Biosynthesis, elicitation and biological activity of isoflavonoid phytoalexins

Phymckmisrry. Vol. 25. Fio 5. pi 972-995. 19% Fmmd in Grd Bnlun

REVIEW ARTICLE NUMBER 16

BIOSYNTHESIS, ELICITATION AND BIOLOGICAL ISOFLAVONOID PHYTOALEXINS

DAVID A. Sk(rrtt and STEPHEN W. BANKst

OO3l-P)22~%S300tOOO

6; 19% I%xgaml Prm Lid

ACI-IVITY OF

Plan1 Palhobgy Department. Umvcrsily of Kenrucky. Lcxmgton. KY 40546-0091. U.S.A.

(Reckrud 4 OClc&er 1985)

Key Wd b&x Leyminosae; phytankxms; isollavonoids; formarion; roxicdy; de ofactIon; tokmnct;dwasc

rcuslancc.

Abstract -1sofIavonoid phytoalexins are a major class of low molecular weight, inhibitory compounds synthesized by certain plants, notably members of the Lcguminosae. Accumulation of thcsc isoflavonoids is often very marked following infection. This review summarizes their biosynthesis, elicitation and biological properties. Consideration is also given IO their inferred function of limiting microbial invasion in plant tissues. --.-

INlRODUCllON

Many tlavonoids are antitnicrobial [l-3]. Thcsc toxic compounds occur in plants conslitutively [4 , following

stress [S], or in both circumstances I [6,7 . Their inhibitory nature has been taken, in part, as evidcnoc that they help protect the producing plants from pathogens [3] and pests [8,9]. In all of this work concerning the putative defensive capacities of Bavonoids, great emphasis has been placed upon isoflavonoids formed in response lo stress, primarily that caused by infection. Thcsc iso- Bavonoids have come to represent the praiominant chemical class amongst the phytoakxins [IO], a biologicallydcfincd term encompassing toxic compounds which accumulate in plants after infection and which may represent a natural mechanism to resist microbial attack.

Post-infactional antimicrobial activity amongst the flavonoids, however. is not restricted to the is&avonoid sub-class. The flavanone, bctagarin, has been rcponcd as a phytoakxin in sugar beet [I 11. Broussonins A and B, which possess a 1.3diphcnylpropanc skeleton, allowing them to bc classi6cd among the simplest flavonoids found naturally, are reputedly phytoakxins occurring in dis- cased shoots of paper mulberry [ 121. Further studies led IO the isolation of eight additional minor phytoakxins from infected cortical tissues: two flavarq four 1.3 diphcnylpropancs and IWO chakoncs [ 133. The resistance of daffodil bulbs to infection by Botryris cinerea involves the production of several antifungal compounds [14]. of which thra have been identified as hydroxyflavans [IS].

The subject of those antimicrobial isoflavonoids that occur constitutively in plants also tits attention, albeit brief. A variety of isoflavonoids have been isolated from apparently healthy trees, where they may contribute to the resistance of woody tissues towards fungi and insects [ 16 181. The antifungal activity of the prenylatcd iso-

t Prcscnt address: Biology Dcparttnctn. Univdty OTT&do,

Toledo. OH 43&X. U S.A.

tlavonc, lutconc, found in immature fruits of Lupinw lureus [I93 and on leaf surfaces of L. 0fLru.s [4]. was considered reason for it to bc a hindrance IO fungal dcvclopmcnt ami, hence., infection. The distinction bctwan the constitutivc prcscncc of antimicrobial isoflavonoids and their formation only after stress is, on occasion, blurred 16.201. Therefore. whik it is often a division of convcnicncc in orienting research programs, it may not always bc of functional significance.

PHVl-OAI..EXlN THF.ORY

Experimental observations consistent with the production of defensive chemicals by plants in response to microbial attach have been reported sinoc at least the early years of this antury, although the studies were few in number and limited in extent [21,22]. Interest in the formation of post-infectional inhibitory compounds as one natural mechanism of disease rcaistancc was spurred by publication of Mulla and B6rgcr.s phytoakxin theory [23]. This proposed that restriction of fungal develop mcnt in plan1 tissues was the result of the accumulation of some toxicchcmical principle by livingalls reacting to the invader. AI that time, no chcmkals wcrc structurally charactcrizcd and the definition was of a biological nature and not of some prccisc chcmkaf dass.

By the late 19SOs, the pioneering ctforts of Miillcr [23,24] inspired others to the chcmkal resolution of the first isotlavonoid phytoakxin (IP), pisatin (Fig I; 26) [25.26], from pea (Pinun sariuum) tissues. Characterization of the related ptcrocarpanoid phytoalcxin, phase&n (Fig I; 27), from ban (Pluurolus d@.@ oaxrra! soon tbcrcaftcr

Ji 27.281. A few other

chcmimls which apparently sat’ cd the phytoakxin concept wcrc idcntiflcd about the same time [22] and a veritable good would follow [2931]. The notion was now well atablishcd of low mokcular weight, toxic oom- pounds, often absent from healthy tissues but formed afIcr tissue damage which, accumulating at a& around

D. A. Sutm and S. W. BANKS

fa

32

Fig, I. Six isoflavonoid phytoakxins and one dctoxitication product. 26 (+t(bR,l IaRbFiultin; 27 ( -b (6&I IaRtphascollin; 28 kiwitone: KHn.sc = kievitone hydraluc; 29 kicvitone hydntr 30 ( - )-(6as.I 1a.D

&ccollin 1; 31 ( - ~(6d.I I~glyccollin II; 32 ( - ~(6aS.I IaS)-glyceollin 111.

infection sites, were presumed to contribute to the iimitation of microbiil development in plants. Of these many chcmiads-and several different dasscs arc involved-the isoflavonoids predominate (IO]. The many research efforts have also rcvatkd an intriguing association, widespread though not restrictive, between IPs and the Lcguminosac [30,32].

FORMAT~DN OF tSOFUVONOt0 t’ttYTOAt.EXINS

Mention has been made of the possibility that phytoakxin accumulation might mr as a result of 5vonoid glycosidc hydrolysis [33]. Levels of the ptero- carpanoid glycosidc, trifolirhizin, deeren& as symptom severity increased in infected red dover roots [34). Although the subccqucnt appearance of the a&cone, ma&Lain-a phytoalexin of red clover foliage [35]- might suggcct trifotirhizin hydrolysis as its source, the data were inconsistent and decreases in trifohrhidn levels were not always rcfkctcd by incrcaus in maackiain. The general presumption has been, for some years now. that phytoakxins arise from remote prccurso rs [36]. This

assumption is basal upon the comparative slowness with which IPs accumulate and the frequent absence of likely immediate pmcurson. Moreover, the lack of any change in 5vonol concentration in beans during phascollin accumulation suggested the stimulation, after infection. of a spccifk route leading to iso5vonoid synthesis [37]. The conviction is still strong that IP formation occurs from remote precursors following appropriate tissue irritation 138, 393.

Implicit in any consideration of phytoakxin production by plantsare two related, yet distinct, issues. First, is the elucidation of pathways rnstrumcntai to the synthesis, in this particular instance, of iso5vonoids with concomitant considerations of the regulatory mechanisms and cnxymology invoivcd. !Sccotx& is the nature of elicitation of phytoakxin biosynthesis, since the response is classically post-infectional [IO].

Bios~t~s~ of ~~~~ phytlwriexins

Pathway eiucidution. Iso5vonoids differ from the 5vonoids in having a rearranged f2diphcnylpropanc

lsoaavolM3id ptl~oakxinr 981

skeleton as opposed to a 1,3diphcnylpropanc skekton, and occur principally in the free state, qtbcr than as glycosidcs. Like the f!avon* the various CksseJ of isoflevonoids arc detcrminal by changes in the oxidation level of the hctaocydic rings. Additional variation is conferred by the elaboration of the aryl group by sazondary reactions: hydroxylation, wthylation and pn- nylation [So].

The biosynthetic pathways which culminate in the formation of IPs may bc conveniently divided into thra parts [33: those early pathways shared with other secondary mctabolitcs, those steps common to tlavonoid and isotlavonoid biosynthesis and those reactions unique to ttu isotlavonoids. Summaries of the presumed routes determining the formation of artain IPs arc presented in WnXxlcs l- 3.

The essential C,, mokcular framework of both Bavo- n&is and isollavonoids results from the convergence of the acetatemalonate and shikimk acid pathways (Schcau 1) [33. Comprehensive studies by Griscbach and collaborators demonstrated that biosynthesis of the iso- &one skeleton from precursors of the general flavormid pathway involved a 13-aryl shift of the B ring, and that this shift took place after the formation of the CI, chakonc intermediate [41 AS].

One of the important probkms to be investigated concerning the biosynthesis of isoftavonoids was the question of whether the chalcone or the isomcrk tlavanone actal a8 the subQtrate for atyl migration. An early experiment to resolve this question took the form of paralkl competitive fcaling expcrioXnts, in which either [ “C]-isoliquiritigcnin (4,Y.Q’~trihydroxychalcone) (Scheme I; I), diluted with an equal amount of unlabclkd ( -bliquiritigenin (7,4’dihydrox@vanone) (Scheme 1; S), or [“CJ-(-)-liquiritigenin, diluted with an equal amount of unlabclkd isoliquiritigenin, were fed to seedlings of Trifolium subrewawum [46]. Isolation ofdaidztin (Fig. 2; 33) and fonnononctin (Scheme 2; 9), followed by determination of the spccitkactivity for each, showed that these were higher from the former mixture, thus indicating that the chakone was the immediate substrate for aryl migration. Similar results were obxrvd for &one and !lavanonc mctabditcs in a subsequent experiment [47]. The relative etE&ncics of cl&cone and flavanone as precursors of daidzcin and formononctin were studied by further competitive feeding experiments. Tbc results strongly favourai the proposal 1481 that chakoncs were the more immediate precursors of isotlavoncs. Indad. it is probabk that only two chaJconcs, isoliquiritigenin @bane I; 7) aA 4.2’,4’,6’-tetrahydroxychakone (Fig. 2;

6

S&me I. Euly step6 in pha~ylpropamid %synthuk I dbmyldxn& PAL - phcnyklaninc xmabonix-lyuc; 2maucmnunicacid;34-hydroxycin~mic~4 4-cmmaroyi Co& 5 nnbnyl Co& CHS, E - dukmc xyn~hasc

6 C,C,C, intcnnalixte; 7 isotiquiritigcnin; CHI - chakmm/tbmone komcruc, 0 liquiritigcnin.

D. A. SMTH ud S. W. BANKS

14 IS

Schanc 2. Stcrcochemisrry of ixoQxvonc reduction during the biosynthais of makqin md vcxtitd. 7 14iquiritigcnin; 9 fommnonetin; IO 7,2’dihydroxy-4’-mcthoxyisofbvone; 11 7,2’dihydroxy4’-mcthoxyb flavanone; 12 7.2’dihydroxy-C~mcthoxyisAavanol; 13carbonium ion; 14 mcdicxrpin; ( -p(hR, I laRb3-hydroxy-

9-mc~hoxyptcmcarpin; 15 vestitol; ( -~(3R~7,2’dihydroxy4’-mcthoxyimflavan.

42). normally act as substrates for aryl migration [49]. Possibk mochanismp for the aryl migration have been

examined by fcaling [2-‘*C-3-‘Hz]-7,4’dihydroxy- Ilavanonc (Fig. 2; 35) to seedlings of Cicer oritlinum Gris&nch and Zilg [SO] found that on incorporation into formononctin, the H: “C ratio fell by 99% but. in contrast, [2-‘*C-2-‘H]-5.7,4’-trihydroxytlavanone (Fig. 2; 36) was incorporated into biochanin A (Fig. 2; 34) with no change in the ‘H : ‘*C ratio. These findings demonstrated that no migration of hcterocyclic protons accompanied the aryl migration. The aryl migration envisaged by Crombie., Dcwick and Whiting [SI], M on an earlier scheme [SZ]. proceeds by d one ckctron transfer from the chakone. A single electron loss forms the spirodicnonc, decomposition of which yields the isolbwone. Conversion of chakones IO isotbvones has ah been acoomplishcd by the protocols of orpic chemistry [SSS], employing thallium III-inducad oxidativc rcarrangmcnts. This type of procedure has recently been utilized in the synthesis of pha.uolIin (Fig. I; 27), though in its racer& form [Ml.

The origin of the acetatcderiva.l ring in isotlPvonoids has been invcstigatd by foaling f 1,2-“Cl-sodium acetate to cuCl,-clicital pea pods [57 as well as to wounded bean cotyledons [58]. Tracer studies to resolve inta- relationships between iso&vonoid cla5scs have been applied in several areas. The first IPs IO be investigated by

these methods were maackiain (Scheme 3; 22) and medicarpin (Scheme 2; 14) which co-occur with formononctin in induced red clover (Trt~oliutn prafmv). Early feeding experiments with CuCl,-treatad scailings established that “C-labclkd isoliquiritigcnin and formononctin were ef%cknt precursors of both maiicarpin and ma&iain, whereas 2’,4’dihydroxy4mcthoxyc~ne (Fig. 2; 37) and ‘H-lab&d da&in were poor precursors [59]. These results were interpreted as being consistent with the proposal that 4’-methylation acoompania the aryl migration step in isolIavonoid biosynthesis. It was observed during these cxpzrimcnts that there was a large pool of formononetin in red clover seedlings which appeared to be stable and was not immbdiately convertal to the ptcrocarpans upon elicitation, indicating that the appearance of maackiain and medicarpin in treated tissue was the result of de IIOI;O synthesis. Later results [60] indkalcd that azdicarpin was produced by a rcdustive sequence in which formononetin appeared IO be Y-hydroxylatcd (Scheme 2; IO), and was then reduced to the corrcspond- ing (3R~isoflavanonc (!Schtmc 2; 1 l), followed by further reduction to the ixohvanol (Scheme 2; 12) This could then Lose water and cyck to form the ptcmcaq.m (Scbanc 2; 14), The pathway to rnuckiain was envisagal [61] as proceeding by reduction of the isoflavone (Schcmc3; 18) to the corresponding isotYavanonc

Idlxvonoid phymxkxins 983

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\ I (I “I, ()

i) IIt.” \ () >

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“’ ()- : 1 “,) 2s

984 D A Swnn and S W. BANKS

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41 42

FI& 2 M~~&~~~uscompoundx fated cxpmamully tn thcelwdat~on of uohvonod beoxynthcrr pathway 33 Dudt*n.uJtunm A.~[2-“C-~‘H,].?.~dlh~dtoxy(L~o~~Y(2-”C.Z-’H]-J.7.4-~flh~rox~~~~. 37 ~.J’dlhydroxy+nwlhoxychrkonz Iu 3-mahoxy-8.9-methykn~~xy~cr~~ 39 3-hydroxy-8.9. mcthyknsd~oxyptcroap&arpdrcM;O 2-hydroxy~B.9-mcrhyknad~ox~~~~~~~.41[6.1 Ia-‘H~)-a~&um.42

4.Y’.4’.6’.lctnhydrox~~e

(Scheme 3; ZOL further rcductron IO the Isoollrvmol and loss of water to form the ptaocupa tn rn analogous soqucna IO that dacnbal for modtcupu~. A similar schcrne. bKd on focdina erpcrimentr has ban proposed for the blogcncxis of ptillin (Ftg I; 27) (621.

The btorynthcux of ptcrocupanr is thought to mvolve a arbomum ion (Scheme 2; 13) Strong support for thu aau from fccdmg crpcnmcntr uxing inducul Kedbngs of Mrd~oqo saraa in whwh vcxtitol (Scheme 2 IS) and medacupln (Scheme 2; 14) were shown to be Inter- convmrbk (631. Ixo&anrand ptaoarpns are Meval to be blosynthcxiad umultana~Jy from a common mterrncdute which has ban rqgutad ax being the arbonium won or its mcxowric caunterput.

The stcrcochcollstry of ixoolvonc reduction dunng the

blosynthcur of pterocarpans has also been stud&. The fadmg of [2-‘HI-Y-hydroxyforatononetm (Scheme 2; 10) to CuCI1-treated sadbny of fenupcck rcvalai an overall rrua addition of hydrogen to the double bond of the lsoRavonc in Its tnnxforautlon to ( -t(6aR.I IaRk lnodrupln [til. Tha IS tn contrast IO the cu addltlon obsavad for the mcarpontiocl of [Z’HJ-fonnononetln

tnto ( +)(6&I IaRbplsatm (Ftg I; 24) (651. Rsatln rqcxcnts an craption among ptcroafpans

becauxc it IS the only one actmgu a phyt&xln that tax a poutlvc optial rotation. It is alxo mtcrestrng that In P. sarivum a aunor phyamltin, ( -b(6aR.l IrRtmudr~~n (Scheme 3; 22). u producd rrmuluncously wth (+ b (6aR.llaR)plutin. By wpplyinp exogenous (-b (6aR.llaR~awckiam to mducd Immature pa pods.

Isotlavonoid phytoxkxins 985

significant quantities of (-~(6&,11aQpisatin (Scheme 3; 25) were synthcsixed. This suggested that the 6a-hydroxylation of maackiain during the biosyntti of pisatin proceedad with retention of configuration BI Cd0 [66]. Confirmation of the direct hydroxylation of ptcrocarpans to form Q-hydroxyptcrocarpans has cow from the results of competitive feeding experiments in which the preferential incorporation of (+)@a.$,1 laZ+

I

JHJmaackiain (Scheme 3; 21) over (-~(6xR.llaR~ r4C]maackiain (Scheme 3; 22) into ( +~(6aR,llaR~

pisatin was observed. Similarty. (+)(6&I IaRm- hydroxymaackiain (Scheme 3; 23) was incorporated into (+)-p&&n lo a much greater extent than (-b (6aS.l MT)&-hydroxyn (Schcrr~ 3; 24) 1673. The possible intermediacy of a ptcrocarpdene or a pterocarp&-cne in the conversion of ptaocarpans to 6a- hydroxypterocarpans has also ban examined. The limited incorporation of [“C]-3hydroxy-8.9-mcthykncdioxyptcrocarp&a-cne (Fig 2; 39) and [“C]-3-mcthoxy-8,9- mcthykncdioxyptcrocarp&cne (Fig 2; 3Q into pisatin compared to the cfhcknt incorporation of (+h [W]-maadriain suggestal that he w was not an intermediate [68]. ‘H-NMR Analysis of pisatin derived from 16.1 la-‘Hr]-tna&iain (Fig 2; 41) in CuCl~- elicited pea pods clearly demonstrated the retention of all ‘H labels and thus confirmed that no ptcrocarp6cnc (Fig. 2; 40) or pterocarp-&t-cnc was involved in the biosynthesis of pisatin [69].

The soybean phytoalexins, collectively known as the glyceollins (Fig I; 30.32) are prcnylatcd Q-hydroxypterocarpans with negative optical rotations. Like pisatin. they have been shown to be derived from the appropri- ately substituted pterazarpan by 6a-hydroxylation with retention of configuration [70,71 , followed by prenylation and, finally, cyclization 172 3 .

It is apparent that an extensive literature exists concem- ing isoflavonoid biosynthesis in general, as well as IP formation in particular, and the reader is referred to Dcwick’s review [l] for a comprehensive treatment of the subject. In addition, a recent lis~ of all naturally occurring isotlavonoidscan be found in the booklet by Ingham [32].

Enzymology and regulurion. For many years it has been known that t_-phenylalaninc ammonia-lyasc (PAL) (Scheme 1) catalyscd the elimination of ammonia from L- phcnylalaninc (Scheme 1; 1) to yield truns-cinnamk acid (Scheme 1; 2bthe tirst committed step in the biosynthesis of all phenylpropanoids. Although increases in extractable PAL activity may precede IP aazumulation [37,73]. the association is sometimes inconsistent and the importance of any regulatory role for this cnzymc in IP formation remains moot. In both cotykdons and hypocotyls of P. vulgaris, phytoakxin elicitation occurred. on occasion, when PAL was suppressed below the wounded control level [74]. Furthermore, whereas kievitonc (Fig I; u)) accumulation cccurrai only in the upper half of treated V&u sinunsis hypocotyls, PAL tiivity increased throughout the hypocotyls 1753. Explanations for incon- sistencies may lie in the presently insutlkient appreciation of the different forms of PAL in plant tissues [76]. Certain isoxymu, for exampk, with critical regulatory roks in isotlavonoid biosynthesis, might be enhanced upon particular tissue damage yet this precise response could be masked by other overall changes in PAL kvels.

In a recent tnzymological investigation [77] concerning glyaollin formation in soybean hypocotyls, stimulated by a glucan elicitor, phytoalexin accumulation was preceded

by a transient rise in the extractable activity of PAL. Furthermore, there was an absolute correlation between iKnascs in PAL activiIy and Ihe eVeMud kd of glyceollin socumulation in elkital seedlings. The results were felt to indicate a rok for this ‘early’ enxymc in the regulation of the phytoakxin response in soybeans, by determining the amount of carbon from phcnylaknine that is diverted into glyccollin biosynthesis. T~K possibility of a rate-limiting step later in the pathway was not. however, dismissad.

Although some studies have been undertaken of the appearance of cinnamic acid Qhydroxylasc, a mixed function monooxygcnasc which catalyses the conversion of rranr&mamic acid to Chydroxycinnamic acid (Scheme 1; 3), in woundad and/or iIluminatai tissues [78-80]. few reports of its induction in systems producing phytoakxins exist 1761. Its aaivity, however, has been detected in association with phauollin production in P. uul(@.s [81] ar~L glyceollin accumulation in soybean (G/y&e nscx) [82]. Likewise, increases in hydroxy- cinnamoyl CoA ligasc (= courmryl CoA l&se) activity have also been reported to accompany phascollin and glyaollin formation [77,81-83].

Chakonc synthase (CHS) (Scheme I), the first enzyme of phenylpropanoid metabolism specific to Ilavo- noid/isofIavonoid biosynthesis, has ban the focus of several different studies, although relatively few have directly corkerned IP production [76]. This cnxymc catalyses chakone formation, despite the initial obscr- vation [84] that IIavanone synthesis occurred-a result due to spontaneous cyclization of the first-formed product (Scbax~ 1; 6). In G. max cotyledons, CHS activity peaked abut 25 hr after phytoakxin elicitation, a time following PAL induction but prior to glyceollin accumulation [85]. Rapid, transient increases in CHS activity preceded phascollin accumulation in P. culguris [8 1,861. Kicvitonc formation by elicitor-treated cell suspension cultures of P. wtgari.5 ah followed transitory rises in PAL and CHS [873. This research monitored 15 enzymes during kicvitonc aazumulation. the results suggesting a highly sckctivc induction of enzymes associated with IP synthesis. Furtkzrtxmre. the findings implied that phytoakxin induction involved tight control mechanisms rather than general changes resulting from substantial metabolic disturbances following infection.

Chakonc isomcrasc (CHI) (Schcn~ 1) catalyses the stcreospa%c iso m&ration of a chalcom (Scheme I; 7) to its corresponding Ilavanonc (Scheme 1; 8) [76]. However, since the synthesis of isoflavonoids has been postulated to occur via direct formation of isotlavoncs from chakoncs [76], any direct role for CHI in the synthesis of IPs seems unncccssaq. Nonetheless, CHI increased in activity in infectal soybeans 1883 as well as in P. uulgoris following phytoalexin elicitation [81.89].

Enzymicstudicsofthc 23-aryl migration that generates isoflavoncs from their chakone/tlavanone precursors met with only limited success [90,91] until. recently, Hagmann and Griscbach [92] reported that a microsomal preparation from soybean cell suspension cultures cata- lysed the r carmngcrncnt of (~naringcnin lo gcnistein. The putative ‘isoflavone synthasc’ involved was detected in cell cultures chalkngal with a phytoakxin elicitor prtpuui from Phyrophthora mrqpcpmno f. sp. glyctwo and appeared IO be an NADPH- and dioxygendcpcndcnt monooxygcnasc. The several-fold increase in isoIlavone synthasc activity following elicitation suggested that the

986 D. A Sun.14 and s. W. BANKS

enzyme might play a rok in regulating glyccollin accumuhttion.

Following iso&vonc formation, various additional structural interconversions and elaborations occur in the generation of individual 1Ps. Only limited enxymic information is available concerning these steps. A 6a- hydroxylasc, believed to be spacifscally involved in glyccollin biosynthesis, has been found in elicitor- chalkngcd soybean seedlings and cell suspension cultures 1711. Cell suspension cultures of soybean and Cker arietinwn havealso yielded O-mcthyltransfcrascs [93,94]. Prenylation is a nacasary step in the biosynthesis of several IPS, such as glyccollins I, II and III [95] and kievitone [%I. Lcubc and Griscbach 1951 reported that the prcnylating enzyme, dimcthylallylpyrophosphate: 3,6a,Ptrihydroxyptcrocarpan dimcthylallyltransferasc, increased in soybean hypocoryls inoculated with Phytophthoro mcga.v~ f. sp. glycituu as well as in soybean cell cultures treated with a phytoakxin elicitor. The increase in ‘prcnyltransferasc’activity had a longer lag phase than those for the increase of PAL or CHS activities, a finding consistent with the later action of the prcnyltransferasc in glyccollin biosynthesis.

Although enzymk studies, in combination with

radiolabclling experimentation to elucidate biosynthetic scquemzcs, allow relationships to be established between protein synthesis and phytoalexin formation, ultimate regulation of phytoakxin accumulation must lie at the level of gene expression. In a general sense. it has been known for some time that the overall rate of RNA synthesis increased in plants following infection and, moreover. that inhibitors of RNA synthesis diminished phytoakxin accumulation as well as the induction of appropriate, biosynthetic cnxymcs [76]. For example, actinomycin D inhibited the synthesis of poly (A)_ containing mRNA in diseased soybeans [97]. Resistance and glyaollin production were also suppressed, suggcst- ing that de noco mRNA synthesis was required for an effective defensive response and, furthermore, that genetic information governing eventual IP accumulation was expressed wh1k the plant resisted infection.

An early. localixcd increase in CHS mRNA activity was evident in bean hypocotyls resisting Collecotrichcun lin- demurhianum [98]. This rise occurred prior 10 the onset of phascollin accumulation. In contrast. in susceptible tissue, there was no induction of mRNA activity in the early stages of infection but, rather, a delayed and widespread increase as l&on development procccdcd. Furthermore, rapid, transient increases in CHS mRNA were noted in cell cultures of Phuseolus wlgaris treated with a high molecular weight elicitor from C. lindemuthionum [99]. The rapid induction of mRNA from very low basal keels was felt to suggest thar the elicitor initially caused a 1ransitory increase in the transcription of 1hc CHS gene(s). alrhough the alternative possibilirics of control over mRNA processing or degradation were not ruled out. Similar increases in mRNA activities related IO phytoalcxin synthesis were also observed after inoculation of soybean seedlings with P. megasperma f. sp. glpiruu or after treatment of cultural soybean cells with a phytoalcxin elicitor preparation [ IOD]. The time course of changes in activi1y were alike for mRNAs coding for enzymes of general phcnylpropanoid metabolism and for CHS mRNA. Cloned cDNA was used IO demonstrate that the induced changes in CHS mRNA ac1ivi1y coincided with changes in the amoun1 of mRNA. These results

suggested that IP synthesis may be regulated by tcm- porary gene activation. Thertforci the regulation of gene expression controlling phytoakxin build-up may be a key. early component in the natural defensive reactions of certain plant tissues.

Some cautionary notes must be cxprcsscd. however. as regards this type of work. First, the results of Rcll et ul. [9%]. discus& above, arc presental in a manner that is inclined to ovcrcmphasizc the apparent increase in CHS, as well as CHS mRNA. activity in the incompatible interaction. Second employing a gltbcan elicitor from ocll walls of P. mcguspernso f. sp. glyciruq Ebcl and coworkers [lo] noted &teases in several cnxymc activities. including those of PAL and CHS. The changes in PAL and CHS activities were correlated with corresponding alterations in mRNA activities encoding these enzymes. However, similar degrees of induction of PAL, CHS and the mRNA activities also occurred in response to an cxtracclluIar polysaccharidc from Xatuhomtmat cumpes- 1ri.s which did not enhance glyccollin levels. This lack of coordination between PAL and CHS activities and the phytoakxin amount might imply that one or more later enzymes play the key regulatory rok(s) in determining glyccollin accumulation. Certainly. as Ebcl and colkagucs [ 1011 point out, great care must be cxcrcixcd in intcrprct- ing induction of PAL and CHS as nacssari ly correlated with phytoakxin synthesis

In the space of a comparatively short number of years, much has been lcamcd about the chemical charactcrix- ation of IPs, their precursors and biosynthetic sequences. These findings have, perhaps narssarily. led and outpaced understanding of the cnxymcs and regulatory mechanisms involvalIntcnsivcuscofthctookofmolc4arbiology. however, should permit far more precise comprehension of the genetic and cnxymic control of IP accumulation. Indeed, efforts in these directions represent some of the most intriguing phytoakxin research presently underway. The review by Dixon et 01. [76] testifies to thcaccomplish- mcnts achieved and the challenges involved.

Elicitation of iso~acwwid phyroaiexim

The obverse topic pertaining to phytoakxin formation, but distinct from biosynthesis as such is resolution of what stimulates the accumulation after particular tissue damage. The expression commonly employed to cn- compass this phenomenon is ‘elicitation’, coming from Keen’s proposal [IO21 that any factor which induced plants to accumulate phytoakxins be termed an ‘elicitor’. Although the phytoalcxin hypothesis wasdcvclopcd in the context of offering. at least, a partial explanation of how some plants might resist fungal colonization, ir became apparent early on that elaboration of phytoalcxms occurred in response 10 factors o1hcr than fungi and. indeed, even 1oabio1icclicitors[!03].Ovcr1hcycars,claritication of the mechanism(s) underlying elicitation has been a major focus for research. and no1 a little controversy [1011106]. Perhaps this fascina1ion with resolving phytoakxin elicitation lies in the promise that it might lead 10 the ability IO activate plant resistance via the application of exotic, but non-toxic. chemicals. Natural rcsisrance in plants might. therefore. play a far greater role in practical disease control than is presently thought IO be the case. Simultaneously. this should lessen the need for pesticide usage. moderating environmental impact prob-

Isol\ovonoid phytoxkxrns 987

kms that presently Confront chemical Control of plant diseases [ 107,IO8).

In the particular ~asc of IPs, definition of how elki- tation occurs is not ~karly rcsolval. One line of thought has been that, by mahanisms not yet understood. plant cells ‘rccognizt’ wall components of fungal invaders and, as a result, latent defenses -including IP formation-are activated. A recent series of papers [109-l 121 rcportal ChaGICfCriZUiOn Of an heXa-@-D-ghJCOpyfanOSy~)-D- gluCito1 from myctlial walls of P. mgasperma f. sp gi~ccinra that evoked phytoakxin accumulation in soybean. In spite of the p&se chemical charaaeritation of the active fraction isolated, its rok as a natural elicitor remains unrrrtain for its release from the fungal walls followed hydrolysis with 2 N trifluoroaCctkaCidat 85” for 2.5 hr [I 121. Furthermore. the isoflavonoid Component(s) presumed to be prod& in the elicitor assay were not individually identified but, rather. a Composite absorb- ana: value at 286nm was used as a measure of their formation. Employing the same host-pathogen system, Keen and Legrand [ 113 ] had previously extractul gly~o- proteins from isolated preparations of the fungal walls with 0.1 N NaOH at 0” for IS hr. Elicitor activity was associated with high molecular weight glycoproteins which appeared to contain only glu~osc and mannose as neutral sugars. Neither boiling nor pronasc treatments destroyed elicitation, whereas periodate treatment did, suggesting that the carbohydrate components were important for activity. The significance of Carbohydrate in the eliciting activity of P. megmperma f. sp. glycinea is undcrscorcd by the observation that an extdular invertase, produced by the fungus and itself a mannan- glycoprofcin. inhibited the activity of a glucan elicitor isolated from the Cell wall of rhis pathogen [ 1141.

Despite the discrepancies in the preparation protocols employed, as well as in the chcmi~al Composition of the elicitors. a Common question emerges from these findings which bears upon their relevance to natural disease resistance. How might release -if release is rcquired+f such eliciting materials, brought about by acid or base treatments in in cirro studies, be ;Lchkvcd in infcctcd plant tissues? A possible explanation would rely upon appropriate enzymic activity. Working once more with soybean and P. mPgaspermo f. sp. glycinea, Keen and Yoshikawa [I IS] determined that host tissues contained &1.3- endoglucanasc isozymcs which rckascd eldtor-active carbohydrates from the myctlial walls. The elicitors, in this instanCe, were gluComannans.

Opposed to the idea of the release of elicitor from a pathogen by a host enzyme. is the separation of host all wall fragments resulting from the activity of a pathogen’s enzyme(s). The plant wall component(s), in turn. elicit phytoalcxin aanrmulation. Just such a situation has been reporlcd and, again, with soybean, although in this instancc the microorganism involved was a bacterium. E;rGtia carozowro [I 161. Heat-labik ‘elicitor* activity in bacterial culture filtrates co-purifkd with a-1,4- cndo~ly~~turon~ acid lyasc attivity. Elicitation was apparently indirect, a result of the enzymic release of active fragments from pa+ polysaccharidcs in the plant all walls.

Roth pathogen and host, therefore, may serve as soutces of IP elicitors. With regard to the pathogen. most work would indicate that a carbohydrate constituent of the oulcr layers triggers phytoalexin accumulation. An eliciting role for some pathogen Component was, historically,

the first situation reportal [ 1 I7 1. The 0th CirCumstanCe is of a host fragment which is itself recognized as an elicitor by that same pknt. This situation was first dcsCribcd by Hargrcavcs and Baiky [I 18) and sub- salucntly reviewed by Baiky [119]. The many obscr- vations that diverse, and apparently unrclatai, abiotic stimuli Could result in IP produaion might, most easily, be rationalized if all were causing tht release of some ‘Constitutive elicitor’ in the plant which initiated phytoalexin formation. The accumulation of 1Ps may be variously evoked by heavy metal salts [73.103,120 , DNA-inte~kting chemicals I21 , antibiotics 122 ,

1 I 1 I herbicides [ 1231. chloroform 124 , surfactants 125 , et hyknc

5 [I IS, I27 1261, sulphur dioxide and ozone [ 1231, freeing , meChanical injury [ 1281 and ultraviolet ir-

radiation [129]. Bailey [119] spazulatcd that healthy plants contain a constitutivc elicitor. sequestered in some non-funetional form, which is released or activated upon Cell damage and stimulates phytoakxin formation. The chemical nature of Hargrcvcs and Baiky’s [ 1181 putative ~nstitutive elicitor is still unresolved.

Hahn and assoCiatea [ 130) reported an ‘endogenous elicitor* from soybean hypocotyls that was shown to be a paztk fragment [ 131). The adjective, endogcnous, was cmploytd bbceusc the elicitor was not active constitutively but, rather, had to be released from Covalent attachment within the all wall to be effective [104]. This distinction from a constitutive elicitor seems finely drawn and, while it might be valid on semantic groundb--although Hargrcavcs and Baiky did not suggest czonstitutive BE- tivity, merely ~~titutive p reKncbeny implication of a different biological function or chcmkal nature may not be merited.

Although IP acszumulation may be evoked by foreign biotic or abiotic fatztors, or by native biotic factors, and enzyrnic release of aftain of these elicitors may be essential to their a&ity. the presence ofcarbohydrate is a nearly Common denominator in active ftactions. Additional, independent work supports this Conclusion. Chitosans, Constituted primarily of @-1+gluCosaminc, elicited pisatin accumulation in peas [ 1323. However, the eliciting activity of this polyortion might have been the indirect result of it injuring plant cells [ 1331 and so activating cndogcnous mazhanisms [ 1043. Sucrose stimulated formation of the chakone, pinostrobin, a phytoakxin in Cajanus cajm [IM]. Plants may, then, respond to particular Carbohydrate components. from external or internal sources, by activating IP formation. Details of the recognitional events involved remain unresolved, although evidence that a receptor site for a branched /?-I$-glu~an elicitor of glyCeollin exists on soybean membranes has been presented [ 1353.

A not unexpected conscqucrux of investigations of phytoakxin elicitation is the development of models encompassing the phenomena apparently involved in this event. Keen [I361 proposed the presence of surfaCe receptors-probably proteins or glycoprotein*-on plant cells that rccognhcd supcr&ial molacuks, the elicitors, of invading micro-organisms. Secondary messengers’ were postulated to transmit this initial stimulus to the nucleus of the host cell contaCted, as well as to neighboring alls via plasmodcstnata De nouo tranauiption was prcsuaud to result in the formation of new mRNA which, in turn, was translated. resulting in the production of enymcs catalysing phytoakxin formation. Accumulation of loyal- iced Conrzntrations of the phytoalexins was assumed to

988 D. A. Sxfrru and S W. BANI[S

prevent, or at last delay, growth of any mkro-organism sensitive to their inhibitory activities. Yoshikawa 11371 claboratcd slightly upon this N emphasizing par- ticukrly a mechanism rekvant to IP aocumulation in soybean infectal with P. megaspmw f. sp. glycinco. In this cast, elicitors wcrc speculated to be rckascd from the fungal cell wail surface as a result of glucanasc activity constitutivcly prcscnt in the host cells.

Such models, whik thought-provoking in dcvcloping theories for the rok of phytoakxins in pathogcncsis, must bc viewed cautiously in tight of the still uncertain status of natural elicitors, the limited info~tion about rcccptor sites and the virtual dearth of data comxming saxmdaq messengers. A posde candidate as a samndary messenger is the gaseous plant growth rcguktor, cthykne. which has been rcportai to elicit pisatin [ 1261. Howcvcr, the cvidencc that it has a primary role in evoking phytoalexin accumulation is unconvincing Although elicitors of glyccollin accumulation also incrca& ethylene formation and PAL activity, chaniarl manipulation of the ethylene rcsponsc showed that it could be both stimulated and rcprcsscd without concomitant effects on glyceollin or PAL [ 1381. Others have suggested that thcsc observations may have been artcfsftual[773, in the scnsc that ethylene may not be nccdcd for mobilization of carbon within the detachcd cotykdonary tissues employed by Paradics et 41. [ 1383. Kimpcl and Kosugc (773 obscmd partial inhibition of glyceollin aazumulation in intact soybean seedlings, when ethykne production was blocked, but no inhibition in seedlings lacking cotykdons. They spazulatc that, while cthyknc may not control the initiation of the glyceollin response, it does mediate the flow of carbon and energy into glyccollin biosynthesis, such that it may be involved as a signal in the removal of rcscrvcs from the cotyledons to sites whcrc phytoakxin formation is oocur- ring Cyclic AMP might represent another candidate as a secondary messenger mediating IP biogenesis, although Hahn and Griscbach [ 1391 did not obtain results consistent with such a possibility.

Gnc other issue pertinent to IP fo~tion concerns the sites whcrc biosynthesis and subsequent build-up occur. Excision of infected tissuesand apparently healthy tissues adjacent to lesion arcas has pcrmittcd determination of the IP kvels accumulating and given a general picture of their localization [3.38,140,141]. Phytoakxin conccn- nations were cot&ted to laion areas or adjacent surrounding tissues, with generally no detcctabk amounts in healthy parts of the plant well rcmovcd from infaction sites, Synthesis and accumulation, sina they often occur in rcsponsc to infection and consequent tissue damage. are frcqucntly associated with necrotic reactions which in- volve tissue browning Nonethckss, Rathtncil and Bendall [142]indicatcd that phascollin formation may rcprcscnt a spa%& stimulation of isoflavonoid metabolism scparatc from the gcncral incrcasc in phenolic compounds as- sociatcd with nazrosis. There sams little doubt that IPs arc synthesizai, de nova. from rcmotc precursors soon after infection in the cells first contacted-prior to their death-and/or in living cells immcdiattly surrounding the region of tissuedamage. Some short-distance transport of the phytoakxins may occur IO account, at least in part, for accumulation in dead ails ar the infection site. The progression ofall dcarh as the laion expands, may result in additional hcalthyallsactivating phytoakxin synthesis until lcsion site is limitcd and spread of the mkroorgan- ism is restricted-unless, of course. the host rcsponsc is

ovcrwMmcd by a highly adapted, viruknt pathogen before an cffbctivc defense can be mounted, as may bc the case with Sckrotinia sclerotiorum infaction of bean kavcs [ 1431. The supposcxl defensive cap&y of 1Ps does not preclude the existence of other constitutivc or ectivatcd rcsistanoe mechanisms functioning in series. or in conozrt, to contain microbial invasion [36,144].

Although this overall picture may bc quite aozurate and is, in faa, supported by substantial data- relatively littk information is available about the prccisc allular location and concentration of phytoakxins. Techniqua have not, generally. bocn dcvclopcd to provide such exact information. Laser microprobe analysis has been cmploycd to detect glyccollin at the allular lcvcl in soybcan (1451. However, the instrumentation is very expensive, elaborate tissucprcparationisncccssaryandaauratequantihcation of glyceolhn in tissue is nof yet possibk with this technique. More rcccntly, Moesta and co-workers [ 1461 rcportal a radioimmunoassay for glyceolhn I that fkrmit- ted quantitative determination of this phytoakxin in 15 m microtomc seaions of infaacd soybean hypocotyl tissues. Together with the detection and quantitation of fungal hyphac by indirect immunoffuoresancc [ 1473. this radioimmunoassay pcrmittcd quantitation of phytoakxin and hyphal colonization in alternate serial cryotomc sections from P. megaspcmcr f. sp. gfycinca-infcctcd soybean root tissue [ 1481. Techniques are now, therefore, close to the point of allowing very precise localization and mcasurcmcnt of IPs in diseased plant tissues. These abilities will help resolve controvcrsics about the rcl- cvancc of phytoakxin aocumulation to natural disease resistance.

BIOLOGICAL ACllVlTlEF OF lSOFLAVONOl0 PHYTOALEXINS

Another csscntial facet to be clarified before a thorough understanding of the natural role of IPs will emerge is whether they do generate a toxic milieu in siw antagon- istic to sensitive organisms. Mtllkr and Bdrgcr 1231 had statal that their conceptual phytoakxin would cause . . . . ‘paralysis’ or the premature death of the fungus” 11491 and this has been the continuing presumption. The years have rcvcakd, in addition, that phytoalexins are not uniquely toxic to fungi, but have inhibitory effects across much of the biological spaztntm. While these rcscarch efforts have disclosed much about the biological properties of phytoakxins in generaI, and IPs in particular, the validity of relating laboratory findings to some natural inhibitory function in plant tissues remains a contentious issue [1X?].

Many different types of bioassay have bun employed to determine the cffccts of IPs (1 511. These have involved studies on the germination of fungal spores growth of germ-tubes, dry weight accumulation in liquid media and mycelial growth on agar surfaces. Antibacterial activity of IPs has been variously Assessed by dispersing phytoakxin in agar subsequently strcakcd with inoculum, applying phytoalexin in organic solvent to bacteria-seeded agar, impregnating phytoakxin in antibiotic assay discs before placing these on sccdcd agar as well as introducing phytoakxin to liquid media and monitoring bacterial

lsoBavonoid phymakxins 989

growth by absorbance. More limited investigations have also been made of phytotoxic cffazts and of animal roxicity.

A general conclusion that has cmergal from this considerable volume of work is that the par&u&r assay conditions employed -the composition and pH of media, the growth stage of the way organism--may have marked effects on the degree of inhibition observed. As exampIts. incorporation of rose bengal into agar modified the response of fungi IO pisatin [ 1521. alkaline conditions rcduad kievitone’s toxicity [ 1531 and, whereas maiicarpin was inhibitory towards spores and germ tubes of SWfnphylium bofryosum [154.155], mycelium was rcla- lively insensitive [I%].

Another generality that can be drawn from these numerous bioassays is that 1Ps do not appear cspazially toxic, with effective doses falling within one order of magnitude, IO-‘..lO’ l M. This cooclusion. however, should be treatcd with considerable caution. Individuals interested in the relevance of phytoalexins IO natural resistance employ bioassays designed IO yield measures of effective concentrations that can be r&ted to disease situations. Bioassay media, for example, have incorporated components of r&van1 plant tissues [141.157]. More pertinent IO considerations of IP m, however, is the frequent UY of plant pathogens in bioassays- organisms which may possess tolcrana mechanisms towards IPs [ 1581. Therefore, an inadvertent bias may have been introduad into many studies whereby potential phytoalcxin toxicity has been substantially under- estimated. Few. if any. IP mode of action studies have been undertaken with a micro-organism deliberately selaztcd for especial sensitivity to the individual phytoalexin being assessed.

Many procedural variables may affect in cirro estimates of phytoakxin toxicity and any one bioassay represents but a model system providing only limited information. Ideally, any IP should be bioassayed in several different systems and against a variety of organisms before d&u- tivc statements are made of its inhibitory nature.

Eflecfs of isq%conoid phytoolexins

The fungitoxicity of IPs is readily apparent from the inhibition they cause to germ-tube elongation. radial mycclial growth and dry weight accumulation [3,154.155,159-1611. Such essentially superficial obscr- vations may be relined through examination of individually affected cells by both light and ekctron microscopy. A variety of cytologitztl effazts have ban noted, including rapid cessation of cytoplasmic streaming. granulation of the cytoplasm, disorganization ofcell contents and brcak- down of the cell membrane [ISI. 153.161-1651. Although damage may often prove fatal to individual fungal cells, cell or colony death is not an inevitable conscqucncc of IP treatment. Skipp and Bailey [I631 noted that phascollin-treated sporelings often regrew from apparently unaffcctal hyphac, particularly from interstitial cells, and maackiain, mcdicarpin. or mixtures of these phytoalcxins, caused only temporary oessation of germ-tube elongation [ 1661.

Plasmalemma disruption, for which cytological cvidcncc has already been cited, is supported by the substantial k&age of electrolytes and mctabolitcs from fungal cells exposed to IPs. Phascollin [ 1651, maackiain [ 1661 and kicvitone [ 1531, all induced loss of “C-lab&d

mctabolitcs The inevitable consequence of uncontrolled leakage of mtabolitcs is loss of myalial dry weight [ 15 I]. Influx, as well as efit4 however, may be a&ted. PhascolIin and kicvitone inhibited the removal of ‘*C-U- glucose from liquid madi [ 153.1651 and, although tua&iain caused no immediate marked effcot on the rate of glucose assimilation by germinated spores, uptake was subsequently reduced [ 1661.

Movement of molazuks across the pIasmaIcmma is not the solitary physiological process in fungi adversely affa!tcd by IPs. Phascollin inhibited oxygen uptake by actively growing mycelium of R&zoctonio s&m’. whereas the cndogcnous respiration of starved mycclium was stimulated [165]. Suppression of exogenous respiration may have been due to insufK&nt uptake of substrate as a consequence of axembrane damage. The stimulation in starved myc&im might aLso have rdlactcd membrane dysfunction, either because it allowed dccompartmentali- xation of potential respiratory substrates within the myalia or bacausc it unsoupkd oxidativc phosphoryhtion. Certainly, sina phascollin faikd to inhibit rapir- ation by isolated mitochondria from Neurosporo CIU.W 11641. some indirect effact of the phytoalcxin seemed likely.

Studies of the antibactcriftl activity of IPs have been more restricted in number and scope than investigations of fungitoxicity [ 1511. Effects may be either bacteriostatic or bactericidal Whereas no inhibitory zones were evident when coumcstrol was applied in antibiotic assay discs to nutrient agar seeded with Psewfomonos spp.. this com- pound did prevent growth in liquid media for 36-48 hr [167]. Tests of eleven isolIavonoids, conducted in both soft agar and liquid media against eight Rhizobim strains [ 1681, indicated that maiicarpin and kicvitone were most toxic, being bactericidal towards R. joponicum and R.

lupini. Phascollin and maackiain were moderately inhibitory, whereas pisatin and counustrol, amongst others, showed little activity. An earlier survey [169] also indicated that kievitonc was more inhibitory than either phaseollin or pisatin.

Gram-negative bcria are usually less sensitive to antibiotics than Gram-positive genera [ 1701. This gencr- alization appears IO include iso8avonoid and Bavonoid phytoalcxins [ 171. 1721. K&tone. phascollin and 7- hydroxytIavan each inhibited six Gram-positive spades at a concentration causing no apparent inhibition of six Gram-negative isolates [151,171]. Govindarajan and Gnanamanickam [I733 noted that kicvitone was inhibitory towards thra Gram-positive human pathogens. Corynebmeriwn dip&eke var. m’tis, Streprococcus hoemoly~icus and Srophylocmcw oureus, suggesting that certain plant isoflavonoids might have some utility in chemotherapy.

If the antifungal and antibacterial activities of IPs may be taken as circumstantial evidence that these compounds might contribute to curtailing microbial development in plant tissues, the argument may reasonably bt expanded to encompass other pests and parasites. A survey of isollavonoids as insect feeding deterrents revealed several with activity and raised the possibility that plants may utilixc the same chemicals to deter insects as well as inhibit micro-organisms [20]. Microscopic nematodes are K- rious plant parasites and there is some evidence that certain of these. too, may be adversely affected by IPs. Glyaollin isomers limited the mobility of Meloidogyne incognita larvae, although the effect appeared to be

990 D. A Sure and S. W. BANKS

ncmatistatic, not nematkidal [ 174.1751. Oxygen uptake by this sensitive nematode was inhibited when phytoakxin was added IO larval suspensions.

Although most of the toxicological studies with IPs have focused on their antimicrobial activities, it is only scnsibte IO question whether their accumulation IO sub- stantialconcentrations in plant tissue might not alsoaITcct plants. In point of fact, phytotoxic effects of IPs are evidenced in several ways Phascollin caused rapid inhibition of respiration, kas growth of suspension cuhures

and cell death [ 1763; a&ted cells of both bean and tobacco appeared granular. Glazncr and VanEtten [ 1773 noted cell death and intial reduced growth of suspension cultures of fL.sedus uureu.9 and P. &g&s following phase&in trcatmcnt. Pisatin inhibited the growth of pea callus cultures [ 1781 and retarded primary root growth in wheat [160]. Leakage of bctacyanin and/or elaztrolytcs occurred after exposure of beetroot discs to kievitone and phasedin, but not pkatin or mcdkarpin [ 153.1791. Root and hypocotyl growth, as well as seal germination in several clover specks [ 1 SO]. was repressed by trifohrhixin.

A point worth bearing in mind, however. is that the toxicity shown by exogenous application of IPs IO plant tissues capable of producing these compounds might not accurately reflect any effects occurring upon natural formation [177]. Nonetheless, even if individual plant c&s are k&d in local areas of high phytoakxin conccn- trations, there is no reason to suppose that such an outcome might not be of overall benefit IO the plant. The lo&i& death of plant cells occurring in the formation of a limited ksion and perhaps due, at kast in part, IO the accumulation of IPs IO phytotoxic kvds may represent a part of the plant’s ‘strategy’ in containing invading micro- organisms My, this notion is not inconsistent with phytoalexins playing a contributory role in hyperscnsi- tivity [ 1811. Dixorganizstion of host ails with concomitant ‘removafofthcscas nutritional sources for pathogen suxlcnana, as a result of IP accumulation, could rtadily be envisaged as hindering extensive microbial colonix- alion of plant Iissues. This situation would seem likely lo be particularly cffcclive against obligate parasites, which require living host cells for successful growth and develop ment. Accepting such an argument, IPs may conoeivably be involved in curtailing viral replication [ 1821 which, presumably, requires organixed and functioning cellular machinery. II is worth bearing in mind, however, that isoBavonoids might not function solely as toxic agents in defensive responses. Suppression of plant growth raises the possibility that these compounds could act as cn- dogenous growth regulators [I].

The paragraphs above represent a briefcommcntary on what is now a considerable literature on the physiological and cytological effects of IPs. Fungi, bacteria. nematodes -- all may be susceptible IO the inhibitory activities of IPs and may have their development in plant tissues curtailed as a c~nscqucnce. Even viral replication may be limited indirectly as a result of the phytotoxic effects of isoflavonoids. Yet this does not quite compkte the roxicolo- gical picture as regards isoflavonoids; a final point merits mention. Toxic consequences may result in animals, including humans. consuming plant tissues containing phytoakxins [183]. Although only a few investigations have addressed this topic, IPs, for exampk, may lysc red blood alls [ 1511 and the e&t may be rapid [ 1651. Coumestrol showed oeslrogenk activity in mouse uterine bioassays and. as may be the case for other isoflavonoids

[3]. it may lead lo infertility or other reproductive problems in mammals fading on aftain plants [3.151,184]. Pisalin

“p rcsscd respiration in isolptcd ral

liver mitochondria [ 185 . Other evidence of the toxicity of Mlavonoids lo animal systems comes from obacrvations that phascollin, phaseollidin and phascollinisoflavan killed water snails and tine shrimp [3, 1861.

Any belief that IR were uniquely fungitoxic chemicals long since disappeared. Nor is there evidence to suggcs~ that they are even sckctively active against fungi, for the concentrations required lo express antifungal activity arc comparabk IO those nadod IO inhibit members of other biological dassca Bacteria, higher plants and a range of animals. as well as isolated cells or organelks arc all vulnerabk IO individual IPs. Furthermore, all death and non-kthal inhibition may OOCUT, indicating that biocidal as well as biostatic activity may be expressed.

Mode of acrion

The sites of action of IPs in the spectrum of organisms alTcctcd seem likely to be similar. for no clsss seems particularly scnsidn nor especially resistant. Clearly. the cytological and physiological consequences of IP trcat- ment represent either dirazt or indirect manifestations of the toxicities of these chemicala Ideally. in order to define a precise mode of action, the carbest effect on the target organism, employing the lowest amount of toxicant needed IO aaomplish inhibition, should be unambigu- ously established. It is probably fair to say that an txpft mode of action has not been unequivocally determined for any IP.

Many of the observations nbenlioncd above could most easily be rationalized by assuming that IPs arc multi-site roxic chcmkals, affecting many metabolic reactions and physiological prarJses as opposed IO site-sckctive compounds which primarily a&t one particular reaction or process [ I5 I, 1871. The considerable range of organisms detrimentally affected. both prokaryotcs and cukaryotu, is certainly suggestive of non-specific activity. Furthermore, the diverse physiological and cytological changes observed soon after trcatmcnt--cs@ally in the fun& which have been most thoroughly invcstigatcd- suggest that many ‘secondary’ cffazts may occur. hdscriminate binding, apparent, as examples, in the substantial uptake of phascollin by bean cells [ 1761 as well as in the moderating effects of detergents, phospholipids and protein on kkvitonc’s toxicity [ 1531, likewise favors multi-site activity. Certainly polyphcnolks, and perhaps even small phenols with multiple phenobc hydroxyls (a description befitting several IPs), may be rather indis- criminate in their binding, judged from the variety of interactions that may occur with proteins and polysaccharidcs [ 1881. There is also a tcliological argument for IPs being multi-site inhibitors These would be more dilKcult for a plant pathogen IO counter, in an evol- utionary scnsc. than sile-sclactive compounds where an alteration in one pathogen gene might induce a change at the specific site of action and so confer resistance [ 1891.

Generalization from the available data indicates dysfunction of membrane systems is. perhaps, parrtilarly instrumental in the Inhibitory action of IPs. Amongst treated fungi, substantial loss of dry weight, leakage, swelling ad bursting of hyphae [151]. are all consistent with membrane damage. particularly damage to the plasmalemma. Results from bacterial studies with glyci-

lsdlrvonoid phymakxins 991

noL glyccollin and coumcstrol indicated that these acted as nonspccifk membrane antagonists that altered the structural integrity of the membrane, thereby causing it IO bc a less el&ient matrix for mcmbrancdcpcndcnt pro- cessts [NO]. The cvidcncc also showed that the effects of thcscthraIPswcreofagcncralnatureandnotdircctcdat a spccilic allular process Bactcriostatic concentrations of glycinol and glyaollin inhibited all allular processes examined. regardless of whether thcsc were coupled to respiration. Lysis of red blood cells, which obviously lack wall* as a consequence of exposure to IPs is also consistent with some action on the cell membrane. The apparent importanct of a lipophilic nature for IPs is additional evidence making a membrane site of action probable. A lipophilic side chain is vital to the antifungal activities of wightconc [I911 and kihitonc [192]; this lipophilic nature likely permits &ective penetration of fungal membranes [ 1933. The plasmalemma seems highly probable as a site ofaction. sina it would likely bc the first membrane encountered by any ex~crnal toxic chemical. Hargrcavcs [ 1791 suggcstcd. however. from plant studies. that the tonoplast may bc primarily a5cctcd by phascollin. Gfcoursc, in those plants naturally producing IPs, their formation presumably would bc intracellular and the tonoplast might, therefore, bc encountered by the phytoalcxins before tbc plasmalanma

Although the balance of the evidence would appecu IO favor a case for 1Ps as multi-site toxicants with likely sites of action in allular membrane systems, site-spccitic action against a particular metabolic process cannot bc dis- missed. Studies with Meloidqyne incogruto indicated that glyccollin inhibited oxygen uptake by this nematode [ 1753. This report also rcvcakd that glyaollin was a potent inhibitor of oxygen uptake by isolatai mitochondria from soybeans and tabk beets and that it did not function as an uncoupler of oxidative phosphoryhtion but, rathcr.asan inhibitor of the electron transport system at some point after the succinatc dchydrogenasc site. Boydston and collcagun [ 1941, who also addressed the issue of glyocolltn inhibition of electron transport by isolatod mitochondria, could not conhrm this obsctva- lion. Their data indicated that glyccollin spccifkally inhibited malate oxidation, acting as a site I inhibitor in a manner similar or identical to rotcnonc. Although thcsc cxpcrimcnts indicated a glyccollin-spazilic SIIC in the mitochondrial electron transport chain associated with the inner membrane, the possibility that glyccollin might also bind to, and ingucncc, other membranes andior enzymes was not ruled out. These invcstigattons of respiratory ctfccts, as Weinstein and Albcrshcim [I901 point out, may not yet have ban extensive enough to provide a convincing argument for a spccifk target for glyaollin’s action and, furthermore, the observed inhibition of electron transport by glyccollin could bc one of several secondary effects resulting from the interaction of glyocollin with all mcmbrancs. Likewise. the action of pisatin as a respiratory uncoupler in ran liver mitochondria [I853 might bc mediated by the disruption of mitochondrial membranes.

The antifungal activity of ptcrocarpanoid phytoakxins was postulatal by Pcrrin and Cruickshank [ 1951 to bc dependent on particular stcric and compositional rcquirc- merits. However, these conclusions wcrc not supported by VanEttcn’s [I%] subsequent investigations. An cvcn more rcccnt study [ 1971 partially corroborates Pcrrin and Cruickshank’s [ 1951 earlier hypothesis. However, Kramer

and collcagucs [ 1971 feel that the fungicidal property of the isoflavonoids is spcc&c to individual fungi, substances and their concentrations, and that no absolute gcncrahz- ation is possible about stnrtural raptircmcnts. It would appear that Ibc structur+an tifungal activity relationships

of IPs, in general, require clati6cation [3,198]. Bakkcr CI cl. [ 1991 conducted an interesting invcstiga-

tion into the possibk role of photodynamism in IP toxkity. They noted that, upon ultraviokt irradiation, phascollin. 3,6a$trihydroxyptcrocarpan, glyccollin, tu- bcrosin and pisatin, but not mcdicarpin, inactivated glucose-&phosphate dchydrogcnasc in an in cirro assay, most likely as a result of free radical formation. Photodcgradation of the phytoakxins occurred almost immcdiatcly upon the onset of illumination and, ulti- mately, the irradiation was highly destructive to all cxccpt mcdicarpin. The relevance of these observations to the in uico activity of IPs is not, as yet, apparent, although the effects clearly merit further study.

Tolerance

There is no doubt that 1Ps cxcrt toxicity towards many, varied organisms. However, not all arc a&ted to the same extent. Diffcrcnccs in sensitivity occur bctwan and within the dilTcrcnt biological classes affected and, indad. cvcn within genera. Allusion has already been made to the apparently grater vulnerability of Gram-positive. as

oppo=d IO Gram-negative. bacteria [ 171, 1721. Phascoltin-tread bean all suspension cultures, despite initial inhibition, eventually achieved substantial growth [ 1761. This long-term recovery was perhaps the result of indtvtdual ails escaping the phytotoxk effects or the consequence of phytoakxin conversion to non-toxic products. Furthcrmorc, glyccollin isomers adversely affected the motility of the nematode. M. incognita, but no observed effect was noted on the related spbcics. Meloidqtg)ne j&o [ I 741.

Although differential sensitivity and/or evidence of recovery from IP treatment. phenomena both indicative

of tolerance mechanisms, have. therefore, been noted with bacteria. plants and animals. most is known concerning fungi [ 1581. Many different fungal types have been investigated in IP bioassays, not only phytopathogcnic specks. but also saprophytcs and zoopathogcns [ I5 I, 2001. DilTcrcnccs in phytoakxin vulnerability amongst fungi have been apparent for many years [l&l] and were at one time reputed to bc associated with plant pathogcnicity. pathogenic fungi being insensitive IO their hosts’ phytoakxin(s) whik non-pathogens were susccp tibk. Such an overall generalization no longer seems warranted for, while instances in accord with this association occur, so. too, do several exceptions [ ISI].

Tolcrancc towards IPs in fungi might have several mechanistic explanations, with paralkls being drawn from fungrcidc research [ZOI, 2021. Insensitive fungi might bc less pcrmcabk to the phytoakxins or possess sites of action with weak phytoakxin atIinitics. In addition. less vulnerable cells might possess a mctabohc bypass for the process inhibited by the phytoakxin or detoxification of the IP might occur before or after entry into the fungal all. Other explanations may also exist and, furthermore, multiple tolerance mcchanistns might bc exprcsscd by an individual organism or all.

The relationship bctwan plant pathogcnkity and comparative insensitivity to host IPs is not always ckar-

992 D. A. Sxm and S. W. BANKS

cut. Nonetheless, there are instances where fungal toler- anoe does seem critical for successful infection. Several papers published by VanEtten and coworkers [203-2081 present a strong case that the ability to tolerate pisatin is essential for the viruknce of Fuwzrium solani f. sp. pbi (ivecfria haemnffxoccaf in pea. Although monooxygenasecatalysed degradation of pisatin by P. sotani f. sp. pisi seems important here, this fungus also appears to possess an inducible, nondegradativc toknnce to pisatin. This nondegradatiw component appears to be due to plasmalemma mod%cation [204].

Detoxification by enzymic conversion of phytoakxins has been the most intensively studied tolerance mechanism [ 151,15e]. Isolation of a fungal enzyme catalysing detoxification of any IP was first accomplished for kievitone hydra&se (KHase) (Fig. I), which mediates t~~o~tion of k&tone (Fig I; trr) to kievitone hydrate (Fig. 1; 29) (x)9.210]. The enzyme was tint isolatai from ceil-free culture filtrates of Fusu&un soloni f. sp. phpwoli [209], but has since been shown to occur in F. s&hi f. sp. p/uaseoli-infected bean tissues. where it ac- cumulates for several days following inoculation [21 I]. Hi~~pu~~ preparations of KHase, which is a pre- dominantly extracellular enzyme [209], have been ob tamed [212]. The molecular weight of KHase was estimated to be 102oo0, isoelectric focusing and afllnity chromatography revealed that KHase was an acidic glycoprotein. Particularly relevant to present considerations, however. were studies undertaken to resolve whether any relationship existed in Fwmium between kievitone detoxification, mediated by Kliase. and virulence to beans. Twenty-eight wild-type isolates of Fusariu~~ and Nectria (N. hucmarococca is the perfect stage of P. soloni [206]) were surveyed for their ability to produce kievitone hydrate and for their virulence towards P. cvkaris (2131. Only three of these isolates poasessad demonstrabk, extracellular KHase activity and these same three isolates were the only ones highly virulent on P. vuiguris. In addition to the wild-type analyses, several variants of a vituknt strain of F. s&ni f, sp. phaseok were tested for KHase activity, virulence to P. rulgaris and sensitivity to kkvitone 12111. All variants with lowered KHaseactivity were more sensitive to the phytoakxin and less virulent to than than the original strain from which they were derived.

These independent studies of two different plant- fungus intuXtion.s (pea--F. sd4ni f sp. pLd and bean--F solmu’ f. sp. phacroll) both indicate that greater tolerance of IPs is associated with more virulent behaviour by individual fungal isolates [203-213].Anothcrcommonfeatureof these investigations, but one that is perhaps more dis- quieting, is the occurrence of, at most, only very low levels of 3,6adihydroxy-89methylcndioxypterocarpan (DMDP) and kkvitont hydrate, the primary metabolites of pisatin and kievitone, respectively, in infected plant tissues. Probably the most logical ~t~re~tion of these observations is that further conversion of the metabolites. catalysod either by the pathogen and/or its host, prevents their accumulation in r&o. Indeed, an intermediate rok for DNDP in fungal detoxification of IPs has already been established [ I58,214,215], though this is not yet the case for kievitom hydrate and some other explanation may apply to its low recovery from Fuwlum-infected beans.

Research into the metabolism of solitary IPs by fungi, whik instructive of the mechanisms involved, may not

represent the most appropriate models of natural situations For example, Phuseoh arlfiarir pfodur~ ~~~cral antifungal stress metabolita following fungal colonization (30). A successful pathogen, such as Fusarium dani f. sp. phawoli. might need to render inactive several of these compounds while invading the plant tissues. Cultural studies indicated that this fungus detoxified dilTerent combinations of the fPs from barn [216,217]. Within 24-30 hr. F. sold f. sp. &se& metabolized and detoxified a mixture of kievitone and phaseollidin as well as a mixture of kievitone, phaseollin and ~Uin~~~n. Only kievitone, however. was subjact to cell-free detoxifkation and, of the various enzymes presumably involved in these stnrtural alterations of the phytoalcxins, apparently only KHase was secreted extracetlularly in an active form. The, as yet unbroken, association between tolerance of kievitone and viruknce to bean in F. sohi f. sp. phawoli, together with the ability of this fungus to cope in culture with multiple treatments of IPs from bean, imply that the accumulation of post-infectional antimicrobial isollavonoids might present a functional. inhibitory environment to any micro organisms without the means to counter these compounds.

Studies of IP tolerance in fungi have, therdore, shed light on the rok these compounds may play in diseased plants, though it is by no means established fact that they do generate effective toxic barrims against the develop ment and spread of micro-organisms in infected tissues A definitive statement on thii matter will require refinement of bioassay systems, to establish the precise toxic poten- tials of IPs in infectal plants, as well as ever more exact understandings of elicitation and the concentrations of phytoalexins occurring at micro-sties in damaged tissues. Research to elucidatt the formation and biological properties of IPs. and the relevance of these phenomena to function, will need to continue to pursue several different avenues.

AcknmtMgmnus-The authors wish to thank John In&am for ~~mt~utthouphtrco~ingthenrantucrip~Potrida Parrott, Den& Mattingly ad Roger E. Dcaton for preparing the typescript.

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