused sizable problems for investigators interested in either natural products or synthetic herbicides. The major difficulties lie in separating secondary effects from primary causes. Although biological effects can be measured in isolated systems, there is always the critical
question of whether the inhibitor actually reaches a particular site in the plant in sufficient concentration to influence a specific reaction, and whether other processes may be affected more quickly. Considerable evidence indicates that allelopathic chemicals can alter the rate of ion uptake by plants. In fact, this may be the mechanism underlying many plant interference responses that have been labeled competition. For example, the phenolic compound salicylic acid can inhibit the uptake of phosphate ions by algae. Recently, Nelson E. Balke's research team at the University of Wisconsin showed decreased potassium ion absorption by oat roots when they are exposed to various phenolic compounds. The team found that both salicylic acid and ferulic acid inhibit potassium ion uptake by oat roots, particularly at low pH, when salicylic acid uptake is greater. Many other studies with whole plants and cell cultures have shown a reduction in uptake of both macro- and micronutrients such as magnesium, calcium,
Allelopathic substances consist of a wide variety of chemical types Allelopathic substances from plants and from their associated microbes range from extremely simple gases and aliphatic compounds to complex multiringed aromatic compounds. Many plant tissues contain relatively high concentrations of cyanogenic glucosides, such as amygdalin, prunasin, dhurrin, and linamarin. When this plant tissue is exposed to hydrolytic enzymes, these compounds liberate hydrogen cyanide, a reaction known as cyanogenesis. The unfortunate plant seed or seedling in the area may suddenly find itself in the equivalent of a gas chamber. Ammonia gas also inhibits seed germination and seedling growth. Toxic levels of ammonia have been known to build up Inside seeds during the germination process. In contrast, ethylene, a gas produced by plant tissues, may stimulate seed germination. It has been injected into the soil, for example, to stimulate germination of witchweed, a serious parasitic plant growing in the southeastern U.S. The weed is induced to germinate before the host crop on which it depends for nutrition is present, and therefore dies. This approach is known as suicidal germination. Weed scientists are searching for other germination stimulators in the hope of getting other weeds to germinate before killing frosts occur in the fall. Plants of the mustard family contain powerful volatile inhibitors of germina-
tion and growth. The so-called mustard oils, allylisothiocyanate and /3-phenethyl-isothiocyanate, are potent inhibitors of germination. Several plant species that inhabit desert or chaparral areas also release volatile chemicals that can inhibit plant growth. Some classical work in this field has been done by Cornelius H. Muller, Walter H. Muller, and associates at the University of California, Santa Barbara. These research workers isolated several volatile monoterpenes, including camphor and cineole, from Salvia shrubs and later demonstrated the inhibitory effects of these compounds. They suggest that these compounds, in part, cause the zone of inhibition where no annual plants grow around Salvia shrubs. These and related volatile compounds also are released by big sagebrush and may contribute to Its dominance on an estimated 2.2 million square kilometers in the western U.S. Many of the acids contained in fruit juices are inhibitors of seed germination. For .example, malic and citric acids, which are common components of apple, tomato, citrus, and grape juices, inhibit seed germination at concentrations of 0.1 to 1.0 %. These acids almost always occur in mixtures and act synergistically to reduce germination more than does one chemical acting by itself. An important role of acids in fruits probably is to prevent seed from germinating inside the fruit.
Organic acids are formed during anaerobic decomposition of plant residues in soil. Research by J. M. Lynch and associates at Letcombe Laboratory in the U.K. has shown that acetic, propionic, butyric, and various other organic acids are produced during plant decay. Lynch believes that, although acetic acid is one of the least toxic organic acids, it still may be the most important allelopathic one in soil because microorganisms can produce large quantities of it. Moreover, under low oxygen conditions, it remains in the soil long enough to inhibit seed germination. Some of the first synthetic herbicides were chlorinated analogs of simple aliphatic acids. The addition of chlorine to these molecules produces greater herbicidal activity and soil persistence. Modifications in the structures of other natural products also may provide characteristics that improve their usefulness as herbicides. Various aromatic acids, aldehydes, and phenols, as well as their derivatives, likewise are implicated in allelopathy. In particular, cinnamic and benzoic acid derivatives have been isolated from a wide range of plants, their residues, and surrounding soils. Among the cinnamic acid derivatives often reported are chlorogenic, p-coumaric, ferulic, and caffeic acids. These compounds may be derived from the aromatic amino acids phenylalanine or tyrosine by a series of Continued on page 39
April 4, 1983 C&EN
37
chemical reactions called the shikimic acid pathway. Vanillic, syringic, and p-hydroxybenzoic acids are common benzoic acids that have been isolated where residues of plants (particularly wheat, corn, sorghum, and oats) have accumulated on the soil. Cooperative work by university and Department of Agriculture researchers at the University of Nebraska led to the identification of a variety of toxic phenolic compounds in soil. Amino acids and their analogs sometimes have been implicated in allelopathy. There is no clear understanding, however, of which ones are most important. A phosphonated amino acid, glyphosate (Monsanto's Roundup), is one of the major synthetic herbicides currently on the market. Recently, a related natural-product herbicide, bialaphos (discovered by the Japanese company Meiji Seika), has been massproduced by microorganisms in batch culture. This illustrates the potential of using living organisms to make herbicides in the future. Several simple lactones (those arising from acetate) are strong inhibitors of seed germination. One of these, parasorbic acid, from the fruit of mountain ash, inhibits seed germination and also has antibacterial action. Another such compound, patulin, is produced by a number of soil fungi, including Pénicillium urticae. This fungus forms large quantities of patulin when growing on residues of wheat. A related fungus produces this compound when growing on apple root residues. Patulin is extremely toxic to plants and animals. Coumarins are lactones of ohydroxycinnamic acid in which side chains often are isoprenoids. Coumarin, esculin, and psoralen (a furanocoumarin) all strongly inhibit seed germination. Such inhibitors are produced by a variety of legumes and cereal grains. For example, coumarin is produced by sweet clover, esculin is produced by timothy roots, and scopoletin or its 7-glucoside, scopolin, is formed by oat roots. The ability of selected oat lines to inhibit weeds has been attributed to their ability to exude scopoletin and related compounds. Only one quinone of plant origin has been found to be toxic to other plants. This is juglone, a powerful inhibitor from black walnut trees. It was identified in
1928 as 5-hydroxynaphthoquinone by Everett F. Davis at Virginia Agricultural Experiment Station in Blacksburg, Va. This chemical is extremely toxic to some herbaceous and woody crop plants, including tomato, alfalfa, and apple. It apparently is released from leaves and fruits, as well as from the bark of roots, and causes sensitive crops in the area to wilt, and, in severe cases, to die. A few of the flavonoids, another diverse group of compounds that are widespread in higher plants, have been implicated in allelopathy. Many researchers, including Elroy L. Rice at the University of Oklahoma, believe that this chemical group may contain a number of important allelopathic compounds. The insecticide rotenone is a naturally occurring isoflavonoid. Flavonoids pose some interesting analytical problems because their structures are complex, often containing glycosidic linkages. The flavonoid phlorizin, isolated from apple roots, is toxic to young apple trees when they are replanted on old orchard sites. Rice and associates have isolated other flavonoids from tall grass prairies and oak forests and have found these compounds to inhibit greatly both nitrifying bacteria and seed germination. The group classified as tannins includes both hydrolyzable and condensed compounds. Several of the more common hydrolyzable tannins are sugar esters of gallic acid, whereas others are complex mixtures of several phenolic acids. Hydrolyzable tannins inhibit seed germination, nitrogen-fixing and nitrifying bacteria, and plant growth. Much more work is needed to determine the importance of these compounds and of the lesser-understood condensed tannins in allelopathic interactions. The alkaloids are cyclic compounds containing nitrogen in their rings or side chains. Little work has been done in recent years on the importance of alkaloids in allelopathy, although Michael Evenari of Hebrew University in Israel pointed out in a review article in 1949 that alkaloids strongly inhibit seed germination. They are particularly important inhibitors of tobacco, coffee, and cocoa seeds. Evenari found that the strongest alkaloid inhibitors are cocaine, physostigmin, caffeine, chinin, cinchonin, cinchonindin, tropa acid, strychnine, berberin, and codeine. Recent work in-
dicates that caffeine can selectively kill several weeds without damaging bean plants. α-Picolinic acid is a microbial alkaloid with toxic action on plants. One of the more active synthetic herbicides on the market, picloram (Dow's Tordon), is a chlorinated picolinic acid derivative. Terpenoids and steroids are built from 5-carbon isoprene units linked together in a variety of configurations that differ in ring closure, functional group, and degree of saturation. The monoterpenoids, which are the major essential oils of plants, also are the principal group of inhibitors in this class of com pounds. The previously mentioned vol atile inhibitors camphor and cineole, along with camphene, dipentene, a-pinene, and β-pinene, are the terpenoids made by the various Salvia species. These also may contribute to allelopathy in eucalyptus and sagebrush. Several fungi also produce terpenoids that de stroy tissue or produce lesions in higher plants. The effect of steroids on higher plants is not well documented. Two com pounds in particular, digitoxigenin and strophanthidin, do have strong antimi crobial action. Some of these chemicals also apparently can alter the perme ability of plant roots. A scientist needs only to consult the current issues of various journals dealing with natural products, such as Phytochemistry or the Journal of Chemical Ecology, to learn of the variety and complexity of chemical structures being isolated from higher plants and micro organisms. Many of the compounds mentioned are allelochemicals with comparatively simple structures. Allelopathy is a relatively young area of study compared to other studies of natural products, such as communica tion compounds, defense chemicals, or medicinals. Only a relatively few chemists working with natural products, using state-of-the-art techniques, have been involved in studies of allelochem icals. Thus, generally only the simple, relatively stable compounds have been recognized to date. The most critical need now in the study of allelopathy is to learn the structure of the many as yet unidentified allelochemicals. I personally hope that more natural-products chemists will join this challenging ven ture.
April 4, 1983 C&EN
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Special Report Allelopathic compounds can be isolated from plants, microbes, and soils Chemical name
Structural formula
Class
Natural source
— β - glucose Dhurrln
Cyanogenic glucoslde
HO-
Allylleothlocyanate
Thlocyanate
CH 2 =CHCH 2 NCS
Camphor
Monoterpene
Sorghum plants
CN
Η
Salvia shrubs
& Acetic acid
Aliphatic acid
Mustard plants
. Decomposing straw
CH3COOH ?H=CH—COOH
Clnnamlc acid
Aromatic acid
Guayule plants
Arbutln
Phenolic compound
Manzanita shrubs
j—glucose
Blalaphos
Amino acid derivative
Î
CH 3
ï Î
H3Q
Π
CH3
Γ
COOH
Microorganisms
-Ρ—CH2CH2—C—C—NH—C—CNH—Ç—1 ΝΗο Η Η
Patulln
Simple lactone
Pénicillium fungus on wheat straw
Psoralen
Furanocoumarin
Psoralea plants OH
Ο
Juglone
Quinone
Black walnut trees
Phlorizin
Flavonoid
Apple roots
glucose
Gallic acid
Tannin
Caffeine
Alkaloid
Spurge plants
CH
3^,A. ../CH3 1 il— CH,
38
April 4, 1983 C&EN
Coffee plants
Special Report a coumarin compound. They also have shown that this compound causes stomata (tiny openings in a plant's outer surface) to close, but it is not clear whether this is a cause or a result of photosynthetic inhibi tion. Chemicals may either stimulate or inhibit respiration, and both actions may be harmful to the energy-pro ducing respiratory process. In the case of enhanced oxygen uptake, the electron transport sequence of oxi dative phosphorylation may be un coupled, resulting in a lack of aden osine triphosphate formation. Juglone, a naphthaquinone from walnut trees, has been shown to un couple oxidative phosphorylation, as do various aromatic acids, aldehydes, flavonoids, and coumarins, all of which reduce energy release. Several compounds isolated from At University of Oklahoma, Elroy L. Rice (right) and Robert L. Parenti, now at soils inhibit respiration in plant Fish & Wildlife Service, carry out research on allelochemicals roots. Juglone is particularly potent in this regard, causing more than a iron, and manganese, in the presence of the compound 90% reduction in the respiration of corn roots after a ferulic acid. one-hour exposure to the chemical. In addition, volatile The root tips of plants contain a meristematic zone terpenes markedly reduce respiration in mitochondria (growing zone) where root cells are dividing rapidly. isolated from oats and cucumbers. Many allelochemicals inhibit mitosis (cell division) in Effects on protein synthesis and membranes plant roots. Coumarin, for example, can block mitosis completely in onion roots within a few hours after Studies monitoring the effects of allelochemicals on treatment. Volatile terpenes from a Salvia species are protein synthesis have used sugars or amino acids la potent inhibitors of mitosis in cucumber seedlings. beled with carbon-14 and traced their incorporation into These compounds also inhibit division of a large number protein. Cinnamic and ferulic acids have been found to of bacterial species isolated from soil. inhibit incorporation of 14C (from glucose) into protein. Ferulic acid and coumarin inhibit the incorporation of The plant growth hormones indoleacetic acid (IAA) labeled phenylalanine into the protein of seeds and and the gibberellins (GA) regulate cell enlargement in young plants. One of the more important groups of plants. IAA is present in plants in both active and inac synthetic herbicides, the acylanilides, also interfere with tive forms and is broken down enzymically by IAA ox protein synthesis in susceptible plants. idase. Several allelopathic compounds influence the activation or breakdown of IAA. For example, moMany biologically active compounds are believed to nohydroxybenzoic acids stimulate IAA oxidase, which function by altering the permeability of membranes. accelerates IAA breakdown. On the other hand, 3,4Plant membranes, unfortunately, are extremely difficult dihydroxybenzoic acid and ferulic acid strongly inhibit to study, although the exudation of compounds from IAA oxidase and thus prevent the breakdown of IAA. A roots or root slices is used as an index of permeability. number of studies support the idea that both the phe Data obtained by Balke and coworkers at the University nolic compounds and flavonoid glycosides play a key of Wisconsin suggest that phenolic compounds, such as role in regulating IAA activity in plants. salicylic acid, enhance the efflux of potassium ions from root tissues by altering the permeability of the plasmaMany commercial synthetic herbicides block the vital lemma (cell membrane). At low pH, both the plasmaprocess of photosynthesis early in the life of a weed lemma and tonoplast (vacuolar membrane) may become seeding. In fact, the seedlings are killed as soon as they leaky, causing massive losses of potassium ions. This are exposed to light. increased membrane permeability ultimately can harm Photosynthetic inhibitors may function as electron the plant. Plant pathogens that induce wilt diseases are transport inhibitors, uncouplers, energy transfer in believed to produce toxins, such as fusaric acid and hibitors, or electron acceptors. Only a few reports have α-picolinic acid, that cause plant membranes to become been published on the influence of natural products on leaky. photosynthetic reactions. Frank A. Einhellig and co workers at the University of South Dakota have dem Various plant enzymes also are inhibited by allelo onstrated that photosynthesis is decreased in several chemicals. For example, the phosphorylase of potatoes plant species soon after they are treated with scopoletin, is inhibited by chlorogenic acid, caffeic acid, and cate40
April 4, 1983 C&EN
chol, and a high concentration of these compounds in potato peels is probably sufficient to completely deactivate that enzyme. Tannins have been reported to inhibit the activity of peroxidase, catalase, cellulase, polygalacturonase, amylase, and other enzymes. Some natural products also may act as herbicides by inhibiting key enzymes necessary for seed germination.
Factors affecting production of allelochemicals Plants vary in their production of allelopathic chemicals depending on the environment in which they are grown and the stresses they encounter. One difficulty faced by researchers is that greenhouse-grown plants produce only limited quantities of inhibitors. Ultraviolet light, because it is absorbed by glass, is absent in a closed greenhouse, and several investigations have shown that such light greatly enhances the production of allelopathic chemicals. For example, when greenhouse light is supplemented by ultraviolet, the chlorogenic acid content of tobacco is increased sixfold and approximates that
Simple trapping system recovers allelochemicals from roots Donor plant
Rock and coarse sand
Nutrient solution
Teflon film
XAD-4 resin
of plants grown outdoors. Sunflower plants receiving supplemental ultraviolet radiation also produce more chlorogenic acids and scopolin. Light quality, intensity, and duration all seem to be important factors regulating the synthesis of allelopathic chemicals. Tobacco plants exposed to red light at the end of the day produce more alkaloids but fewer phenolics than those exposed to far-red radiation. Red light is more effective than white light in stimulating formation of lignin in potato tubers. Long days seem to enhance the content of phenolic acids and terpenes in many plants. Nutrient deficiencies also may influence the production of allelochemicals, particularly the phenolics and the scopolin-related chemicals. Deficiencies of boron, calcium, magnesium, nitrogen, phosphorus, potassium, and sulfur all enhance the concentrations of chlorogenic acids and scopolin in a variety of plants. Levels of chlorogenic acids decrease in some plants that are deficient in magnesium or potassium. Water shortages alone or in combination with other stresses can substantially increase the concentration of chlorogenic and isochlorogenic acids in plants. In sunflowers, for example, the combination of drought stress and nitrogen deficiency produces about a 15-fold increase in these compounds. Heat and chilling also influence the production and release of allelochemicals. Many other factors can affect the synthesis or release of chemicals by plants. Exposure to herbicides or natural inhibitors may increase the production of scopolin-related compounds. The type and age of plant tissues are extremely important, because compounds may be preferentially stored in certain tissues or produced at a specific point in the plant's life cycle. Within a species, differences may exist in the amount of toxin produced by different varieties. For example, Peter K. Fay and William B. Duke at Cornell University have found great differences in the ability of various oat lines to exude scopoletin and related compounds. While on sabbatical leave at Cornell, I found that some types of cucumber seedlings could greatly inhibit weed germination, but others had no effect or even stimulated weed growth. More recently, while working at the University of California, Davis, I found that some alfalfa cultivars have much higher concentrations of seed germination inhibitors than others. These findings suggest that plant types can be selected on the basis of their efficiency as producers of inhibitors or that inhibitor production can be induced by applying the proper stresses to a plant.
Impact of allelochemicals on plant growth This relatively simple device, developed by Chung-Shin Tang and Chin-Chung Young at the University of Hawaii, allows undisturbed plant roots to exude chemicals into the rock and sand media. The continuously circulated solution, which contains mineral nutrients required for growth, elutes allelopathic substances from the media, then passes through an XAD-4 adsorptive resin, which traps the allelochemicals. The resin column then can be eluted to remove the chemicals for bioassay and characterization
The succession of various plant species in old fields and in cut-over forests has intrigued ecologists for many decades. The appearance and disappearance of species and the changes in dominance by species over a period of time have been attributed to many factors, including physical changes in the habitat, seed production and dispersal, competition for resources, or combinations of all these. Rice and coworkers at the University of Oklahoma have presented extensive evidence that allelopathy may April 4, 1983 C&EN 41
Special Report Allelopathic chemicals can act on several sites within a plant cell Inhibition of enzyme activity throughout the cell
Microtubules Cell wall
Alteration of permeability of plasma membrane (plasmalemma)
Inhibition of cell division in chromatin Nucleolus
Tonoplast (vacuolar membrane)
Nucleus Inhibition or stimulation of respiration in mitochondria
Vacuole Inhibition of cell expansion
Inhibition of protein synthesis with ribosomes
Alteration of nutrient uptake at plasma membrane (plasmalemma)
Inhibition of photosynthesis in chloroplast Endoplasmic reticulum Effects on membranes may include the chloroplast, nuclear, and mitochondrial membranes, as well as the tonoplast and plasmalemma
play an important role in the disappearance of the weeds that initially invade abandoned fields. For example, Johnsongrass and annual sunflower contain inhibitors that prevent germination and growth of many other weed species. These inhibitors, however, have little or no effect on the germination or growth of triple awn grass, which often follows them in succession. Rice's group also find that crabgrass may inhibit the germination and growth of several other species of annual weeds. Several weed species tend to predominate on agricultural lands. The literature indicates that about 50 weed species throughout the world may possess allelopathic properties. Recently; Prasanta C. Bhowmik and Jerry D. Doll of the University of Wisconsin reported that residues of several weeds inhibit soybean growth. Stephen B. Horsley at the Northeastern Forest Experiment Station in Warren, Pa., has presented extensive evidence that reforestation problems also may be linked to allelopathy. Some logged-over sites of the Allegheny Plateau in northwestern Pennsylvania have remained essentially treeless (but not plantless) for about 80 years. Several plants, including grasses, ferns, goldenrods, and asters, growing there produce toxins that inhibit the establishment of the black cherry seedlings that normally reforest these sites. Another important role in allelochemicals is their regulation of nitrogen cycling in soils. Plants can use nitrogen, one of the most important nutrients required for healthy plant growth, in both its nitrate and ammonium forms. There are two distinct advantages, however, in keeping nitrogen in the ammonium form in soil. Whereas positively charged ammonium ions are strongly adsorbed and held by soil colloids, they do not readily leach away with rain or irrigation water, as do 42
April 4, 1983 C&EN
nitrate ions. In addition, plants that absorb nitrate must reduce it through several steps before the nitrogen can be incorporated into amino acids. Because this requires considerable energy, inhibition of nitrification conserves energy as well as nitrogen itself. Rice and associates believe that plant succession selects those plants that inhibit nitrification and conserve ammonium nitrogen for their use. They find that Nitrosomonas and Nitrobacter, two bacterial species responsible for nitrification reactions in soil, are scarce in areas where succession has proceeded to the climax vegetation stage. Rice also has assayed many plants from old fields for their ability to inhibit the nitrogen-fixing bacteria Azotobacter and Rhizobium. The latter bacterial group is particularly important to legume crop plants because it infects them to produce nodules and then proceeds to fix atmospheric nitrogen used for crop growth. Extracts, root exudates, and decaying residues of several important weed species inhibit the nodulation of legumes by Rhizobium. Among these are western ragweed, large crabgrass, prostrate spurge, and annual sunflower. Nitrogen is deficient and nodulation absent in legume crops planted in residues of certain perennial weed species common to Michigan. Inhibition of nitrogenfixation may be another way that weeds interfere with crop growth. This is an agricultural problem that clearly deserves much more research. In many environments, plants tend to pattern themselves as pure stands or as individuals spaced in rather specific densities or configurations. Many species show obvious zones of inhibition around which few, if any, dissimilar plants can grow.These patterns cannot be explained adequately by competition alone and probably are caused by a combination of allelopathy, competition, and environmental factors. This phenomenon occurs
with herbaceous plants, as well as with woody shrubs and trees. Muller and associates at the University of California, Santa Barbara, have determined that a mustard species can form almost pure stands after invading annual grasslands in coastal southern California. Extensive research indicates that this takes place because inhibitors present in the dead stalks and leaves of the mustard plants prevent the germination and growth of other plants. Observations of this type led William B. Duke and myself to speculate about 10 years ago that we should be able to exploit the same phenomenon with crops. In other words, we should be able to achieve almost pure stands of crops (over weeds) by use of an allelopathic method. An obvious place where this could benefit many of us would be in the grass that makes up our lawns. Such an approach warrants research, because little is known now about the allelopathic effects of turf grasses. Many trees and shrubs have zones of inhibited plant growth around them that are difficult to explain by factors other than chemical mechanisms. In addition to several desert and chaparral species, black walnut, black locust, red pine, sycamore, and eucalyptus seem to exert selective herbicidal action on herbaceous vegetation in their immediate vicinity. Considerable evidence indicates that chemical inhibitors are responsible for the ability of some seeds to resist decay and to remain dormant in the soil for many years. One reason weeds are so persistent is that their seeds can lie dormant in the soil for many years but still remain viable. The classic seed burial studies of William J. Beal and of various botanists who continued his work at Michigan State University have shown that seeds of at least one weed, moth mullein, can remain dormant in soil for 100 years. Three other species germinated after 80 years of burial. Unsaturated lactones and phenolic compounds, in particular, are powerful antimicrobial agents present in many seeds. Fruits and seeds also contain diverse germination inhibitors, including phenolic compounds, benzoic acids, flavonoids, and their glycosides. Several plants are known to release into the soil various inhibitors that cause problems with the successful replanting of crops. Perhaps the best known of these problems involves the replanting of fruit trees, particularly peach and apple. In both cases, there appear to be several reasons for the toxic effects on replanted trees, but the release of toxins by decaying root residues has been demonstrated clearly. Peach roots contain high concentrations of amydalin, which is degraded in soil to benzaldehyde and hydrogen cyanide. Both chemicals inhibit the growth of a variety of plants, including peaches. Phlorizin, a phenolic compound in apple roots, and a number of its decomposition products in soil also have been implicated in problems with replanting apple trees. At the beginning of this century, several researchers noted that croplands upon which sorghum was repeatedly grown became unproductive, at least partly because of chemical effects..More recent studies indicate that sorghum residues can reduce weed growth greatly, but
do allow a number of crops, particularly legumes, to grow productively.
Allelopathy in agriculture Trying to put allelopathy to practical use has been an exciting and fruitful research project for my associates and me during the past eight years. Originally, we hypothesized that we might select and breed crop plants with allelopathic traits that could inhibit their associated weeds. Although we did find genotypes of cucumber that inhibit some important weeds in the laboratory and greenhouse, they did not perform consistently under field conditions. Also, a high degree of variability occurred from plant to plant, even within genetic lines. Wild types of crops, collected from sites around the world where the plants are indigenous, were more inhibitory than varieties that are grown commercially. This approach still has not been subjected to thorough research and needs attention by plant breeders. To my knowledge, only oats, soybeans, and sunflowers (in addition to the cucumbers we studied) have been searched for allelopathic traits. These traits hold a great deal of promise for controlling weeds among plants that are grown in high-density monocultures, such as turf-
A research assistant at University of California, Davis, examines ultraviolet absorbance recording of eluates from a column used to separate possibly allelopathic compounds in dwarf spikerush, an aquatic plant April 4, 1983 C&EN
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Special Report grasses, forage grasses, and legumes. In addition, repeated culture of allelopathic plants might reduce weed populations. Gerald R. Leather at the Department of Agriculture's weed science laboratory in Frederick, Md., recently used sunflowers to demonstrate such a reduction. Some of our best successes have been achieved with annual rotation of allelopathic crops or in companion plantings of them with perennial crops. In these systems, we capitalize on the toxic action of both the living crop and its residues. Sometimes, we rely on freezing weather to kill the allelopathic plants and thus produce the desired residues. Other plants must be severely inhibited or killed with contact herbicides so that they do not later harm the following crop. In our experiments, the crops that have shown the best weed suppression are sorghums, wheat, rye, oats, and barley. We have suppressed up to 95% of several important weeds, although other weeds are virtually unaffected. Similarly, crops vary in their response to the allelopathic residues. The large-seeded legume crops, such as peas and beans, and the cucurbit vegetables, such as cucumbers and melons, respond favorably. On the other hand, several species of smaller-seeded crops, such as lettuce, tomato, and radish, have been injured. This indicates that allelopathic chemicals are selective in their action, much like many of the synthetic herbicides. This approach to weed control may be particularly timely, because there now is considerable enthusiasm
Jane Barnes and Alan R. Putnam at Michigan State use high-pressure liquid chromatography to separate and detect compounds in their work on allelopathy among farmers for reducing tillage and for using surface plant residues to conserve soil and moisture. Such a system would have great impact, particularly in developing countries. The evidence indicates that a complex of chemicals contributes to allelopathic effects from crop residues. Frederic R. Lehle in my laboratory has isolated from sorghum at least eight different fractions of various chemical classes that show inhibitory action. Some of these compounds are extremely toxic, but others require rather massive doses to inhibit germination or growth. Microbial products also may contribute to the toxicity of sorghum residues in the field. We already have isolated from soil several microorganisms that can produce herbicidal compounds. Allelopathic plants also may provide a strategy for vegetation management in aquatic systems. The diminutive spikerush displaces vigorous and unwanted aquatic plants, such as pondweeds, in California canals and drainage ditches, according to Peter A. Frank, Richard R. Yeo, and their Department of Agriculture coworkers at Davis, Calif. Working with spikerush, Kenneth L. Stevens and colleagues of the Department of Agriculture laboratory at Berkeley, Calif., have isolated dihydroactinidiolide, a chemical that may contribute to the plant's allelopathic action. And Lars W. J. Anderson at Agriculture's lab in Davis has demonstrated recently that this compound can alter the structure of leaves in germinating pondweeds.
Directions of future research A scientist at Michigan State studies a microorganism that produces herbicidal compounds 44
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I expect a flurry of activity in allelopathy research during the next decade, particularly in agriculture. Ecologists have shown that allelopathy is a mechanism by which plants can help regulate their densities and
distributions. Agronomists, horticulturists, foresters, and others should now determine how to use this strategy to manage their systems better. Natural products can be extremely useful, either for imparting weed resistance to plants or suppressing weeds. They can be highly detrimental to crop growth and nitrogen fixation or perhaps be useful as inhibitors of nitrification. At present, we do not understand thoroughly the impact of weeds on our crops, although obviously they often severely interfere with growth. We know too little about how best to rotate crops or about which vegetables to plant near each other in our gardens. I visualize that some revolutionary cultural systems will be developed to exploit natural products. For example, perhaps veg etation could be planted in an apple orchard that would fix nitrogen for the trees, conserve ammonium nitrogen, repel or inhibit some insect and disease pests of the trees, harbor agents for biocontrol of insect and disease pests, inhibit weed growth, maintain good soil structure, and prevent soil erosion. This is a dream perhaps, but I am confident that progress will be made toward achieving such systems. Major research efforts should be directed toward de veloping methods to increase weed seed decay and to stimulate or inhibit weed seed germination. As far as I am aware, no successful attempts have been made to enhance weed seed decay under field conditions, al though it can be accomplished in the laboratory by re moving the microbial inhibitors from the seed. The se lection of a microorganism that can destroy weed seeds is one possible approach. Another is to adsorb or inac tivate the inhibitor that may protect the seed against decay.
Seed dormancy is a complex phenomenon involving several compounds, including abscisic acid, gibberellic acid, cytokinins, and ethylene. Seeds may germinate as a result of an external chemical stimulus. For example, germination of the seeds of witch weed (a parasitic weed) is enhanced by the chemical strigol, which is exuded by the roots of susceptible host plants at concentrations as low as 10~ 15 M. Ethylene also stimulates germination of witchweed and is used by soil injection to cause suicidal germination of the weed seed before the host-crop plants develop. Undoubtedly, many other compounds could be used in this manner. One major unanswered question regarding allelopa thy is whether the chemicals involved are end products of metabolism or actually are synthesized by the plant for specific purposes. Some investigators believe that these compounds are either by-products of metabolism or are waste products tucked away into the cell vacuoles to prevent autotoxicity. No one has proved that allelopathic chemicals are specifically synthesized as the result of external stimuli, although related secondary chemi cals seem to be continuously synthesized and degraded in healthy plant cells. Allelopathy research holds enormous excitement for the organic chemist and biochemist. Many allelopathic compounds remain to be isolated and identified. Perhaps the natural plant or microbial products can be modified slightly to give them greater activity, selectivity, or persistence. Perhaps plants or microbes can be manip ulated genetically to make them better producers of the desired product. Some research efforts along these lines are already under way in major chemical companies. I am expecting some fascinating results! D
Suggested readings
Alan R. Putnam is professor of horticulture at the Pesticide Research Center of Michigan State University. He earned a B.S. from the University of New Hampshire and both an M.S. and Ph.D. from Michi gan State University. His past 18 years at Michigan State have been devoted to teaching and to research on improved strategies for weed control and on studies of weed biology and weed crop interactions. He has conducted studies on conservation tillage and no-tillage systems for a variety of horticultural crops and has investigated many nonconventional approaches to weed control. As author or coauthor of more than 100 articles on weed control, Putnam currently is preparing a book chapter on allelopathy. He teaches an undergraduate course in principles of weed science and a graduate course in plant interactions in agroecosystems. Put nam's research has been funded by many government agencies and industry groups. Funds for his most recent allelopathy and microbial herbicide projects have come from the Department of Agriculture's Competitive Grants Program and Dow Chemical.
Altieri, Μ. Α., Doll, Jerry D., PANS, 24, 495 (1978). Conn, Eric E., Ann. Rev. Plant Physiol., 31, 433 (1980). Harborne, Jeffrey B., "Phytochemical Ecology," Academic Press, London, 1972. Harper, James R., Balke, Nelson E., Plant Physiol., 68, 1349 (1981). Leather, Gerald R., WeedScL, 31, 37 (1983). Patrick, Ζ. Α., Soil Sci., 111, 13 (1972). Pickett, Steward T., The Biologist, 55, 49 (1973). Putnam, Alan R., Duke, William B., Science, 185, 370 (1974). Putnam, Alan R., Duke, William B., Ann. Rev. Phytopath., 16, 432 (1978). Rice, Elroy L., "Allelopathy," Academic Press, New York, 1974. Rice, Elroy L, Bot. Rev., 45, 15 (1979). Swain, Tony, Ann. Rev. Plant Physiol., 28, 479 (1977). Swain, Tony, Harbone, Jeffrey B., Van Sumere, Chris F., "Recent Ad vances in Phytochemistry," Plenum Press, New York, 1977. Tang, Chung-Shih, Young, Chin-Chung, Plant Physiol., 69, 155 (1982). Tukey, Harold B. Jr., Bot. Rev., 35, 1 (1969). Whittaker, R. H., Feeny, P. P., Science, 171, 757 (1971).
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