REVIEWARTICLE

Flavonoids: Their Structure, Biosynthesis and Rolein the Rhizosphere, Including Allelopathy

Leslie A. Weston & Ulrike Mathesius

Received: 29 December 2012 /Revised: 18 January 2013 /Accepted: 23 January 2013 /Published online: 9 February 2013# Springer Science+Business Media New York 2013

Abstract Flavonoids are biologically active low molecularweight secondary metabolites that are produced by plants,with over 10,000 structural variants now reported. Due totheir physical and biochemical properties, they interact withmany diverse targets in subcellular locations to elicit variousactivities in microbes, plants, and animals. In plants, flavo-noids play important roles in transport of auxin, root andshoot development, pollination, modulation of reactive ox-ygen species, and signalling of symbiotic bacteria in thelegume Rhizobium symbiosis. In addition, they possess an-tibacterial, antifungal, antiviral, and anticancer activities. Inthe plant, flavonoids are transported within and betweenplant tissues and cells, and are specifically released intothe rhizosphere by roots where they are involved in plant/-plant interactions or allelopathy. Released by root exudationor tissue degradation over time, both aglycones and glyco-sides of flavonoids are found in soil solutions and rootexudates. Although the relative role of flavonoids in allelo-pathic interference has been less well-characterized than thatof some secondary metabolites, we present classic examplesof their involvement in autotoxicity and allelopathy. We alsodescribe their activity and fate in the soil rhizosphere inselected examples involving pasture legumes, cereal crops,and ferns. Potential research directions for further elucida-tion of the specific role of flavonoids in soil rhizosphereinteractions are considered.

Keywords Plant interference . Roots . Exudation .

Rhizosphere . Secondary metabolites . Phenolics

Introduction

Flavonoids are low molecular weight secondary metabolitesthat are produced by plants, and generally are described asnon- essential for plant survival, unlike primary metabolites.Secondary products are biologically active in many ways,and over 10,000 structural variants of flavonoids have beenreported (Williams and Grayer, 2004; Ferrer et al., 2008);their synthesis appears to be ubiquitous in plants andevolved early during land plant evolution, aiding in plantprotection and signalling (Pollastri and Tattini, 2011; Delauxet al., 2012). Due to their physical and biochemical proper-ties, flavonoids also are able to interact with many diversetargets in subcellular locations to elicit various activities inmicrobes, plants and animals (Taylor and Grotewold, 2005;Buer et al., 2010). Although flavonoids have many roles inplants, including their influence on the transport of auxin(Brown et al., 2001; Wasson et al., 2006; Peer and Murphy,2007), they also play important roles in modulating thelevels of reactive oxygen species (ROS) in plant tissues(Taylor and Grotewold, 2005; Agati et al., 2012), and pro-vide colouring to various tissues including flowers (Davieset al., 2012). In addition, they are required for signallingsymbiotic bacteria in the legume rhizobium symbiosis(Djordjevic et al., 1987; Zhang et al., 2009), and are impor-tant in root and shoot development (Buer and Djordjevic,2009).

In relation to their role in allelopathy and the inhibition ofseedling root growth, the activity of flavonoids as regulatorsof auxin transport and degradation is likely to be of partic-ular importance. Depending on their structure, flavonoidscan impact the breakdown of auxin by IAA oxidases and

L. A. Weston (*)EH Graham Centre, Charles Sturt University, Wagga Wagga,NSW 2678, Australiae-mail: leweston@csu.edu.au

U. MathesiusDivision of Plant Science, Research School of Biology,Australian National University, Canberra, ACT 0200, Australiae-mail: ulrike.mathesius@anu.edu.au

J Chem Ecol (2013) 39:283–297DOI 10.1007/s10886-013-0248-5

peroxidases (Furuya et al., 1962; Stenlid, 1963; Mathesius,2001) and also affect polar auxin transport (Stenlid, 1976;Jacobs and Rubery, 1988; Peer and Murphy, 2007), therebyimpacting root growth of target species. Some isoflavonoidphytoalexins act as cofactors to auxin in adventitious rootdevelopment, although the mode of action of these mole-cules remains unknown (Yoshikawa et al., 1986). In addi-tion, flavonoids show affinity for many enzymes and otherproteins in plants and animals, including those required formitochondrial respiration. In this case, certain flavonoidscontribute to inhibition of NADH oxidase and the balanceof reactive oxygen species (Hodnick et al., 1994, 1988),thereby impacting respiration.

In animal systems, plant-produced flavonoids are impor-tant dietary components, and are known to possess a broadrange of properties including antibacterial, antifungal, anti-viral, and anticancer activity (Taylor and Grotewold, 2005;Soto-Vaca et al., 2012). Many flavonoids also have servedas templates in the development of new pharmaceuticals(Cutler et al., 2007). Interestingly, flavonoids in planta canbe transported within and between tissues and cells, andoften are released into the rhizosphere where they are in-volved in plant to plant interactions, specifically allelopathicinterference (Hassan and Mathesius, 2012). They can bereleased by root exudation or through tissue degradationover time, and although both aglycones and glycosides offlavonoids are found in root exudates, their relative role inallelopathic interference, specific activity and selectivity,and mode(s) of action remain less well-characterised(Berhow and Vaughn, 1999; Weston and Duke, 2003;Levizou et al., 2004; Hassan and Mathesius, 2012). Thisreview describes the diversity of flavonoids produced byhigher plants, their biosynthesis and transport, their roles inthe rhizosphere, and gives particular emphasis to their re-cently described roles in allelopathic interference with otherplants. We also outline potential research directions for thefuture to further elucidate the specific role of flavonoids insoil-rhizosphere interactions.

Flavonoid Structure, Function, and Biosynthesisin Plants

Flavone ring structures are found in fruits, vegetables,grains, nuts, stems, leaves, flowers and roots and are ubiq-uitous throughout nature, playing an integral role in plantgrowth and development (Harborne, 1973). The term flavo-noid generally is used to describe a broad collection ofnatural products that possess a C6-C3-C6 skeleton, or morespecifically a phenylbenzopyran function (Marais et al.,2007). The typical flavone ring is the backbone of flavonoidstructure, or the nucleus of diverse flavonoid molecules(Fig. 1). The flavonoid biosynthetic pathway now is well

elucidated, compared to biosynthetic pathways of othersecondary products (Dixon and Steele, 1999; Winkel-Shirley, 2001). Flavonoids are synthesized through thephenylpropanoid or acetate-malonate metabolic pathway,which also is well-described in Arabidopsis. Interestingly,unlike legumes, Arabidopsis lacks chalcone reductase andisoflavone synthase enzymes, so therefore it cannot pro-duce one subset of flavonoids, the isoflavonoids (Buer etal., 2007, 2010,).

Arabidopsis mutants (Peer et al., 2001) and transgeniclegumes with modified branches of the flavonoid pathway(Yu et al., 2003; Subramanian et al., 2005, 2006; Wasson etal., 2006) now are available and provide a unique tool forstudying the role of flavonoids in rhizosphere interactions.Interestingly, flavonoids have similar precursors to thoseutilized for lignin biosynthesis but exhibit a number of basalstructures that result in generation of diverse structuresincluding flavones, flavonols, flavan-3-ols, flavanones, iso-flavanones, isoflavans, and pterocarpans (Fig. 1).Substitution by glycosylation, malonylation, methylation,hydroxylation, acylation, prenylation, or polymerizationleads to diversity in this family and has important impactupon function, solubility, and degradation (Dixon andSteele, 1999; Winkel-Shirley, 2001; Zhang et al., 2009).

In higher plants, flavonoid synthesis begins when en-zyme complexes form on the cytosolic side of the endoplas-mic reticulum (Jorgensen et al., 2005), which then maylocalize to the tonoplast for subsequent glycosylation andstorage in the vacuole (Winkel, 2004). In specific tissues,flavonoid synthesis and accumulation often is located indistinct cells (Fig. 2). Subcellularly, flavonoids have beenfound in the nucleus, the vacuole, cell wall, cell membranesand the cytoplasm (Hutzler et al., 1998; Erlejman et al.,2004; Saslowsky et al., 2005; Naoumkina and Dixon,2008). While flavonoid glycosides stored in the vacuoleprobably do not generally have active roles, their releasedaglycone counterparts could have functions in the plantcytoplasm, e.g., in regulation of enzyme activity, formationof reactive oxygen species, and auxin transport (Taylor andGrotewold, 2005; Naoumkina and Dixon, 2008). In somestudies, flavonoid glycosides also have been found to haveactive roles, e.g., in regulation of IAA oxidase, which couldlead to changes in auxin accumulation (Furuya et al., 1962;Stenlid, 1968). Accumulation of flavanols (catechins) oftenhas been observed in nuclei, especially in gymnospermspecies. Their roles could include the regulation of geneexpression through chromatin remodelling and effects onenzymes and protein complexes that regulate gene expres-sion (Feucht et al., 2012).

In root tissues, flavonoids can accumulate at the root tipand in root cap cells from where they can be exuded orsloughed off into the soil (see below). Flavonoids also arelocalized in specific cell types of the root (Fig. 2), and can

284 J Chem Ecol (2013) 39:283–297

be readily studied by use of fluorescent imaging due to theirautofluorescence (Bayliss et al., 1997; Hutzler et al., 1998;Mathesius et al., 1998). Roots typically produce many di-verse flavonoids and these are stored as glycosides or agly-cones and released both by root exudation and tissuedecomposition or leaching (Rao, 1990). Root–produced fla-vonoids play roles in signalling to microbes and otherplants, as well as in protection from soil pathogens, andtheir accumulation in roots is highly dependent on bioticand abiotic environmental conditions (Rao, 1990).

Flavonoid Transport, Exudation and Potential Rolesin the Rhizosphere

While flavonoid accumulation can be cell- and tissue-specific, there is evidence for intra- and intercellular flavo-noid movement, in some cases through active transport.Intracellular movement is most likely to occur via vesiclemediated transport or through membrane bound transportersof the ABC (ATP binding cassette) or MATE (MultidrugAnd Toxic Extrusion compound) families (Zhao and Dixon,2009). In Arabidopsis, application of flavonoid aglycones tothe root or the shoot leads to their transport across severalcell layers, suggesting that they can be easily transportedbetween cells (Buer et al., 2007). This long distance

transport is likely to be catalyzed by members of the ABCtransporter families because application of ABC transporterinhibitors reduced long distance auxin transport (Buer et al.,2007). However, the specific molecular mechanisms ofinter- and intracellular flavonoid transport require furtherstudy.

Flavonoids have been shown to be readily exuded intothe rhizosphere, although few studies have been able toquantify their exudation into soil. Flavonoid exudation hasbeen shown to increase in response to various microbialsignal molecules of symbionts and pathogens (Schmidt etal., 1994; Armero et al., 2001) and to abiotic stress includingnitrogen, temperature, and water stress (Coronado et al.,1995; Dixon and Paiva, 1995; Juszczuk et al., 2004).Flavonoids also can be passively released from decompos-ing root cap and root border cells directly into the rhizo-sphere (Hawes et al. 1998; Shaw et al. 2006). AlthoughABC transporters have been implicated in their exudation,detailed mechanisms are not well understood. ABC trans-porter mutants of Arabidopsis showed altered root exudateprofiles, which contained changes in flavonoids includingphytoalexins, as well as organic acids and sugars (Badri etal., 2008, 2009, 2012) An ATP-dependent ABC transporteralso has been shown to be involved in the exudation ofgenistein, an isoflavonoid, from soybean root plasma mem-brane vesicles (Sugiyama et al., 2007). Similarly, silencing

Phenylalanine

4-coumaroyl CoA + 3 x malonyl CoA

tetrahydroxy chalcone trihydroxy chalcone

liquiritigenin

Isoflavonoids

naringenin

3-OH-flavanones

flavan-3,4-diols

3-OH-anthocyanidins

Anthocyanins

Aurones

Phlobaphenes

Flavones

Flavonols

Condensed tannins

isoflavone

Chalcone synthase

Isoflavone synthase

Isoflavone reductase

Isoflavone synthase

Dihydroflavonol reductase

Flavone synthase I + II

Flavonol synthase

Medicarpin

O

OOH

OH

OH

OH

OH

Quercetin

O

OOH

OH

OH

OH

Luteolin

O

OCH3

O

OH

OOH

OH O

OH

Vestitone reductase

Chalcone isomerase

Genistein

Fig. 1 Simplified overview of the flavonoid biosynthetic pathway. Note that specific branches of the pathway might be plant species-specific.Major enzymes and end products are highlighted

J Chem Ecol (2013) 39:283–297 285

of the ABCG-type transporter MtABCG10 from Medicagotruncatula Gaertn. recently was shown to significantly re-duce accumulation of several isoflavonoids in roots and rootexudates, and this was accompanied by enhanced suscepti-bility of the roots to the pathogen Fusarium oxysporum(Banasiak et al., 2013). However, because ABC transportersare likely to have multiple substrates, future studies willhave to demonstrate the specific role of (iso)flavonoids inthe altered exudates of these types of mutants..

Flavonoid persistence in the soil varies with environmen-tal conditions and is influenced strongly by the presence ofsoil microbes, some of which can metabolize or modifyflavonoids (Hartwig and Phillips, 1991; Rao and Cooper,1994,1995). Flavonoids also can become unavailable due toabsorption to soil particles and organic matter (Shaw and

Hooker, 2008). Their mobility in soil varies greatly withchemical composition, e.g., glycosylation, which deter-mines their water solubility. In turn, the presence of flavo-noids in soil can alter soil composition and nutrientavailability through their activity as antioxidants and metalchelators. Chelation and reduction of metals in the soilimpact nutrient availability, especially phosphorus and iron.For example, an isoflavonoid identified from Medicagosativa L. root exudates dissolved ferric phosphate, enhanc-ing phosphate and iron availability (Masaoka et al., 1993).The flavonoids genistein, quercetin and kaempferol alteriron availability by reducing Fe(III) to Fe(II) and by chelat-ing unavailable iron from iron oxides (Cesco et al., 2010).

Some of the more well-known biological roles of flavo-noids in the rhizosphere include the activation of nod genesfrom symbiotic rhizobia, chemo-attraction of rhizobia andnematodes, inhibition of pathogens, and activation ofmycorrhizal spore germination and hyphal branching(Table 1). These functions can indirectly affect thegrowth of conspecifics through the regulation of nitrogenfixation and mycorrhization, as well as their susceptibility topathogens (Fig. 3).

Legume root exudates contain species-specific flavo-noids, specifically flavones, that activate the nodulationgenes of their respective symbionts by binding to the tran-scriptional activator NodD (Redmond et al., 1986; Petersand Long, 1988; Cooper, 2004; Peck et al., 2006). Thisleads to Nod factor synthesis and subsequent infection andnodulation of the legume host. However, some flavonoids,especially isoflavonoids, also inhibit nod gene induction(Zuanazzi et al., 1998). Some flavonoids that induce nodgenes, specifically luteolin and apigenin, have dual actionsas chemo-attractants, with different flavonoids attracting dif-ferent Rhizobium species (Aguilar et al., 1988; Dharmatilakeand Bauer, 1992). Flavonoid exudation by the host changesduring different stages of the symbiosis, presumably fine-tuning Nod factor synthesis during nodule development andcolonisation (Dakora et al., 1993). Flavonoid composition inthe rhizosphere around legume roots also can be altered byrhizobia, which metabolise and alter the structure of flavo-noids over time (Rao and Cooper, 1994, 1995).

Flavonoids and other phenolic compounds also specifi-cally repel soil-dwelling plant parasitic nematodes and affecthatching and migration. For example, the flavonols kaemp-ferol, quercetin, and myricetin acted as repellants for theroot lesion nematode Radopholus similis and the root knotnematode Meloidogyne incognita, whereas the isoflavo-noids genistein and daidzein and the flavone luteolin actedonly on R. similis (Wuyts et al., 2006). Kaempferol, quer-cetin, and myricetin also inhibited motility of M. incognitaand kaempferol inhibited egg hatching of R. similis, whereasother nematodes were not affected by any of these com-pounds. It was demonstrated that nematode-resistant plant

A

B

C

D

p

c

c

Fig. 2 Flavonoid accumulation in roots is cell type specific. a Flavo-noid accumulation in root tips of Medicago truncatula. Blue andorange autofluorescence is due to the presence of flavonoids. Notethe significant accumulation of flavonoids in the root tip and in rootcap cells (orange, arrow). b Flavonoid accumulation in a mature whiteclover (Trifolium repens L.) root. Note the accumulation of differentflavonoids exhibiting different emission wavelengths in different celltypes, e.g. pericycle (p) and cortex (c). c Flavonoid accumulation in ayoung but differentiated root section of white clover in cortex cells (c).d Flavonoid accumulation in a section through the root tip of whiteclover showing flavonoid accumulation in nuclei of meristematic cells(light blue) and in the cytoplasm of epidermal and outer cortical cells(yellow, arrow). All images were taken using fluorescence microscopywith UV excitation. Magnification bars are 500μm in a and 100μm inb, c and d

286 J Chem Ecol (2013) 39:283–297

cultivars contained increased amounts of flavonoids, spe-cifically the isoflavonoids and the pterocarpan medicarpin,in alfalfa (M. sativa L.) (Baldridge et al., 1998; Edwardset al., 1995)

Flavonoids also inhibit a range of root pathogens, espe-cially fungi (Makoi and Ndakidemi, 2007). Generally, iso-flavonoids, flavans, or flavanones have been found as themost potent antimicrobials. These defense compounds caneither be induced upon pathogen attack (phytoalexins) or bepreformed (phytoanticipins), while others are exuded intothe soil (Armero et al., 2001). In this role, flavonoids havebeen shown to act as antimicrobial toxins (Cushnie andLamb, 2011) and anti- or pro-oxidants (Jia et al., 2010).Pterocarpans, end products of the isoflavonoid pathway,including medicarpin, pisatin and maackiain also have anti-microbial properties (Naoumkina et al., 2010). For example,

pisatin from pea provided protection from pathogenic fungiand oomycetes (Pueppke and Vanetten, 1974). The likelymechanism of action against fungi is through inhibition ofelongation of fungal germ tubes and mycelial hyphae(Blount et al., 1992; Higgins, 1978).

During pathogen attack in a resistant plant species, phy-toalexins are thought to become oxidized, leading to forma-tion of toxic free radicals that can stimulate cell death duringa hypersensitive response (Heath, 2000). Flavonols alsocontribute to resistance against pathogens. For example,quercetin is antimicrobial and inhibits the ATPase activityof DNA gyrase in bacteria (Plaper et al., 2003; Naoumkinaet al., 2010). Carnation (Dianthus caryophyllus) also hasbeen shown to mount a significant defense against Fusariumoxysporum by formation of the fungitoxic flavonol triglyco-side of kaempferide (Curir et al., 2005).

Table 1 Examples of flavonoids of different biosynthetic branches of the pathway and their roles in the rhizosphere

Flavonoid name Flavonoid class Function Reference

Luteolin Flavone Nod gene and chemotaxis inducer in rhizobia Cooper (2004); Peters and Long (1988)

Nematode repellant Wuyts et al. (2006)

7,4′dihydroxy flavone Flavone Nod gene and chemotaxis inducer in rhizobia Cooper (2004); Djordjevic et al. (1987);Redmond et al. (1986); Tsai andPhillips (1991)

Hyphal branching stimulator

5,7,4′-trihydroxy-‘,5’dimethoxyflavone

Flavone Allelopathic inhibitor of seedling growth Kong et al. (2004, 2007)

Quercetin Flavonol Nod gene inducer Cooper (2004)

Iron chelator Cesco et al. (2010)

Nematode repellant and motility inhibitor Wuyts et al. (2006)

Antimicrobial Naoumkina et al. (2010)

Hyphal branching stimulator Tsai and Phillips (1991)

Allelopathic inhibitor of seedling growth Rice (1984)

Kaempferol Flavonol Nod gene and chemotaxis inducer in rhizobia Cooper (2004)

Iron chelator Cesco et al. (2010)

Nematode repellant, egg hatching and motilityinhibitor

Wuyts et al. (2006)

Allelopathic inhibitor of seedling growth Levizou et al. (2004); Rice (1984)

Myricetin Flavonol Nod gene inducer Cooper (2004)

Nematode repellant Wuyts et al. (2006)

Formonenetin Isoflavonoid Nod gene inhibitor Djordjevic et al (1987)

Hyphal branching inhibitor Tsai and Phillips (1991)

Genistein Isoflavonoid Nod gene inducer Cooper (2004)

Iron chelator Cesco et al. (2010)

Nematode repellant Wuyts et al. (2006)

Daidzein Isoflavonoid Nod gene inducer Cooper (2004)

Nematode repellant Wuyts et al. (2006)

Coumestrol Isoflavonoid Nod gene inducer Cooper (2004)

Morandi et al. (2009)Hyphal branching stimulator

Medicarpin Isoflavonoid-derivedpterocarpan

Antimicrobial phytoalexin Naoumkina et al. (2010)

Maackiain Isoflavonoid-derivedpterocarpan

Antimicrobial phytoalexin Naoumkina et al. (2010)

J Chem Ecol (2013) 39:283–297 287

Other known roles of flavonoids in the rhizosphere in-clude effects on arbuscular mycorrhizal fungi, which form abeneficial symbiosis with the majority of land plants underconditions of phosphorus deficiency (Harrison, 2005).Hyphae of the mycorrhizal fungi are attracted to root exu-dates, and in some cases this has been attributed to thepresence of flavonoids, which stimulate hyphal branchingand presymbiotic growth towards the host (Siqueira et al.,1991; Scervino et al., 2005a, b, 2006, 2007; Steinkellner etal., 2007). In Medicago truncatula Gaertn., the hyper-accumulation of coumestrol, a potent hyphal stimulator, iscorrelated with hyperinfection by the symbiont (Morandi etal., 2009). However, both hosts and non-hosts also havebeen reported to exude flavonoids that inhibit hyphalbranching (Tsai and Phillips, 1991; Akiyama et al., 2010).Exudation of flavonoids from the host also is phosphorus-regulated (Akiyama et al., 2002), similar to the dependenceof flavonoid accumulation on nitrogen availability inlegumes forming nitrogen-fixing symbioses (Coronado etal., 1995). As one can see from review of the plantliterature, the role of flavonoids in plant defense in therhizosphere and other physiological processes is complexand will no doubt be the subject of additional study aswe continue to evaluate regulation of these processesfrom a molecular perspective.

Role of Flavonoids in Allelopathy and Plant Defense

Allelopathy is defined as the direct or indirect effect ofsecondary products produced by a donor plant upon a re-ceptor plant; these interactions are most often classified asdetrimental or harmful, but allelopathic interactions also canbe described as beneficial (Rice, 1984). The importance ofallelopathy in agriculture is increasingly recognized(e.g.,see papers in this issue by; Kato-Noguchi and Peters,2013; Shulz et al., 2013; Weston et al., 2013; Wothingtonand Reberg-Horton, 2013; Zhang et al., 2013), and allelo-pathic interactions are often used for weed suppression oradditional management of weeds in diverse cropping sys-tems (Weston, 1996; Xuan and Tsuzuki, 2002; Weston andDuke, 2003;).

Flavonoids have been reported in the literature for over50 years as chemical mediators involved in allelopathicinteractions in the soil rhizosphere. Many plants producecopious quantities of diverse flavonoids from their livingroots, and our capacity to identify these metabolites in tracequantities has improved dramatically in recent years, hencemany recent publications have focused on the role of flavo-noids in allelopathy. As a result, flavonoids are more fre-quently implicated in allelopathic interactions in the soilrhizosphere as they have been identified in significant

O

OOH

OH

OH

OH

OH

Quercetin (flavonol): Antioxidant, chelatorof iron in soil, nematode repellant, phytoinhibitor

O

OOH

OH

OH

OH

Luteolin (flavone): Nod gene inducer

Coumestrol(coumestan, isoflavonoid):Stimulator of VAM hyphal branching, Nod gene inhibitor

Maackiain(pterocarpan, isoflavonoid): antimicrobial phytoalexin

Kaempferol (flavonol)Phytoinhibitor, auxin transport inhibitor, nematode repellant

p-coumaric acid and other simple phenolics: Phytoinhibitors

O

OOH

OH

OH

OH

OH

Fig. 3 Overview of flavonoid functions in the rhizosphere

288 J Chem Ecol (2013) 39:283–297

concentrations in many bioactive root exudates. Both simpleand more complex phenolics, including flavonoids, are re-leased from decomposing plant tissues as leachates andthrough the process of microbial degradation and transfor-mation in soil. In this review, we examine several casestudies of allelopathy in both natural and managed eco-systems, including plants of diverse taxonomic origin, todemonstrate how allelopathic interference is potentiallymodulated by the presence of bioactive flavonoids inthe rhizosphere.

Alfalfa and Clover Autotoxicity The perennial legumes al-falfa (Medicago sativa L.) and red or white clover (Trifoliumpratense L. or Trifolium repens L.) are widely used intemperate regions as high quality pastures and fodder plantscontaining substantial levels of protein (Oleszek andJurzysta, 1987; Hancock, 2005). These crops also are im-portant for their contributions of large quantities of organicmatter to the soil, improvement of soil structure, and en-hanced water infiltration following establishment. Alfalfagenerally contributes about two fold higher levels of organicdry matter in comparison to the forage crops of red or whiteclover. Most alfalfa and certain clovers typically are estab-lished as perennials, and as such they tend to be fairlyresistant to weed infestation over time. However, as pasturesage, both established alfalfa and clover pasture stands oftenexhibit significant reductions in plant counts and productiv-ity. This phenomenon, known as autotoxicity, severely lim-its the ability of producers to renovate declining pastures(Tesar, 1993; Hancock, 2005). When renovating these pas-tures, the planting of successive crops also frequently leadsto poor stands in crops immediately following establishedclover or alfalfa. In the past, the cause of this phenomenonwas thought to be depletion of soil moisture and nutrients orbuild-up of soil pathogens during perennial crop growth, butnow there is evidence that these crops also exhibit phyto-toxicity or allelopathy, through production and release oftoxic secondary metabolites (see also this issue Huang et al.,2013). In the case of alfalfa or lucerne, autotoxicity can limitthe development and productivity of the crop itself(Cosgrove and Undersander, 2003; Hancock, 2005) andresult in permanent morphological reductions in root devel-opment and shoot growth (Jennings, 2001; Jennings andNelson, 2002) (Fig. 4.)

Since these perennial legumes are known to be bothautotoxic and allelopathic (Hedge and Miller, 1992), numer-ous investigators have attempted to identify the allelochem-icals responsible for phytotoxicity, with limited success.(Oleszek and Jurzysta, 1987) reported the release of water-soluble allelochemicals from alfalfa and red clover rootextracts, which inhibited fungal and seedling growth in avariety of soils with different textural properties over time.They concluded that although the extracts contained

numerous phytoinhibitors including saponins that were sol-uble in water or alcohol, the presence of medicagenic acidalong with other unidentified water- soluble inhibitors wasassociated with inhibition.

Other investigators also have found that alfalfa plantsreleased water-soluble allelochemicals from fresh leaves,stems and crown tissues as well as dry hay, older roots,and seeds (Klein and Miller, 1980; Hedge and Miller,1992; Tsuzuki et al., 1999; Xuan and Tsuzuki, 2002). In acultivar study, the Japanese cultivar Lucerne was deter-mined to be most inhibitory to seedling germination andgrowth when compared to other cultivars in laboratory andgreenhouse experimentation (Xuan and Tsuzuki, 2002).Miller (1996) and Chung and Miller (1995) also observedsignificant cultivar differences in phytotoxicity of alfalfaextracts upon seedling growth, with Pioneer 5472 the mostsuppressive when compared with other American alfalfacultivars.

Hancock has speculated on the evolutionary role or pur-pose of autotoxicity in alfalfa (also known as lucerne) orclover. Alfalfa, along with other perennial pasture legumes,is believed to have developed and evolved in the northernand eastern coastal regions of the Mediterranean. Duringthe period in which evolution was thought to have oc-curred, these areas likely experienced hot dry conditionsand resource limitations (Hancock, 2005). Under condi-tions such as these, Hancock and others postulated that acompetitive advantage would arise if other plants, includ-ing alfalfa seedlings, could be prevented from establish-ing near mature plants, specifically through autotoxicity(Jennings, 2001)(Figs. 4, 5 and 6).

In many legumes it has been well established that phe-nolics, including flavonoids, are routinely produced and

Fig. 4 Established stand of alfalfa (Medicago sativa L.) in Australianpaddock. Note the concentric space around each alfalfa plant, withlittle to no other vegetation in this concentric ring. Older alfalfa standsoften exhibit autotoxicity and allelopathy over time and individualplant growth becomes limited by presence of adjacent plants. Phototaken by P. A. Weston, Australia

J Chem Ecol (2013) 39:283–297 289

released from developing roots during seed germination andseedling establishment (Mandal et al., 2010) . As described,these compounds play various critical roles in mediatinglegume Rhizobium symbiosis, but also play important rolesin stimulation or inhibition of other soil organisms or prop-agules (Buer et al., 2010; Mandal et al., 2010; Hassan andMathesius, 2012). Interestingly, the same phenolic acids thatstimulate rhizobial defense and IAA production also functionas potent inhibitors of seed germination and seedling growth,and include p-coumaric acid, vanillic acid, ferulic acid, gallicacid, p-hydroxybenzoic acid, and aldehyde and other cinnamicacid derivatives (Rice, 1984; Weston and Duke, 2003; Batishet al., 2006). Flavonoids typically produced by alfalfa seed-lings also promote spore germination of Glomus spp., impor-tant AM fungi that beneficially infect plants. Quercetin, 4’–7-dihydroxyflavone and 4′–7-dihydroxyflavanone were shownto be especially stimulatory to spore germination in AM fungi(Tsai and Phillips, 1991).

Quercetin, luteolin, and other substituted flavones andflavanones are released by germinating seeds and livingroots of alfalfa and related legume crops over time, and their

interactions with fungal pathogens suggest that they alsomay be important for protection in plants against soil-borne pathogens while stimulating VAM fungi (Hartwig etal., 1991). Somewhat surprisingly, we have found that nosystematic soil-based studies have attempted to characterizethe bioactive constituents produced by exudation or releasedby older established Medicago sativa roots into the rhizo-sphere, nor has anyone attempted a practical determinationof their concurrent impacts on seedling establishment, path-ogen suppression, VAM fungi or rhizobacterial growth anddevelopment. These organisms all are commonly found intemperate soils, and are likely to be impacted by secondaryproducts such as flavonoids in the rhizosphere. Given thelikelihood that a complex mixture of water-soluble com-pounds is released by mature alfalfa roots, secondary prod-ucts in a complex mixture could play either stimulatory orinhibitory roles in the rhizosphere, and these roles maychange depending on available concentrations in the soilwater solution. As we currently have the ability to accuratelydetect trace quantities of bioactive secondary products andsensitively perform metabolomic profiling by using both gasor liquid chromatography (GC or LC) coupled to mass spec-trometry, it is now possible to fully characterize productionand turnover of flavonoids and other secondary products inthe rhizosphere of mature alfalfa stand. Future studies couldbe designed to investigate alfalfa autoxicity under field con-ditions, in both young and mature alfalfa stands to furtherdetermine the role of specific flavonoids and phenolics in thisdetrimental phenomenon.

Flavonoid production in roots of perennial legumes alsohas been shown to be strongly regulated by nitrogen supply.Under nitrogen limiting conditions, flavonoid biosynthesisgenes such as chalcone synthase and isoflavone reductaseare upregulated and show enhanced expression, indicatingthe nitrogen nutrition status of the plant plays a role inimpacting secondary product production (Coronado et al.,1995). This also is the case for other secondary plant prod-ucts such as the hydroxamic acids BOA and DIMBOAproduced by Secale cereale L. (Mwaja et al., 1995). Thebioavailability of soil nitrogen could thus also play a criticalrole in the regulation of allelopathy or autoallelopathicinteractions in established legume stands.

In perhaps the most interesting field study outlining thefate of flavonoids over time, Fomsgaard and colleagues usedsensitive LC-MS/MS techniques to profile a diverse groupof over 20 flavonoids released from living and decomposingwhite clover stands in Denmark, in situ and after soil incor-poration of the clover as a green manure (Carlsen et al.,2012). As the authors report, numerous studies have impli-cated allelochemicals produced by white clover with weedsuppression, as well as negative interactions associated withallelopathy or replant/pathogenesis problems followingwhite clover establishment. This ground-breaking study

Fig. 5 and 6 In contrast to an established alfalfa (Medicago sativa L.)stand with limited weed infestation present (right), an adjoiningpaddock in the absence of alfalfa shows significant infestation of bothgrass and broadleaf weeds (left). Photos taken by P. A. Weston,Australia

290 J Chem Ecol (2013) 39:283–297

evaluated the pattern of flavonoid release from living clovergrown under field conditions and also from leachates fol-lowing incorporation of green cover crops into field soil.

Their findings help to explain the potential for allelopa-thy and autoallelopathic interactions associated with estab-lished white clover stands. Specifically, the flavonoidaglycones formononetin, medicarpin, and kaempferol pre-dominated in soil analyses, with glycosides of kaempferoland quercetin also present at relatively high concentrations.Kaempferol persisted for days in field soil surroundingliving or incorporated clover stands. These aglyconesand related constituents have specifically been noted topossess substantial phytoinhibitory activity (Rice, 1984).Kaempferol and kaempferol-3-O-L-arabinofuranoside stim-ulated seed germination at low concentrations but inhibitedseedling growth at higher concentrations (Hai et al., 2008);these compounds also are present in walnut (Juglans regiaL.) leaf extracts. The Carlsen study (2012) also noted thathighest concentrations of flavonoids in clover crops wereassociated with presence of clover flowers, in comparison toleaves, stems or roots in soil degradation studies. Several ofthe flavonoids identified are also known inhibitors of fungalgrowth, while others are associated with stimulation ofmicrobial growth in the rhizosphere (Mandal et al., 2010).In a recent study, Sosa and colleagues (2010) noted thatflavonoid aglycones derived from Cictus landanifer alsopersisted for very long periods of time in soil before degra-dation, even with no further addition of plant leachates.

Based on these interesting studies, we suggest that addi-tional experimentation is required to determine 1) mobilityof these compounds in various soil types and profiles, 2)location of maximal concentrations (likely to be nearestliving roots, for example), and 3) half-life of major flavo-noids and their glycosides in living soils. The application ofcomprehensive metabolic and proteomic profiling per-formed from similarly designed experimentation withlegumes growing in a field setting will most certainly aidin further defining the roles of flavonoids, phenolics, andtheir related degradation products in the rhizosphere.Although many flavonoids have been implicated in allelo-pathic inhibition of seedling growth and radicle elongationsuch as kaempferol and 6-methoxy-kaempferol, and rham-netin and isorhamnetin (Levizou et al., 2004), the mode ofaction of these inhibitors has not often been studied(Berhow and Vaughn, 1999).

In studies that evaluate the activity of selected flavonoidsimplicated in allelopathic interactions, some of the flavo-noid mixtures tested caused inhibition of root growth, re-duction in frequency of cell division in root meristematicregions, and suppression in the formation of root hairs andstatocytes in root cap cells (Levizou et al., 2004). Furtherevaluation of primary root formation in lettuce indicated thateriodictyol, naringenin, and quercetin 3,3-dimethylether

induced strong ageotropic responses. The loss of normalgravitropic orientation could result from the activity of someflavonoids as polar auxin transport inhibitors, and resultingperturbations in growth could impact ability to acquireresources and competitive interference. As mode of actionstudies are limited, additional studies with specific flavo-noids and mixtures are needed to further define their impacton root growth and morphology. However, as Levizou et al.(2004) suggest, the lack of dose dependent inhibitionresponses with many flavonoids indicates that activity ishighly concentration dependent; some of these compoundscould be inhibitory or stimulatory depending on availableconcentration in the soil/water solution in the rhizosphere.

Legume root exudates also contain allelochemicals activeagainst the parasitic weed Striga, which constitutes a majorconstraint to African agriculture with yield losses up to100 % in large parts of sub Saharan Africa (Gressel et al.,2004). The forage legume Desmodium uncinatum was iden-tified as an effective intercropping plant because it inhibitspost-germination and attachment of Striga, and this inhibi-tion was mimicked by several (iso)flavonoid glycosidesidentified from its root exudates (Hooper et al., 2010;Khan et al., 2010). Interestingly, some of these isoflavonesstimulated Striga germination, and this could later be po-tentially exploited to cause ‘suicidal’ germination of Striga.In Africa, the use of Desmodium as an intercrop plant hasbeen a cheap and successful strategy for smallholder farmersto control Striga infestations in their fields, with addedadvantage that some of the isoflavonoids are also activeagainst stem borers affecting the crops (Khan et al., 2006) .

The Role of Flavonoids in Barley and Rice AllelopathyBarley (Hordeum vulgare L.) is an important and ancientcereal crop grown as a source of protein for human andanimal consumption in temperate regions of the world.Barley was reported to exhibit weed suppression over2,000 years ago (Bertholdsson, 2004; Kremer and Ben-Hammouda, 2009). More recently, barley has been notedto exhibit allelopathic activity when used as a rotational cropwith succeeding small-seeded crops (Bertholdsson, 2004). Itwas also noted to suppress successive crops of bread anddurum wheat (Triticum aestivum L. and T. durum L.,respectively) (Kremer and Ben-Hammouda, 2009). Certaincultivars of barley also exhibit strong autotoxic tenden-cies, especially when grown under droughty conditionsin the field (Lovett and Hoult, 1995; Kremer and Ben-Hammouda, 2009). Repeated plantings of barley have prov-en to be high risk for the development of autoallelopathy,particularly in droughty areas, especially with certain culti-vars. Barley residues also were weed suppressive in trialsconducted in the US, Canada and Europe. Spring barleyresidues were particularly inhibitory to newly germinatedweed seedlings (Putnam and DeFrank, 1983). Despite the

J Chem Ecol (2013) 39:283–297 291

considerable literature pointing to the fact that barley ismore allelopathic under certain field conditions, very limitedresearch has yet been performed to identify the phytotoxinsassociated with interference. Kremer and Ben-Hammouda(2009) compiled the results of numerous field and green-house experiments involving barley and found that 44 dif-ferent allelochemicals were potentially associated withtoxicity in these studies. These were primarily identifiedfrom leaf, stem and seed extracts, but a number of com-pounds also were identified from root exudates or extracts.Potential allelochemicals included alkaloids, phenolics, fla-vonoids, cyanoglucosides, polyamines and hydroxamicacids, indicating that barley possesses a diversity of bioac-tive secondary products capable of eliciting numerous rhi-zosphere interactions. The alkaloids hordenine and graminewere exceptionally disruptive of cellular processes in affectedplants, and five phenolic acids were also implicated in barleyautoxicity studies.

Barley was noted to produce several unique flavonoids inits tissues, with most studies reporting existence of thesecompounds in shoot residues, as often only shoot tissueswere assayed in reported experiments. Unusual flavonoidsidentified included lutonarin, saponarin, and isovitexin, aswell as catechin and cyanidin. Potential modes of actioninclude inhibition of cell growth, disruption of ATP forma-tion, and interference with auxin function (Berhow andVaughn, 1999). Traditional breeding and selection may beuseful for enhancement of allelopathic activity; Bertholdsson(2005) showed that enhanced early vigor and weed suppres-sive tendencies could be selected for as useful traits inbarley germplasm (Bertholdsson, 2005). Older land raceswere likely to possess greater suppressive or allelopathictendencies than newly developed cultivars (Bertholdsson,2004) and could be a source of future germplasm forselection of enhanced weed suppression.

Research on rice (Oryza sativa L.) allelopathy has beenunderway for many years (Weston, 1996, 2005; Weston andDuke, 2003; see also this issue Kato-Noguchi and Peters,2013; Gealy et al., 2013; Fang et al., 2013; Worthington andReberg-Horton, 2013). This tropical or semi-tropical cerealhas been known to exhibit weed suppressive tendencies thatare cultivar dependent. Currently, rice cultivars have beenselected for enhanced allelopathic activity in both the USand China (Weston and Duke, 2003; Kong et al., 2004). Thepresence of momilolactone A and B in rice roots andshoots, as well as root exudates has been considered criticalto allelopathic activity in certain rice cultivars (Kato-Noguchi et al., 2002). However, a bioactive flavone(5,7,4′-trihydroxy-3′,5′-dimethoxyflavone), a cyclohexe-none, and a mixture of long-chain and cyclic hydrocarbonswere isolated from allelopathic rice residues, accessionPI312777 by the Kong laboratory in China. Both the fla-vones and cyclohexenone were particularly inhibitory to

weed seeds, including barnyardgrass (Echinochloa crus-galli L.), several Cyperus spp., and also to spore germina-tion of several soil-borne fungal pathogens. The combina-tion of both compounds resulted in greater inhibitoryactivity to weed propagules. Both compounds were shownto be released into the soil rhizosphere, after degradation orleaching of rice residues, and infestation of barnyardgrassaround living rice resulted in greater release of these sec-ondary products in soil compared to weed free plots (Konget al., 2004, 2007). Silencing the PAL pathways in rice byRNA interference (RNAi) leads to decreased phenolics pro-duction and root exudation and subsequent effects on soilmicrobial populations (see this issue, Fang et al., 2013).

Interestingly, a review of the literature related to flavo-noid activity shows that many of the same compoundsassociated with reduction in seed germination or seedlinggrowth also are active on soil pathogens, indicating somebroad spectrum activity of both commonly reported and rareplant flavonoids. In additional research performed by theKong laboratory (Kong et al., 2007), two flavone glycosideswere identified in extracts obtained from the allelopathicrice cultivar. Glycosides could not be detected in rice soilsas they degraded, but both aglycones were more stable, withhalf-lives of up to 24 h in living rice soils. The flavonoidaglycones were less mobile in the soil profile as compared toglycosides, indicating the aglycones are likely to play amore important role in suppression of plant growth or soilpathogens over time.

Flavonoid Production in Ferns is Associated WithAllelopathy Ferns often have been observed to form densemonocultural stands in conditions of low light in forestunderstories. Ferns are the oldest plant taxa and exhibitconsiderable morphological diversity, but despite taxonomicinterest in their origin and spread, few studies have beenconducted from a chemotaxonomic perspective. Ferns ex-hibit a rich array of secondary metabolites, and many exhibitstrong allelopathic tendencies, forming distinct concentricspheres of inhibition around mature plants. In an interestingstudy performed with Pityrogramma ferns, bioactive flavo-noids were identified in fern foliage, including high concen-trations of chalcones and dihydrochalcones (Star, 1980).Some ferns produce flavonoids in their roots, and othersproduce high concentrations in powders, called farinas, ofwhite, yellow, or gold on the abaxial sides of their fronds. Inhigh concentrations, these powders flake off fern fronds andfall to the soil surface, thus interacting with germinating weedseeds, and potentially contributing to the sphere of inhibitionsurrounding established ferns (Cooper-Driver, 1980).

In past literature, angiosperms were characterized on thebasis of their flavonoids; specifically, primitive woodyplants containing flavonols and proanthocyanaidins, herba-ceous advanced plants containing a mixture of flavonols and

292 J Chem Ecol (2013) 39:283–297

flavones, and highly advanced herbaceous plants containingonly flavones (Cooper-Driver, 1980). Flavonoid distributionin ferns showed a similar pattern with the most primitiveherbaceous fern containing flavones and biflavones but notflavonols and proanthocyanidins. The mixture of flavonoidsencountered in many ferns have proven to be effectiveantimicrobial agents, preventing disease in ferns and alsoplaying unknown roles in the rhizosphere as defense com-pounds. The role of flavonoids in fern ecology requiresfurther investigation, in light of new findings regarding theirrole in chemical signalling and allelopathic interactions. Itwould be useful to revisit the chemotaxonomic surveysperformed prior to 1980, given our increased sensitivityand capacity to detect and identify secondary metabolitesin planta and in surrounding soils, and the information wehave now garnered about flavonoids diversity in legumesand other higher plant species.

Future Research Directions

Major gaps in our knowledge of flavonoid exudation andfunction in the rhizosphere, particularly these involvingallelopathic interactions, include the detailed mechanismsof flavonoid exudation and the identification of transportmechanisms and transporter proteins specific to flavonoidexudation. Future study of the regulation of flavonoid trans-porters by abiotic and biotic rhizosphere signals will beimportant to gain information on flavonoid release rates/fluxover time. Additionally, measurements of actual flavonoidconcentrations in real soil environments are largely lacking.This potentially could be be accomplished by solid phaseroot zone extraction using micro-extraction techniques inspecific rhizosphere locations to determine spatial and tem-poral changes in flavonoid exudation (Mohney et al., 2009;Weidenhamer et al., 2009). Such an approach might allowmore precise estimations of flavonoid breakdown andmovement in the soil. Flavonoid-inducible reporters alsocould be expressed in bacterial cells and used as livingbiosensors in the soil. The rhizobium nodD proteins ofvarious species might prove to be potential bio-sensors ofspecific flavonoids around roots. Furthermore, mutants andtransgenic plants with altered flavonoid metabolism or exu-dation will continue to be used to study the effect of en-hanced or repressed flavonoid exudation on rhizosphereorganisms and this should continue to spawn a new flurryof research in the soil rhizosphere. Mass spectrometric iden-tification and quantification of flavonoids from root exu-dates also could be used to screen for mutants with alteredflavonoid exudation, and both metabolomic and proteomicprofiling will undoubtedly improve our knowledge of exu-dation processes in higher plants. For the many plant speciesfor which no transgenic approach is currently feasible, the

generation of mutants with high throughput sequencingtechniques may prove useful (see also this issue Duke etal., 2013).

Large knowledge gaps also remain in our understand-ing of how flavonoids act as allelopathic compounds.This review has indicated that it will be important toidentify molecular targets of flavonoids in plant speciesthat are inhibited, thereby unravelling mechanisms ofhow allelopathic plants that produce flavonoids are pro-tected from autotoxicity. So far it is unclear whetherflavonoids are taken up by target species, as suggestedby experiments in Arabidopsis in vitro (Buer et al.,2007), whether the uptake varies among species, whetherplants utilize transporter proteins for flavonoid uptake,and whether flavonoids have different molecular targetsin various species. It also is possible that flavonoidsexuded into the rhizosphere are first modified by soilmicroorganisms (Rao and Cooper, 1994) before becomingbioactive as allelochemicals. Flavonoids could be takenup into sensitive plants by traditional uptake and trans-port processes as well as by fungal networks involvingmycorrhizae (Barto et al., 2012).

Acknowledgements The authors are grateful to the AustralianResearch Council for funding for a Future Fellowship to UM(FT100100669) and New South Wales Office of Medical and ScienceResearch for funding a Biofirst Life Sciences Research Fellowship toLAW. The authors also acknowledge the helpful reviews receivedduring the review process.

References

AGATI, G., AZZARELLO, E., POLLASTRI, S., and TATTINI, M. 2012.Flavonoids as antioxidants in plants: Location and functionalsignificance. Plant. Sci. 196:67–76.

AGUILAR, J. M. M., ASHBY, A. M., RICHARDS, A. J. M., LOAKE, G. J.,WATSON, M. D., and SHAW, C. H. 1988. Chemotaxis ofRhizobium legminosarum biovar phaseoli towards flavonoidinducers of the symbiotic nodulation genes. J. Gen. Microbiol.134:2741–2746.

AKIYAMA, K., MATSUOKA, H., and HAYASHI, H. 2002. Isolationand identification of a phosphate deficiency-induced C-glycosylflavonoid that stimulates arbuscular mycorrhiza formationin melon roots. Mol. Plant-Microbe. Interact. 15:334–340.

AKIYAMA, K., TANIGAWA, F., KASHIHARA, T., and HAYASHI, H. 2010.Lupin pyranoisoflavones inhibiting hyphal development in arbus-cular mycorrhizal fungi. Phytochemistry 71:1865–1871.

ARMERO, J., REQUEJO, R., JORRIN, J., LOPEZ-VALBUENA, R., andTENA, M. 2001. Release of phytoalexins and related isoflavonoidsfrom intact chickpea seedlings elicited with reduced glutathione atroot level. Plant. Physiol. Biochem. 39:785–795.

BADRI, D. V., CHAPARRO, J. M., MANTER, D. K., MARTINOIA, E., andVIVANCO, J. M. 2012. Influence of ATP-binding cassette trans-porters in root exudation of phytoalexins, signals, and in diseaseresistance. Front. Plant. Sci. 3:149.

BADRI, D. V., LOYOLA-VARGAS, V. M., BROECKLING, C. D., DE-LA-PENA, C., JASINSKI, M., SANTELIA, D., MARTINOIA, E., SUMNER,

J Chem Ecol (2013) 39:283–297 293

L. W., BANTA, L. M., STERMITZ, F., and VIVANCO, J. M. 2008.Altered profile of secondary metabolites in the root exudates ofArabidopsis ATP-binding cassette transporter mutants. Plant.Physiol. 146:762–771.

BADRI, D. V., QUINTANA, N., EL KASSIS, E. G., KIM, H. K., CHOI, Y. H.,SUGIYAMA, A., VERPOORTE, R., MARTINOIA, E., MANTER, D. K.,and VIVANCO, J. M. 2009. An ABC transporter mutation alters rootexudation of phytochemicals that provoke an overhaul of naturalsoil microbiota. Plant Physiol 151:2006–2017.

BALDRIDGE, G. D., O’NEILL, N. R., and SAMAC, D. A. 1998. Alfalfa(Medicago sativa L.) resistance to the root-lesion nematode,Pratylenchus penetrans: Defense-response gene mRNA and iso-flavonoid phytoalexin levels in roots. Plant. Mol. Biol. 38:999–1010.

BANASIAK, J., BIAŁA, W., STASZKÓW, A., SWARCEWICZ, B.,KEPCZYNSKA, E., FIGLEROWICZ, M., and JASINSKI, M. 2013. AMedicago truncatula ABC transporter belonging to subfamily Gmodulates the level of isoflavonoids. J. Exp. Bot.. doi:10.1093/jxb/ers380.

BARTO, E. K., WEIDENHAMER, J. D., CIPOLLINI, D., and RILLIG, M. C.2012. Fungal superhighways: Do common mycorrhizal networksenhance below ground communication? Trends. Plant. Sci.17:633–637.

BATISH, D. R., SINGH, H. P., KOHLI, R. K., and DAWRA, G. P. 2006.Potential of allelopathy and allelochemicals for weed manage-ment, pp. 209–256, in H. P. Singh, D. R. Batish, and R. K.Kohli (eds.), Handbook of Sustainable Weed Management. FoodProducts Press, Binghamton.

BAYLISS, C., CANNY, M. J., and MCCULLY, M. E. 1997. Retention insitu and spectral analysis of fluorescent vacuole components insections of plant tissue. Biotech. Histochem. 72:123–128.

BERHOW, M. A. and VAUGHN, S. R. 1999. Higher Plant Flavonoids:Biosynthesis and Chemical Ecology, pp. 423–438, in K. M. M. D.Inderjit and C. L. Foy (eds.), Principals and Practices inPlant Ecology- Allelochemical Interactions. CRC Press, BocaRaton.

BERTHOLDSSON, N.-O. 2004. Variation in allelopathic activity over 100years of barley selection and breeding. Weed. Res. 44:78–86.

BERTHOLDSSON, N.-O. 2005. Early vigour and allelopathy - Twouseful traits for enhanced barley and wheat competitivenessagainst weeds. Weed. Res. 45:94–102.

BLOUNT, J. W., DIXON, R. A., and PAIVA, N. L. 1992. Stress responsesin alfalfa (Medicago sativa L).16. Antifungal activity of medicar-pin and its biosynthetic precursors—implications for the geneticmanipulation of stress metabolites. Physiol. Mol. Plant Pathol.41:333–349.

BROWN, D. E., RASHOTTE, A. M., MURPHY, A. S., NORMANLY, J.,TAGUE, B. W., PEER, W. A., TAIZ, L., and MUDAY, G. K. 2001.Flavonoids act as negative regulators of auxin transport in vivo inArabidopsis. Plant Physiol. 126:524–535.

BUER, C. S. and DJORDJEVIC, M. A. 2009. Architectural phenotypes inthe transparent testa mutants of Arabidopsis thaliana. J. Exp. Bot.60:751–763.

BUER, C. S., IMIN, N., and DJORDJEVIC, M. A. 2010. Flavonoids: Newroles for old molecules. J. Integ.. Plant. Biol. 52:98–111.

BUER, C. S., MUDAY, G. K., and DJORDJEVIC, M. A. 2007. Flavonoidsare differentially taken up and transported long distances inArabidopsis. Plant Physiol. 145:478–490.

CARLSEN, S. C. K., PEDERSEN, H. A., SPLIID, N. H., and FOMSGAARD,I. S. 2012. Fate in soil of flavonoids released from white clover(Trifolium repens L.). Appl. Environ. Soil. Sc 2012:1–10.

CESCO, S., NEUMANN, G., TOMASI, N., PINTON, R., and WEISSKOPF, L.2010. Release of plant-borne flavonoids into the rhizosphere andtheir role in plant nutrition. Plant Soil 329:1–25.

CHUNG, I. M. and MILLER, D. A. 1995. Differences in autotoxicityamong seven alfalfa cultivars. Agron. J. 87:596–600.

COOPER-DRIVER, G. 1980. The role of flavonoids and related com-pounds in fern systematics. Bull. Torrey. Botan. Club. 107:116–127.

COOPER, JE. 2004 Multiple responses of rhizobia to flavonoids duringlegume root infection, pp 1–62 in: Callow JA (ed) Advances inBotanical Research Incorporating Advances in Plant Pathology,Vol 41.

CORONADO, C., ZUANAZZI, J. A. S., SALLAUD, C., QUIRION, J. C.,ESNAULT, R., HUSSON, H. P., KONDOROSI, A., and RATET, P.1995. Medicago sativa root flavonoid production is nitrogenregulated. Plant Physiol. 108:533–542.

COSGROVE, D. and UNDERSANDER, D. 2003. pp. 1–2, Seeding AlfalfaFields Back into Alfalfa. Extension Publication. Focus on Forage.University of Wisconsin, Madison.

CURIR, P., DOLCI, M., and GALEOTTI, F. 2005. A phytoalexin-likeflavonol involved in the carnation (Dianthus caryophyllus)-Fusarium oxysporum f Sp dianthi pathosystem. J. Phytopathol.153:65–67.

CUSHNIE, T. P. T. and LAMB, A. J. 2011. Recent advances in under-standing the antibacterial properties of flavonoids. Int. J.Antimicrob. Agents 38:99–107.

CUTLER, S. J., VARELA, R. M., PALMA, M., MACIAS, F. A., andCUTLER, H. G. 2007. Isolation, Structural Elucidation andSynthesis Of Biologically Active Allelochemicals for PotentialUse as Pharmaceuticals, pp. 1–398, in Y. Fujii and S. Hiradate(eds.), Allelopathy: New Concepts and Methodology. NationalInstitute for Agro-environmental Sciences, Tsukuba.

DAKORA, F. D., JOSEPH, C. M., PHILLIPS, D. A .(1993) Rhizobiummeliloti alters flavonoid composition of alfalfa root exudates, p.335 in: Palacios Rea (ed) New Horizons in Nitrogen Fixation.Kluwer Academic Publishers

DAVIES, K. M., ALBERT, N. W., and SCHWINN, K. E. 2012. Fromlanding lights to mimicry: The molecular regulation of flowercolouration and mechanisms for pigmentation patterning. Funct.Plant Biol. 39:619–638.

DELAUX, P. M., NANDA, A. K., MATHE, C., SEJALON-DELMAS, N., andDUNAND, C. 2012. Molecular and biochemical aspects of plantterrestrialization. Perspect. Plant. Ecol. Evolu. Systemat. 14:49–59.

DHARMATILAKE, A. J. and BAUER, W. D. 1992. Chemotaxis ofRhizobium meliloti towards nodulation geneinducing compoundsfrom alfalfa roots. Appl. Environ. Microbiol. 58:1153–1158.

DIXON, R. A. and PAIVA, N. L. 1995. Stress-induced phenylpropanoidmetabolism. Plant. Cell. 7:1085–1097.

DIXON, R. A. and STEELE, C. L. 1999. Flavonoids and isoflavonoids—a gold mine for metabolic engineering. Trends Plant Sci. 4:394–400.

DJORDJEVIC, M. A., REDMOND, J. W., BATLEY, M., and ROLFE, B. G.1987. Clovers secrete specific phenolic compounds which eitherstimulate or repress nod gene expression in Rhizobium trifolii.EMBO J. 6:1173–1179.

DUKE, S.O., BAJSA, J., and PAN, Z. 2013. Omics methods for probingthe mode of action of natural and synthetic phytotoxins. J. Chem.Ecol. 39:333–348.

EDWARDS, R., MIZEN, T., and COOK, R. 1995. Isoflavonoid conjugateaccumulation in the roots of lucerne (Medicago sativa) seedlingsfollowing infection by the stem nematode (Ditylenchus dipsaci).Nematologica 41:51–66.

ERLEJMAN, A. G., VERSTRAETEN, S. V., FRAGA, C. G., and OTEIZA,P. I. 2004. The interaction of flavonoids with membranes:Potential determinant of flavonoid antioxidant effects. FreeRadic. Res. 38:1311–1320.

FANG, C., ZHUANG, Y., XU, T., LI, Y., LI, YUE, and LIN, W. 2013.Changes in rice allelopathy and rhizosphere microflora by inhib-iting rice phenylalanine amonia-lyase gene expression. J. Chem.Ecol. 39:204–212.

294 J Chem Ecol (2013) 39:283–297

FERRER, J. L., AUSTIN, M. B., STEWART, C., and NOE, J. P. 2008.Structure and function of enzymes involved in the biosynthesis ofphenylpropanoids. Plant. Physiol. Biochem. 46:356–370.

FEUCHT, W., TREUTTER, D., and POLSTER, J. 2012. Flavanols in nucleiof tree species: Facts and possible functions. Trees-Struct Funct26:1413–1425.

FURUYA, M., GARLSTON, A. W., and STOWE, B. B. 1962. Isolationfrom peas of co-factors and inhibitors of indolyl-3-acetic acidoxidase. Nature 193:456–457.

GEALY, D., MOLDENHAUER, K., and DUKE, S. (2013). Root distributionand potential interactions between allelopathic rice, sprangletop(Leptochloa spp.), and barnyardgrass (Echinochloa crus-galli) basedon 13C isotope discrimination analysis. J. Chem. Ecol. 39:186–203.

GRESSEL, J., HANAFI, A., HEAD, G., MARASAS, W., OBILANA, B.,OCHANDA, J., SOUISSI, T., and TZOTZOS, G. 2004. Major hereto-fore intractable biotic constraints to African food security thatmay be amenable to novel biotechnological solutions. CropProtect. 23:661–689.

HAI, Z., JIN-MING, G., WEI-TAO, L., JING-CHENG, T., XING-CHANG,Z., ZHEN-GUO, J., YAO-PING, X., and MING-AN, S. 2008.Allelopathic substances from walnut (Juglans regia L.).Allelopathy J 21:425–432.

HANCOCK, D. W. 2005. Autotoxicity in Alfalfa (Medicago sativa L.):Implications for Crop Production. University of Kentucky,Lexington KY, pp 1–17

HARBORNE, J. B. 1973. Phytochemical Methods. Chatman and HALL,LONDON.

HARRISON, M. J. 2005. Signaling in the arbuscular mycorrhizal sym-biosis. Annu. Rev. Microbiol. 59:19–42.

HARTWIG, U. A., JOSEPH, C. M., and PHILLIPS, D. A. 1991. Flavonoidsreleased naturally from alfalfa seeds enhance growth rate ofRhizobium meliloti. Plant Physiol. 95:797–803.

HARTWIG, U. A. and PHILLIPS, D. A. 1991. Release and modificationof nod gene-inducing flavonoids from alfalfa seeds. PlantPhysiol. 95:804–807.

HASSAN, S. and MATHESIUS, U. 2012. The role of flavonoids in root-rhizosphere signalling: Opportunities and challenges for improv-ing plant-microbe interactions. J. Exp. Bot. 63:3429–3444.

HAWES, M. C., BRIGHAM, L. A., WEN, F., WOO, H. H., ZHU, Z. 1998.Function of root border cells in plant health: Pioneers in therhizosphere. Ann. Rev. Phytopathol. 36:311–327.

HEATH, M. C. 2000. Hypersensitive response-related death. Plant Mol.Biol. 44:321–334.

HEDGE, R. S. and MILLER, D. A. 1992. Concentration dependencyand stage of crop growth in alfalfa autotoxicity. Agron. J. 84:940–946.

HIGGINS, V. J. 1978. The effect of some pterocarpanoid phytoalexinson germ tube elongation of Stemphylium botryosum.Phytopathology68:339–345.

HODNICK, W. F., DUVAL, D. L., and PARDINI, R. S. 1994. Inhibition ofmitochondrial respiration and cyanide-stimulated generation ofreactive oxygen species by selected flavonoids. Biochem.Pharmacol. 47:573–580.

HODNICK, W. F., MILOSAVLJEVIC, E. B., NELSON, J. H., and PARDINI,R. S. 1988. Electrochemistry of flavonoids—relationships be-tween redox potentials, inhibition of mitochondrial respirationand production of oxygen radicals by flavonoids. Biochem.Pharmacol. 37:2607–2611.

HOOPER, A. M., TSANUO, M. K., CHAMBERLAIN, K., TITTCOMB, K.,SCHOLES, J., HASSANALI, A., KHAN, Z. R., and PICKETT, J. A.2010. Isoschaftoside, a C-glycosylflavonoid from Desmodiumuncinatum root exudate, is an allelochemical against the develop-ment of Striga. Phytochemistry 71:904–908.

HUANG, L., SONG, L., XIA, X., MAO, W., SHI, K., ZHOU, Y., YU, J.2013. Plant-soil feedbacks and soil sickness: From mechanisms toapplication in agriculture. J. Chem. Ecol. 39:232-242.

HUTZLER, P., FISCHBACH, R., HELLER, W., JUNGBLUT, T. P., REUBER,S., SCHMITZ, R., VEIT, M., WEISSENBOECK, G., and SCHNITZLER,J. P. 1998. Tissue localisation of phenolic compounds in plants byconfocal laser scanning microscopy. J. Exp. Bot. 49:953–965.

JACOBS, M. and RUBERY, P. H. 1988. Naturally occuring auxin trans-port regulators. Science 241:346–349.

JENNINGS, J. A. 2001. Understanding Autotoxicity in Alfalfa.Wisconsin Forage Council: 2001 Proceedings. University ofWisconsin, Madison WI.

JENNINGS, J. A. and NELSON, C. J. 2002. Zone of autotoxic influencearound established alfalfa plants. Agron. J. 94:1104–1111.

JIA, Z. H., ZOU, B. H., WANG, X. M., QIU, J. A., MA, H., GOU, Z. H.,SONG, S. S., and DONG, H. S. 2010. Quercetin-induced H(2)O(2)mediates the pathogen resistance against Pseudomonas syringaepv. Tomato DC3000 in Arabidopsis thaliana. Biochem. Biophys.Res. Commun. 396:522–527.

JORGENSEN, K., RASMUSSEN, A. V., MORANT, M., NIELSEN, A. H.,BJARNHOLT, N., ZAGROBELNY, M., BAK, S., and MOLLER, B. L.2005. Metabolon formation and metabolic channeling in the biosyn-thesis of plant natural products. Curr. Opin. Plant Biol. 8:280–291.

JUSZCZUK, I.M., WIKTOROWSKA, A., MALUSA, E., and RYCHTER, A. M.2004. Changes in the concentration of phenolic compounds andexudation induced by phosphate deficiency in bean plants(Phaseolus vulgaris L.). Plant Soil 267:41–49.

KATO-NOGUCHI, H., INO, T., SATA, N., and YAMAMURA, S. 2002.Isolation and identification of a potent allelopathic substance inrice root exudates. Physiol. Plant. 115:401–405.

KATO-NOGUCHI, H. and PETERS, R. (2013). The role of momilactonesin rice allelopathy, J. Chem. Ecology. 39:175–185.

KHAN, Z. R., MIDEGA, C. A. O., BRUCE, T. J. A., HOOPER, A. M., andPICKETT, J. A. 2010. Exploiting phytochemicals for developing a’push-pull’ crop protection strategy for cereal farmers in Africa. J.Exp. Bot. 61:4185–4196.

KHAN, Z. R., PICKETT, J. A., WADHAMS, L. J., HASSANALI, A., andMIDEGA, C. A. O. 2006. Combined control of Striga hermonthicaand stemborers by maize-Desmodium spp. intercrops. CropProtect 25:989–995.

KLEIN, R. R. and MILLER, D. A. 1980. Allelopathy and its role inagriculture. Commun. Soil Sci. Plant Anal. 11:43–56.

KONG, C. H., XU, X., ZHOU, B., HU, F., ZHANG, C., and ZHANG, M.2004. Two compounds from allelopathic rice accession and theirinhibitory activity on weeds and fungal pathogens. Phytochemistry65:1123–1128.

KONG, C. H., ZHAO, H., XU, H., WANG, P., and GU, Y. 2007. Activityand allelopathy of soil of flavone O-glycosides from rice. J. Agric.Food Chem. 55:6007–6012.

KREMER, R. J. and BEN-HAMMOUDA, M. 2009. Allelopathic Plants.19. Barley (Hordeum vulgare L.). Allelopathy J 24:225–241.

LEVIZOU, E. , KARAGEORGOU, P. K. , PETROPOULOU, G.,GRAMMATIKOPOULOS, G., and MANETAS, Y. 2004. Induction ofageotropic response in lettuce radical growth by epicuticularflavonoid aglycones of Dittrichia viscosa. Biol. Plant. 48:305–307.

LOVETT, J. V. and HOULT, A. H. C. (EDS.) 1995. Allelopathy and Self-Defense in Barley. American Chemical Society, Washington DC.

MAKOI, J. H. J. R. and NDAKIDEMI, P. A. 2007. Biological, ecologicaland agronomic significance of plant phenolic compounds in rhizo-sphere of the symbiotic legumes. Afr. J. Biotechnol. 6:1358–1368.

MANDAL, S. M., CHAKRABORTY, D., and DEY, S. 2010. Phenolic acidsact as signaling molecules in plant-microbe symbioses. Plant.Signal. Behav 5:359–368.

MARAIS, J. P. J., DEAVOURS, B., DIXON, R. A., and FERREIRA, D. 2007.The Stereochemistry of Flavonoids, pp. 1–35, in E. Grotewold(ed.), The Science of Flavonoids. Springer Press, New York.

MASAOKA, Y., KOJIMA, M., SUGIHARA, S., YOSHIHARA, T., KOSHINO,M., and ICHIHARA, A. 1993. Dissolution of ferric phosphates byalfalfa (Medicago sativa L.) root exudates. Plant Soil 155:75–78.

J Chem Ecol (2013) 39:283–297 295

MATHESIUS, U. 2001. Flavonoids induced in cells undergoing noduleorganogenesis in white clover are regulators of auxin breakdownby peroxidase. J. Exp. Bot. 52:419–426.

MATHESIUS, U., BAYLISS, C., WEINMAN, J. J., SCHLAMAN, H. R. M.,SPAINK, H. P., ROLFE, B. G., MCCULLY, M. E., and DJORDJEVIC,M. A. 1998. Flavonoids synthesized in cortical cells during nod-ule initiation are early developmental markers in white clover.Mol. Plant-Microbe. Interact. 11:1223–1232.

MILLER, D. 1996. Allelopathy in forage crop systems. Agron. J.88:854–859.

MOHNEY, B. K., MATZ, T., LAMOREAUX, J., WILCOX, D. S., GIMSING,A. L., MAYER, P., and WEIDENHAMER, J. D. 2009. In situ siliconetube microextraction: A new method for undisturbed sampling ofroot-exuded thiophenes from marigold (Tagetes erecta L.) in soil.J. Chem. Ecol. 35:1279–1287.

MORANDI, D., LE SIGNOR, C., GIANINAZZI-PEARSON, V., and DUC, G.2009. A Medicago truncatula mutant hyper-responsive to mycor-rhiza and defective for nodulation. Mycorrhiza 19:435–441.

MWAJA, V., MASIUNAS, J. B., and WESTON, L. A. 1995. Effects offertility on biomass, phytotoxicity and allelochemical content ofcereal rye. J. Chem. Ecol. 21:81–96.

NAOUMKINA, M. and DIXON, R. A. 2008. Subcellular localization offlavonid natural products: A signalling function? Plant. Signal.Behav. 3:573–575.

NAOUMKINA, M. A., ZHAO, Q. A., GALLEGO-GIRALDO, L., DAI, X. B.,ZHAO, P. X., and DIXON, R. A. 2010. Genome-wide analysis ofphenylpropanoid defence pathways. Mol. Plant. Pathol. 11:829–846.

OLESZEK, W. and JURZYSTA, M. 1987. The allelopathic potential ofalfalfa root mediacagenic acid glycosides and their fate in soilenvironments. Plant Soil 98:67–80.

PECK, M. C., FISHER, R. F., and LONG, S. R. 2006. Diverse flavonoidsstimulate NodD1 binding to nod gene promoters in Sinorhizobiummeliloti. J. Bacteriol. 188:5417–5427.

PEER, W. A., BROWN, D. E., TAGUE, B. W., MUDAY, G. K., TAIZ, L.,and MURPHY, A. S. 2001. Flavonoid accumulation patternsof transparent testa mutants of Arabidopsis. Plant Physiol.126:536–548.

PEER, W. A. and MURPHY, A. S. 2007. Flavonoids and auxin transport:Modulators or regulators? Trends Plant Sci. 12:556–563.

PETERS, N. K. and LONG, S. R. 1988. Alfalfa root exudates andcompounds which promote or inhibit induction of Rhizobiummeliloti nodulation genes. Plant Physiol. 88:396–400.

PLAPER, A., GOLOB, M., HAFNER, I., OBLAK, M., SOLMAJER, T., andJERALA, R. 2003. Characterization of quercetin binding site onDNA gyrase. Biochem. Biophys. Res. Commun. 306:530–536.

POLLASTRI, S. and TATTINI, M. 2011. Flavonols: Old compounds forold roles. Ann. Bot. 108:1225–1233.

PUEPPKE, S. G. and VANETTEN, H. D. 1974. Pisatin accumulation andlesion development in peas infected with Aphanomyces euteiches.Fusarium solani f. sp. pisi or Rhizoctonia solani. Phytopathology64:1433–1440.

PUTNAM, A. R. and DEFRANK, J. 1983. Use of allelopathic cover cropsto inhibit weeds. Crop Protect. 2:173–182.

RAO, A. S. 1990. Root flavonoids. Bot. Rev. 56:1–84.RAO, J. R. and COOPER, J. E. 1994. Rhizobia catabolize nod gene-

inducing flavonoids via C-ring fission mechanisms. J. Bacteriol.176:5409–5413.

RAO, J. R. and COOPER, J. E. 1995. Soybean nodulating rhizobiamodify nod gene inducers daidzein and genistein to yield aromaticproducts that can influence gene-inducing activity. Mol. Plant-Microbe Interact. 8:855–862.

REDMOND, J. R., BATLEY, M., DJORDJEVIC, M. A., INNES, R. W.,KEUMPEL, P. L., and ROLFE, B. G. 1986. Flavones induce expres-sion of nodulation genes in Rhizobium. Nature 323:632–635.

RICE, E. L. (ED.) 1984. Allelopathy. Academic, Orlando.

SASLOWSKY, D. E., WAREK, U., and WINKEL, B. S. J. 2005. Nuclearlocalization of flavonoid enzymes in Arabidopsis. J. Biol. Chem.280:23735–23740.

SCERVINO, J. M., PONCE, M. A., ERRA-BASSELLS, R., BOMPADRE, M. J.,VIERHEILIG, H., OCAMPO, J. A., and GODEAS, A. 2006.Glycosidation of apigenin results in a loss of its activity on differentgrowth parameters of arbuscular mycorrhizal fungi from the genusGlomus and Gigaspora. Soil Biol. Biochem. 38:2919–2922.

SCERVINO, J. M., PONCE, M. A., ERRA-BASSELLS, R., BORNPADRE, J.,VIERHEILIG, H., OCAMPO, J. A., and GODEAS, A. 2007. The effectof flavones and flavonols on colonization of tomato plants byarbuscular mycorrhizal fungi of the genera Gigaspora andGlomus. Can. J. Microbiol. 53:702–709.

SCERVINO, J. M., PONCE, M. A., ERRA-BASSELLS, R., VIERHEILIG, H.,OCAMPO, J. A., and GODEAS, A. 2005a. Flavonoids exclusivelypresent in mycorrhizal roots of white clover exhibit a differenteffect on arbuscular mycorrhizal fungi than flavonoids exclusivelypresent in non-mycorrhizal roots of white clover. J. Plant. Interact.1:15–22.

SCERVINO, J. M., PONCE, M. A., ERRA-BASSELLS, R., VIERHEILIG, H.,OCAMPO, J. A., and GODEAS, A. 2005b. Flavonoids exhibit fungalspecies and genus specific effects on the presymbiotic growth ofGigaspora and Glomus. Mycol. Res. 109:789–794.

SCHMIDT, P. E., BROUGHTON, W. J., and WERNER, D. 1994. Nod-factors of Bradyrhizobium japonicum and Rhizobium sp.NGR234 induce flavonoid accumulation in soybean root exudate.Molec. Plant. Microbe. Interact. 7:384–390.

SHAW, L. J. and HOOKER, J. E. 2008. The fate and toxicity of theflavonoids naringenin and formononetin in soil. Soil Biol.Biochem. 40:528–536.

SHAW, L. J., MORRIS, P., HOOKER, J. E. 2006. Perception and modifi-cation of plant flavonoid signals by rhizosphere microorganisms.Env. Microbiol. 8:1867–1880.

SHULZ, M., MAROCCO, A., TABAGLIO, V., and MACIAS, F.A. 2013.Benzoxazinoids in rye allelopathy—From discovery to applica-tion in sustainable weed control and organic farming. J. Chem.Ecol. 39:154–174.

SIQUEIRA, J. O., SAFIR, G. R., and NAIR, M. G. 1991. Stimulation ofvesicular-arbuscular mycorrhizae formation and growth of whiteclover by flavonoid compounds. New Phytol. 118:87–93.

SOTO-VACA, A., GUTIERREZ, A., LOSSO, J. N., XU, Z. M., and FINLEY,J. W. 2012. Evolution of phenolic compounds from color and flavorproblems to health benefits. J. Agric. Food Chem. 60:6658–6677.

SOSA, T., VALARES, C., ALIAS, J. C., and LOBON, N. C. 2010.Persistence of flavonoids in Cictus landanifer soils. Plant Soil337:51–63.

STAR, A. E. 1980. Frond exudate flavonoids as allelopathic agents inPityrogramma. Bull. Torrey Botan. Club 107:146–153.

STEINKELLNER, S., LENDZEMO, V., LANGER, I., SCHWEIGER, P.,KHAOSAAD, T., TOUSSAINT, J.-P., and VIERHEILIG, H. 2007.Flavonoids and strigolactones in root exudates as signals in sym-biotic and pathogenic plant-fungus interactions. Molecules12:1290–1306.

STENLID, G. 1963. The effects of flavonoid compounds on oxidativephosphorylation and on the enzymatic destruction of indoleaceticacid. Physiol. Plant. 16:110–121.

STENLID, G. 1968. On the physiological effects of phloridzin, phloretinand some related substances upon higher plants. Physiol. Plant.21:882–894.

STENLID, G. 1976. Effects of flavonoids on the polar transport ofauxins. Physiol. Plant. 38:262–266.

SUBRAMANIAN, S., GRAHAM, M. Y., YU, O., and GRAHAM, T. L. 2005.RNA interference of soybean isoflavone synthase genes leads tosilencing in tissues distal to the transformation site and toenhanced susceptibility to Phytophthora sojae. Plant Physiol.137:1345–1353.

296 J Chem Ecol (2013) 39:283–297

SUBRAMANIAN, S., STACEY, G., and YU, O. 2006. Endogenous iso-flavones are essential for the establishment of symbiosisbetween soybean and Bradyrhizobium japonicum. Plant J.48:261–273.

SUGIYAMA, A., SHITAN, N., and YAZAKI, K. 2007. Involvement of asoybean ATP-binding cassette—Type transporter in the secretionof genistein, a signal flavonoid in legume-Rhizobium Symbiosis(1). Plant Physiol. 144:2000–2008.

TAYLOR, L. P. and GROTEWOLD, E. 2005. Flavonoids as developmentalregulators. Curr. Opin. Plant Biol. 8:317–323.

TESAR, M. B. 1993. Delayed seeding of alfalfa avoids autotoxicityafter plowing or glyphosate treatment of established stands.Agron. J. 85:256–263.

TSAI, S. M. and PHILLIPS, D. A. 1991. Flavonoids released naturallyfrom alfalfa promote development of symbiotic Glomus spores invitro. Appl. Environ. Microbiol. 57:1485–1488.

TSUZUKI, E., MIURA, M., SAKAKI, N., and YOSHINO, T. 1999. Study onthe control of weeds by using higher plants. Rep. Kyushu BranchCrop Sci. Soc. Japan 65:39–40.

WASSON, A. P., PELLERONE, F. I., and MATHESIUS, U. 2006. Silencingthe flavonoid pathway in Medicago truncatula inhibits root nod-ule formation and prevents auxin transport regulation by rhizobia.Plant Cell 18:1617–1629.

WEIDENHAMER, J. D., BOES, P. D., and WILCOX, D. S. 2009. Solid-phaseroot zone extraction (SPRE): A new methodology for measure-ment of allelochemical dynamics in soil. Plant Soil 322:177–186.

WESTON, L. A. 1996. Utilization of allelopathy for weed managementin agroecosystems. Agron. J. 88:860–866.

WESTON, L. A. 2005. History and current trends in the use of allelop-athy for weed management. HortTechnology 15:529–534.

WESTON, L., ALSAADAWI, I.S., and BAERSON, S.C. 2013. Sorghumallelopathy—From ecosystem to molecule. J. Chem. Ecol.39:142–153.

WESTON, L. A. and DUKE, S. O. 2003. Weed and crop allelopathy.Crit. Rev. Plant Sci. 22:367–389.

WILLIAMS, C. A. and GRAYER, R. J. 2004. Anthocyanins and otherflavonoids. Nat. Prod. Rep. 21:539–573.

WINKEL-SHIRLEY, B. 2001. Flavonoid biosynthesis. A colorful modelfor genetics, biochemistry, cell biology, and biotechnology. PlantPhysiol. 126:485–493.

WINKEL, B. S. J. 2004. Metabolic channeling in plants. Annu. Rev.Plant Biol. 55:85–107.

WORTHINGTON, M. and REBERG-HORTON, S. C. 2013. Breeding cerealcrops for enhanced weed suppression: Optimizing allelopathy andcompetitive ability. J. Chem. Ecol. 39:213–231.

WUYTS, N., SWENNEN, R., and DE WAELE, D. 2006. Effects of plantphenylpropanoid pathway products and selected terpenoids andalkaloids on the behaviour of the plant-parasitic nematodesRadopholus similis, Pratylenchus penetrans and Meloidogyneincognita. Nematology 8:89–101.

XUAN, T. D. and TSUZUKI, E. 2002. Varietal differences in allelopathicpotential of alfalfa. J. Agronomy and Crop Science 188:2–7.

YOSHIKAWA, M., GEMMA, H., SOBAJIMA, Y., and MASAGO, H. 1986.Rooting cofactor activity of plant phytoalexins. Plant Physiol.82:864–866.

YU, O., SHI, J., HESSION, A. O., MAXWELL, C. A., MCGONIGLE, B.,and ODELL, J. T. 2003. Metabolic engineering to increase isofla-vone biosynthesis in soybean seed. Phytochemistry 63:753–763.

ZHANG, J., SUBRAMANIAN, S., STACEY, G., and YU, O. 2009. Flavonesand flavonols play distinct critical roles during nodulation ofMedicago truncatula by Sinorhizobium meliloti. Plant J.57:171–183.

ZHANG, H., MALLIK, A., and ZENG, R. 2013. Control of Panamadisease of banana by rotating and intercropping with Chinesechive (Allium tuberosum Rottler): Role of plant volatiles. J.Chem. Ecol. 39:243–252.

ZHAO, J. and DIXON, R. A. 2009. MATE transporters facilitate vacu-olar uptake of epicatechin 3 ′-O-glucoside for proanthocyanidinbiosynthesis in Medicago truncatula and Arabidopsis. Plant Cell21:2323–2340.

ZUANAZZI, J. A. S., CLERGEOT, P. H., QUIRION, J. C., HUSSON, H. P.,KONDOROSI, A., and RATET, P. 1998. Production of Sinorhizobiummeliloti nod gene activator and repressor flavonoids fromMedicagosativa roots. Mol. Plant-Microbe Interact. 11:784–794.

J Chem Ecol (2013) 39:283–297 297