Weed Science Society of America

Strategies for Using Transgenes to Produce Allelopathic Crops Author(s): Stephen O. Duke, Brian E. Scheffler, Franck E. Dayan, Leslie A. Weston, Eiji Ota Reviewed work(s): Source: Weed Technology, Vol. 15, No. 4 (Oct. - Dec., 2001), pp. 826-834 Published by: Weed Science Society of America and Allen Press Stable URL: http://www.jstor.org/stable/3988566 . Accessed: 20/04/2012 12:41
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Weed Technology. 2001. Volume 15:826-834

Symposium Strategiesfor Using Transgenesto Produce AllelopathicCrops'

STEPHEN 0. DUKE, BRIAN E. SCHEFFLER,FRANCK E. DAYAN, LESLIE A. WESTON, and EIJI OTA2 Abstract: Crop allelopathy has seldom been used effectively by farmersin weed management.Traditional breeding methods have not been successful in producinghighly allelopathiccrops with good yields. Genetic engineering may have the potential for overcoming this impasse. Crops have been made resistantto insects, pathogens, and herbicides with transgenes,but biotechnology has not produced crops that control weeds with allelochemicals. The strategiesfor producingallelopathiccrops by biotechnology are relatively complex, usually involving multiple genes. One can choose to enhance production of allelochemicals already present in a crop or to impart the productionof new compounds. The first strategy involves identificationof the allelochemical(s), determinationof the enzymes and genes encoding them, and the use of genetic engineering to enhance their production. The latter strategy employs altering existing biochemical pathways by insertions of transgenes to produce new allelochemicals. With either strategy, there are potential problems with tissue-specific promoters, autotoxicity, metabolic imbalances, and proper movement of the allelopathic compound to the rhizosphere. Nomenclature: Glufosinate; barley, Hordeum vulgare L.; celery, Apium graveoens L. var. Dulce (Miller) Pers.; cucumber,Cucumis sativa L.; diffuse knapweed, Centaureadiffusa Lam #3 CENDI.; maize, Zea mays L.; potato, SolanumtuberosumL.; rice, Oryzasativa L.: sorghum,Sorghumbicolor (L.) Moench # SORVU; sudangrass,Sorghumsudanese (Piper) Stapf;tomato,Lypersiconesculentum L.; wheat, Triticumaestivum L. Additional index words: Allelochemical, genetic engineering, phytotoxin, transgene. Abbreviations: DIMBOA, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one;PCR, polymerasechain
reaction.

INTRODUCTION The ethnobotanical lore indicates that people have been aware of some plants "poisoning" the soil for other species for many years. The relationshipof this phenomenon to agriculturewas generally negative, since weeds are usually more allelopathic than crops. Although allelopathy has been the focus of considerable research during the past century, its role in weed managementis still extremely limited. During the last half of the 20th century, weed management became almost completely dependenton syntheticherbicides.This was due to many factors, but was primarilydue to the relatively low cost
' Received for publication December 12, 2000, and in revised form December 28, 2001. 2 Research Leader, Plant Molecular Biologist, and Plant Physiologist, USDA, AgriculturalResearch Service, P.O.Box 8048, University,MS 38677; Associate Professor, Cornell University, Ithaca, NY 14853; Senior Scientist, Kubota Corporation, Ryugasaki, Japan. Corresponding author's E-mail: sduke@olemiss.edu. I Lettersfollowing this symbol are a WSSA-approvedcomputercode from Composite List of Weeds, Revised 1989. Available only on computer disk from WSSA, 810 East 10th Street, Lawrence, KS 66044-8897.

of effective weed control with herbicides. The introduction of herbicide-resistant crops, combined with the advantages of inexpensive, nonselective herbicides, promises to make farming dependent on herbicides for the foreseeable future (Duke 1998; Hess and Duke 2000). Conversely, use of insecticides, nematocides, and fungicides is decreasingbecause crops can be made resistant to these pests by either conventional breeding or use of transgenesimpartingpest resistancetraits.For these reasons, the proportionof pesticides sold that are herbicides continues to grow, now approachingapproximately75% of the marketin North America (Anonymous 1998). There are mountingconcerns over the human and environmental healthrisks of pesticides.Increased regulatory requirements, includingthe mandatory reregistration old of herbicides,are causing attritionof the existing herbicide arsenal.In addition,increaseddiscovery and development costs and reduced investmentsin discovery researchare slowing the introduction new herbicides.Herbicideopof tions for some minor crops are alreadyseverely limited.

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Alternativeweed control technologies have not yet been adequatelydevelopedto meet growing needs. Crop interferenceis a potential method of biocontrol of weeds, using the crop as the biocontrol agent. Interference is a combinationof the processes of competition for resources and production of phytotoxins that suppress competitors (allelopathy). Research to supportthe allelopathy approachhas been conducted for several decades, but as a result of this research, there are still no commercial cultivars differentiatedfrom others by their allelopathic properties. Germplasmof barley (Hordeum vulgare L.) (Lovett and Hoult 1995), cucumber (Cucumis sativa L.) (Putnamand Duke 1974), rice (Oryza sativa L.) (Olofsdotteret al. 1999), sorghum [Sorghumbicolor U.) Moench # SORVU] (Nimbal et al. 1996a), and wheat (Triticumaestivum L. (Wu et al. 1999) have been screened for allelopathic potential. Significant variation was found in each of these crops, demonstratingthat germplasm for allelochemical production exists within some crop species. In a few cases, allelopathictraitshave been manipulatedby traditionalgenetic methods (e.g., Wu et al. 1999). Nevertheless, the level of allelopathic activity provided has apparentlynot been sufficient or consistent enough to provide adequate weed management in the field. Otherwise, we assume that such a cultivar would be commercialized,at least for organicfarming. Chavez et al. (1999) found with certain allelopathic rice varieties that herbicideuse can be reduced,but farmers have not yet implementedthis practice. Nursery, ornamental, and organic farmers in the eastern United States routinely use sorghum X sudangrass [Sorghum sudanese (Piper) Stapfl hybridcover crops as a rotational procedureto reduce annualweed populations(Weston 1996). More effective cultivarchoices are needed to fully integratethis technology into commercial settings. Transgeneshave been used to make crops resistantto pathogens (e.g., Beachy and Bendahmane2000), insects (e.g., Fitt and Wilson 2000), and herbicides(Duke 1996; Hess and Duke 2000). However,they have not been used to make crops that strongly suppress growth of weeds, either by allelopathicor competitive means. In this paper, we provide genetic engineering strategies that could be used to create or improve allelopathictraitsof crops. We also discuss the many potential difficulties of such an endeavor. RESEARCHAPPROACHES There are two methods of creatinga more allelopathic crop: (1) enhancement of existing allelopathic potential and (2) insertionof genes to produceallelochemicalsthat
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Figure 1. Scheme for bioassay-directedisolation of bioactive products from plants.

are not found in the crop. Either approachis potentially much more complicated than creating an herbicide-resistant crop or producinga crop with resistanceto insects or pathogens. In the first case, one must decide what compound(s) already made by the crop would be effective as an herbicide if produced and exuded into the soil in sufficient amounts. Proving that a compound produced by a crop is phytotoxic is relatively simple, but there are few cases in which there is adequateproof thatthe compoundcoming from one plant actually inhibits growth of surrounding plants in a natural or agriculturalsetting. In most cases, apparentallelopathic effects associated with particular species are based upon field observationsand laboratory tests of crude extracts of that species. Identification of the specific compounds involved is rare. Many putative allelochemicals have been discovered by looking for known phytotoxic compounds in plants. If an unknown compound is responsible for allelopathy, this approachwill not be productive.The most definitive approach to determination of potential allelochemicals producedby a plant is that of bioassay-directedisolation (Figure 1). Our laboratory has routinely used this approach, and we have published several reviews that describe this method and many of its variations in detail (Duke et al. 2000a, 2000b, 2000c; Rimandoet al. 2001). At this time, the potentialallelochemicalsfrom the available germplasm of most major crops have not been determined.In most cases where allelochemicalshave been
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CROPS DUKE ET AL.: STRATEGIESFOR USING TRANSGENES TO PRODUCE ALLELOPATHIC

identified, there is incomplete knowledge of their biosynthetic pathways. Thus, a molecular biology approachfor enhancement of existing biochemical pathwaysfor allelochemicalproduction is limited in many crops by the paucity of information on the chemical structureof allelochemicals or by incomplete knowledge of the biochemical pathway of those allelochemicals that are known. The intensive plant genome programsfor majorcrops could be helpful in providing some of the missing information.However, allelochemicals are secondaryproductsand, in some cases, are quite species specific. Indeed, much of modern chemotaxonomy is based on unique secondary product profiles. Thus, the discovery of the genes associatedwith the synthesis of the allelochemicals of some species may be quite difficult. A laborious approach to identification of genes encoding key enzymes for allelochemical productionis to purify the enzymes and work back to the genes (Canel 1999). This approachis complicatedby enzymes of secondary metabolism that are usually present in low abundance, except in specialized cells or tissues. After purification, the protein can be sequenced to obtain the gene sequence, or it can be used as an antigen to raise antibodies that can be used to screen a cDNA expression library for a clone expressing the protein of interest. In the former case, degenerativeoligonucleotides based on the protein sequence can be used in PCR reactions that amplify the desired segment of DNA. The first example of this for an enzyme involved in secondary metabolism was the cloning of the gene for strictosidine synthase (Kutchan 1989). An advantage of either of these approaches is that the isolated gene clearly encodes a critical protein involved in the allelochemical of interest. The disadvantageof this approachis that discovery, isolation, and purificationof enough of the enzyme for sequencing, or even antibody production,is usually a difficult and labor-intensiveendeavor. The other approachis to identify genes and determine or verify what enzymes they encode. Mutationsresulting in lesions in a biochemical pathway can also be used to identify genes controlling synthesis of allelochemicals. Mutagenesis using transposable elements has been a valuable technique for gene isolation. Transposableelements are mobile segments of DNA that can move within the genome. They can disrupt gene function when they are inserted into an active gene, usually resultingin a recessive phenotype. Screening for a mutation in the biosynthetic pathway of the allelochemical must be performed on thousands of individual plants. This is not a
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trivial matter if there is no rapid screen for the trait. an Furthermore, active and characterized eltransposable ement system is necessary in the species of interest in orderto use this method. The most characterized system is that of maize (Zea mays L.). Introducinga modified system from maize (Bancroftet al. 1992) into the desired plant via transformation been accomplishedwith rice has (Izawa et al. 1997; Shimamoto et al. 1993), a species with considerable allelopathic potential (Olofsdotter et al. 1999). A set of gene clones can be generatedby differential screening techniques to see if they are involved in allelochemical biosynthesis. Several methods use differential screening as gene selection criteria.All of them use mRNA that is isolated from tissues that either are, or are not, expressing the trait of interest, which in our case is productionof an allelochemical. Those mRNAs that are found in tissues producingan allelochemical,but are not found in nonproducing tissues, are identified. These mRNAs are then cloned and characterized. Two methods commonly used in differential screening are differential display and subtractivehybridization.With either method, problems can be avoided by testing isolated clones to see if they are truly differentiallyexpressed. The biggest problemwith differentialscreeningis that two tissues must be identified that differ in the trait of interest, with as few other differences as possible. Because most allelochemicals of interest in an agricultural setting will probably be expressed in root tissue, roots are an obvious choice. Roots and other plant organs would have many genes expressed in common for general cellular functions, but there will be a large number of differences in gene expression that will be difficult to sort through to determine which specific genes are associated with the allelochemical. Ideally, comparingroot tissue that is producing the allelochemical(s) with root tissue that is not producing it would be most desirable. However, generating tissue that is comparable to producing tissue but is not producing the compound could be difficult. Comparisonsof tissues or cell types within the root may allow for identificationof similar cells that are differentiallyproducing allelochemicals. Differences may also be found at various developmentalstages or in differentroot types. Alternatively,differentenvironmental conditions in which the allelochemical is expressed in one condition, but not the other, may allow for differential screening. In this case, cloned or inbred lines should be used to reduce genetic differences unrelated to the trait of interest. Even after identificationof genes throughtransposon
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tagging, differentialscreening, or other techniques,proof that a particulargene represents a key step in the biochemical pathway of an allelochemical must be accomplished. A high level of homology with genes or proteins from databases may provide useful leads to their function, but this is not definitive proof. Several approaches can be used to prove that a cloned gene is associated with a specific step of a biochemical pathway. This can be accomplished most directly by expressing the clone, isolating the producedprotein, and demonstratingits enzymatic activity on precursor(s) of the allelochemical. This is only feasible if the clone representsa structural, not a regulatory, gene and if it can be expressed in its active form. An indirect approach is to demonstratethat a clone correspondsto a mutatedgene (generatedby transposon tagging or mutagenesis) that affects synthesis of the compound of interest. Another indirect approach is to make an antisense or overexpression construct of the clone in question and transformit back into the plant. When a plant is transformedwith a functional antisense or overexpression construct, the expression of the correspondinggene is altered.With antisenseconstructs,expression of the targeted gene is reduced, whereas with overexpressionconstructs,expression can be reduced or increased(Jorgensenet al. 1996). In either case, the level of production of the allelochemical should be directly affect. Work with 2,4-dihydroxy-7-methoxy-1,4-benzoxazin3-one (DIMBOA), a compound associated with insect, pathogen, and even herbicide resistance in maize (Frey et al. 1997; Niemeyer 1988), is an example of how modern molecular genetics can identify genes involved in secondary productbiosynthesis without taking the direct biochemical approach(Frey et al. 1995, 1997). Conventional genetics was used to isolate a mutationat the Bxl gene, which resultedin a lack of DIMBOA accumulation in homozygous recessive plants (bxl/bxl). Using the Mutator transposableelement, bxllbxl plants were put througha directed transposon-tagging program,and isolated mutationswere then screened by segregationanalysis. The screening process looks for the cosegregation of the mutated locus and the Mutator transposableelement. With this approach,the Bxl locus was isolated and found to have DNA homology to tryptophansynthase alpha.This enzyme convertsindole-3-glycerolphosphate to indole, an importantintermediateproductin the DIMBOA biosynthesis pathway.This was confirmedthrough genetic complementationtests and biochemical analysis. In other studies, subtractivescreening of high vs. low
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DIMBOA-producing plants resulted in the isolation of a cDNA clone encoding for a particularP450 monooxygenase (Frey et al. 1995). Through DNA homology, three other P450-dependent monooxygenase loci were cloned. The four loci (Bx2-5) were expressed in a yeast expression system. The expressed proteins were then tested on the various intermediatesof DIMBOA to determine which gene was responsible for each biochemical step (Frey et al. 1997). Identificationof a gene that is associated with production of an allelochemical does not ensure that it encodes a limiting step in biosynthesis. Biochemical analysis of the pathway and the role of the enzyme encoded by the gene can provide clues to this question. Changing the promoter of the rate-limiting gene so that it is expressed at a slightly increased level would be a simple method of increasingpathwayactivity. This would avoid the phenomenon of cosuppression associated with high levels of gene expression (e.g., Que et al. 1997). Increasing the expression of a gene encoding one gene in the pathway would result in anotherenzyme of the pathway becoming rate limiting. Thus, one could not expect to engineer huge increases in the compound of interest by increasing the expression of only one gene unless it is a regulatory gene that controls several steps in a biosynthetic pathway. In this case, enhancementin its expression level can increase the output from all of the controlled genes, probablyresulting in a greaterincrease in productthan increasing the expression of only one gene encoding a structural protein(Bradleyet al. 1998; Scheffler et al. 1994). The approachof using existing allelochemicals is outlined in Figure 2. The second approachto genetically engineering allelopathy into a crop is to introduce a new biochemical pathway. This could involve the relatively simple process of introducinga one-step conversion of an existing secondary compound or the very complex task of introducing a multienzyme pathway that the plant does not have. With this strategy,identificationof allelochemicals and genes for their productionwould not be a problem. An example of a potential strategy for this approachis to use the microbial genes for bialaphos or phosphinothricin production.Many crops have been transgenically altered with the bar or pat gene to be resistant to the synthetic form of phosphinothricin,glufosinate (Lydon and Duke 1999). This has been done to renderthe crops resistantto glufosinate or so that glufosinate can be used as a selection agent for successful transgene transformations. The microbial genes for the bialaphos biosynthesis are available (Lydon 1996). Bialaphos is a protox829

DUKE ET AL.: STRATEGIESFOR USING TRANSGENES TO PRODUCE ALLELOPATHIC CROPS

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Figure 2. Scheme for determinationof appropriate existing genes in a potentially allelopathic plant for genetic manipulationof the allelopathic trail.

root- or hairseic roo

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in, meaning that it is convented to phosphinothricinin the targetweed. Bialaphos producedby fermentationhas been sold in eastern Asia as an herbicide. It might be possible to introducegenes for bialaphosproductioninto crops that have alreadybeen made resistantto it. Ideally,
the compound would be produced only in roots, where it would be exuded into the soil. Although, this herbicide does not have a high level of soil activity, the crop would

produce it on a continuousbasis that might be sufficient for weed control. After a potential gene that might be used to enhance allelochemical productionis identified, its expression in a crop must be introducedor increased by transformation. The transgenewill probablyhave to be introduced into the target plant using DNA constructsin which the gene is under the control of a tissue-specific promoter. In the case of allelochemicals for use in agriculture,a

the promoterof choice. Such promotersare not yet readily available in the public sector. Crop species of the genus Sorghum might be manipulated by genetic engineering to enhance their existing allelopathic potential. Sorghum spp. are often used as summerannualcover crops, partly because of their ability to suppress weeds (Forney and Foy 1985). Sorghum spp. cover crops are known to exude, from living root hairs, oily droplets composed of the allelopathic compound sorgoleone and many minor constituents. These compoundsare potent inhibitorsof photosynthesis(Gonzalez et al. 1997; Nimbal et al. 1996b; Rimando et al. 1998), respiration(Rasmussen et al. 1992), and plastoquinone synthesis (G. Meazza, personalcommunication) in higher plant systems. Root exudates of sorghum contain between 85 and 90% pure sorgoleone, and the concentrationof this naturalproduct in soils in which sorghum has grown can easily reach 10 to 100 ,uM(Netzley et al. 1988). Although the biosynthetic pathway of the cyclic quinone portion of the sorgoleone molecule has been reported to be the polyketide pathway (Fate et al. 1990), only a very small amount of radiolabeledacetate could be incorporatedin the quinone head of sorgoleone. Alternative biosynthetic pathways are those of the homogentisate pathway associated with p-benzoquinone biosynthesis (Luckner 1984) and a pathway in which a long-chain fatty acid has one end cyclized. We intend to clarify the sorgoleone biosyntheticpathwayby using two approaches.One is by differential screening to identify genes that are expressed in sorghumroots that have sorgoleone-exuding root hairs vs. those without root hairs. The second is to more conclusively identify the sorgoleone pathway by using high sorgoleone-producingroot cultures. We have started the former strategy using a commercial kit, as well as with self-designed sets of primersprovidingthoroughcoverage of the sorghumgenome. With this polymerase chair reaction (PCR)-based method, the material goes through two rounds of substraction. In initial experiments, about 50 clones were differentially expressed. About 50% of the clones had homology to previously characterized genes. Another 25% had homology to expressed sequence tags of unknown function, and the remaining25% had no homology to any DNA or protein sequences. Of the clones with homology to known genes, at least two appear to be related to functions with the polyketide pathway. Also, a gene encoding a mixed-function oxidase has recently been identified as a differentially expressed gene associated with sorgoleone synthesis (Yanget al. 2001). Such
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enzymes are involved in the productionof oxidized ring structures, such as the quinone end of the sorgoleone molecule. POTENTIAL DIFFICULTIES

ways when manipulatingsecondary metabolism. Coordinated expression of a set of genes involved in the trait of interest with a single promoter may overcome this potential problem. Autotoxicity. Plants can make antimicrobialsand compounds to provide protectionfrom insects and other herbivores without danger of autotoxicity because the molecular target sites in these nonplantorganismsare quite differentfrom those of phytotoxins.With plant-produced phytotoxins (whether used in allelopathy or not), autotoxicity is a potential problem for which a mechanism of avoidancemust be in place. Mechanismsof avoidance include sequestration,secretion, resistance at the target site, or metabolic inactivation. Sequestrationcan occur in the vacuole, subcuticular space of glandular trichomes, or other sites that preclude contact with cytoplasm. Many compounds involved in plant-plant allelopathic interactions are probably secreted into the environmentthroughthe roots. Although they are secreted into the soil, they can still pose a threat if reabsorbed. Therefore,without an additionallevel of defense, secretion may be of limited use. The simplest mechanism of autotoxicity avoidance is resistance at or absence of the molecular target site. We are unaware of any examples of this mechanism,perhapsbecause the moleculartarget sites of few allelochemicals are known. Metabolic inactivationis probablya common mechanismof autotoxicity avoidance. Two approachesto this can be used by the plant. The compound can be irreversiblychemically modified in cells in which it could do harm, or it could be synthesized in a form that requiresmetabolic activation to become a phytotoxin. Bioactivation of protoxins is common with phytotoxic naturalcompoundsproduced by microbes. For example, cornexistin (Amagasa et al. 1994), hydantocidin (Siehl et al. 1996), and bialaphos (Lydon and Duke 1999) are inactive at the in vitro level because they must be converted to phytotoxins in the plant. The plant can use combinationsof these approaches. For example, many phytotoxic secondarycompounds are stored in vacuoles conjugated with other molecules (e.g., sugars or amino acids) that reduce their phytotoxicity. Although we know very little about this aspect of allelopathy, plant species that produce allelochemicals used to poison competing plant species must have evolved mechanisms to avoid autotoxicity. When increasing productionof existing allelochemicals or introducing new ones into a plant with transgenes, autotoxicity avoidance is a majorconcern. Absence of the target site or metabolic inactivation are the more desirable
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Allelochemicalsand Resource Partitioning.What is

the cost to a plant of partitioninga significant amount of its resources into secondary products such as allelochemicals? Can this be done without reducing yield? Is this metabolic cost the reason that crops bred intensively for high productivity have few indications of any allelopathic activity at all? Gershenzon's (1994) detailed analysis of the resources needed for secondary metabolism concludes that the metabolic cost of most secondary compoundsis higher than that of most compoundsfound in primarymetabolism.Thus, there must be a significant survival benefit to the plant for this relatively high expenditure of resources. In a natural setting, a yield reduction due to resource partitioninginto allelochemicals could be beneficial in many situations,but it may not be to a farmer.The acceptablebalance between allelochemical-based weed control and yield will be dependent on the crop and the cropping situation. For example, for cover crops used for green manure and weed suppression, the balance might tilt toward a more allelopathic variety. Whether this tradeoff will be acceptable in an agroecosystem, in which yield, ratherthan propagation, is the primaryconcern, will probablydepend on the species and the allelochemical. The metabolic cost of allelopathy can be minimized if the allelochemical(s) produced by a plant has high unit activity as a phytotoxin. Metabolic Imbalances. Alterationof biosynthetic pathways can have unintended consequences. Plants have been selected by nature to survive and propagate the species. Humans have also selected other traits, while maintaining these fundamental traits. The metabolic pathways of plants interact in complex ways that have been maintainedthroughouteons of selection. Redirection of metabolic intermediateseasily can create shortages of needed compounds or accumulationof toxic intermediates.An example of this is the work of Canel et al. (1998) in which overexpression of tryptophancarboxylase in periwinkle [Catharanthus roseus (L.) G. Don], in an attempt to improve production of certain medicinal alkaloids, led to a general significantly reduced growth, apparentlydue to disruption of metabolism by reducingtryptophanpools. This study and others (e.g., Yao et al. 1995) indicate that one must consider metabolite flux and interdependencyof metabolic pathVolume 15, Issue 4 (October-December) 2001

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mechanisms of resistance. The target site must be characterized for the former approach, and genes encoding an enzyme that deactivatesthe phytotoxinmust be found for the latter strategy.The level of resistanceneeded will be a factor that must be considered, regardless of the approachtaken. Autotoxicity may be more likely to be a problem when introducing a new compound into a plant than when increasing the production of an existing allelopathic compound.In the lattercase, the plant would have an inherentmechanismfor such avoidance.However,the level of protection might be insufficient for greatly increased levels of the compound. If so, enhancementof the native protectivemechanismor introductionof a new mechanism of resistance would be options. Resistance to Allelochemicals. Evolution of resistance is a growing problemwith syntheticherbicidesand could pose a problem for long-term or intensive use of allelopathy for weed suppression.Just as resistance to synthetic herbicides has evolved, resistance to allelochemicals producedby crops might also evolve. A recentpaper suggests that one reason that diffuse knapweed (Centaurea diffusa Lam # CENDI) is a more virulent weed in North American than in Eurasia is that the weeds in its geographical origin have evolved resistance to allelochemicals that it might make (Callaway and Aschehoug 2000). Several factors influence the probability of evolved resistance. In most cases, the more persistentthe selection pressure,the more rapidly resistance evolves. Thus, the continuous nature of the selection pressure by allelochemicals might increase the probabilityand speed of resistanceevolving. The relatively weak phytotoxicityof most characterized allelochemicals in field situations could influence the speed of evolution; however, the effect would be a function of the moleculartarget site and other factors. The molecular target site of the allelochemical can have a strong influence on the rate of appearance of resistance. With synthetic herbicides, evolution of resistance is much more rapid with certainmolecular target sites (e.g., acetolactatesynthase: Saari and Maxwell 1997) than to those with other sites of action (e.g., EPSP synthase: Dyer 1994). Little is known of molecular sites of allelochemicals. There is considerable variation in resistance to allelochemicals; however, few studies, such as those of Wieland et al. (1999), have investigated the mechanisms of resistance. Whether these differences in susceptibility are due to an evolved response to presence of the allelochemical or to unrelated factors is unknown. A rough estimate of the proba832

bility and speed of the evolution of resistance to a phytotoxin can be produced by treating a large population of mutagenized seeds with the lowest concentrationof the toxin normallyrequiredof 100%mortalityto see the frequency of resistance (e.g., Padgette et al. 1996). This has been done with few synthetic herbicides and, to our knowledge, with no naturalphytotoxins. Risk Assessment. As with any othertransgeniccrop, the potential risk to human health and to the environment must be evaluated before a crop that has been transgenically modified to be more allelopathic can be released. Human health issues include the toxicity of the allelochemical and its metabolic intermediates, allergenic properties of any transgenes introduced into the crop, and unexpected metabolic effects that would result in other toxicants or in decreased nutritionalvalue of the crop. The most importantenvironmentalrisk is that of horizontaltransferof the transgeneinto relatedwild species (Hancock et al. 1996), thus giving the recipient species a competitive edge over other native plants. Enhancementof existing allelochemicalproductionin crop species such as sorghum and rice or the transferof genes encoding their allelochemical propertiesto other crops might pose little risk to humanhealth because they are already widely consumed crops with no known health risks. Nevertheless, germplasm of crops such as potato (SolanumtuberosumL.), tomatos (LypersiconesculentumL.), and celery [Apiumgraveolens L. var.dulce (Miller) Pers.] contain genes for producingnaturalproducts that are quite toxic in sufficientconcentration. Crops engineeredto producetheir allelochemicalsonly in roots and to excrete them into the soil would not likely be a problem if the harvested portion of the crop is not the root. Even with root crops, if the plant excretes the compound into the soil almost as fast as it is produced,concentrationsin the root might still be low enough to be of negligible concern. Introgressionof herbicide resistance transgenesfrom crop species to wild species poses little environmental risk because these traits would not give the wild species any competitive advantageover other species in natural ecosystems in which the herbicide is not used. However, transferof an allelopathic trait could give the recipient species a significant competitive advantage in natural ecosystems. This risk can be contained by the use of reproductivebarffersin the transgeniccrop and by using the transgenic crop only in geographic areas in which the crop species has no wild relative with which it can interbreed.Furthermore,in most cases allelopathy (including autotoxicity avoidance) is probablya multigenic
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trait, requiring the transferof several genes to produce an allelopathic crop. If the transgenesare located at different locations within the genome, the probabilityof all these genes being simultaneously incorporated into a wild species genome is exceedingly small. The fewer the genes used to confer or regulate an allelopathy trait, the more carefully the risk of introgressionshould be evaluated. CONCLUSIONS Introductionto or improvement of allelopathic traits of crops through genetic engineering is likely to be a formidable task. Even if successful, such a value-added trait is not likely to completely replace other weed management practices because the effectiveness of allelopathy will be dependent on many factors. Nevertheless, if such traits could even marginally reduce herbicide use or tillage, the monetary savings to farmers over time would be significant, and the reduced environmentalimpact would be highly desirable.In underdevelopedcountries, hand weeding is still a necessary family chore carried out mainly by women and children,preventingthem from spending their time on education and more worthwhile pursuits. The introduction of highly weed-suppressive crops could benefit these societies in many intangible ways. LITERATURE CITED
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