A survey of antifungal compounds from higher plants, 1982–1993

Pergamon

0031-9422(94)EO287-3

Phytockmistry,

Vol 37, No

1, pp. 19-42, 1994 Eheviet Science Ltd Printed in Gnat Britain. oml-9422/94 sz4.cm+om

REVIEW ARTICLE NO. 92 A SURVEY OF ANTIFUNGAL

RENBE J.

COMPOUNDS 1982-1993

FROM HIGHER PLANTS,

GRAYERand JEFFREY B. HARBORNE

Department of Botany, University of Reading, Whiteknights, Reading RG6 2AS, U.K. (Received 30 March 1994)

IN HONOUR

OF PROFESSOR

Key Word Index-Flowering

ROBERT HEGNAUER’S

SEVENTY-FIFTH

BIRTHDAY

plants; antifungal agents; constitutive compounds; phytoalexins; second-

ary metabolites.

Abstract-Recent work on the characterization of antifungal metabolites in higher plants is reviewed. Interesting new structures are discussed and the distribution of those substances in different plant families is outlined. Distinction is made between constitutive antifungal agents and phytoalexins, which are specifically formed in response to fungal inoculation. The literature survey covers the 12 years since 1982.

INTRODUCTION

into the active antifungal substance after infection by means of a short and simple biochemical reaction, such as enzymic hydrolysis, e.g. cyanogenic glycosides which release toxic HCN after infection or leaf damage. This process of activation only takes a short time, since the enzyme(s) needed for the reaction are already present in the uninfected plant, though stored in a different compartment. Damage or infection of the plant brings together the enzyme and inactive form of the compound to produce the active post-inhibitin. In contrast, phytoalexin production may take two or three days, as by definition it first requires the synthesis of the enzyme systems needed for their biosynthesis. For some compounds it is difficult to determine whether they are phytoalexins or constitutive antifungal compounds (especially inhibitins and post-inhibitins) as the distinction between them is not always clear. Moreover, the same compound may be a preformed antifungal substance in one species and a phytoalexin in another. For instance, the flavanone sakuranetin (1) is constitutive in blackcurrant leaves (Ribes nigrum, Grossulariaceae) [3], but induced in rice leaves (Oryza sativa, Gramineae) [43. Additionally, some compounds may be phytoalexins in one organ and constitutive in another of the same plant species, e.g. momilactone A which is induced in rice leaves as a phytoalexin [S], but occurs constitutively in rice seeds [6]. In the literature review below we have distinguished between preformed antifungal compounds and phytoalexins, but the former are not further subdivided into prohibitins, inhibitins and post-inhibitins, since it is not always possible to infer from the data given

A fungal spore landing on the leaf surface of a plant has to combat a complex series of defensive barriers set up by the plant before it can germinate, grow into the plant tissues and survive. The arsenal of weapons against the fungus includes physical barriers (e.g. a thick cuticle) and chemical ones, i.e. the presence or accumulation of antifungal metabolites. These can be preformed in the plant, the so called ‘constitutive antifungal substances’, or they are induced after infection involving de novo enzyme synthesis, the ‘induced antifungal constituents’ or ‘phytoalexins’. Since the latter compounds can also be induced in plants by means of abiotic factors, e.g. UV irradiation, Ingham [l] defines phytoalexins as ‘antibiotics formed in plants via a metabolic sequence induced either biotically or in response to chemical or environmental factors’. Constitutive antifungal substances were called ‘prohibitins’ by Schmidt [2], but Ingham [1] restricts this term to those pre-infectional plant metabolites which are normally present in concentrations high enough to inhibit most fungi. In other plant species, the concentration of an antifungal substance may normally be low, but may increase enormously after infection in order to combat attack by micro-organisms; Ingham called this type of constituent an ‘inhibitin’. A third type of constitutive compound which he called a ‘post-inhibitin’, is defined as ‘an antimicrobial metabolite produced by plants in response to infection, but whose formation does not involve the elaboration of a biosynthetic pathway within the tissues of the host’. Post-inhibitins are normally present in the plant in an inactive, bound form, but are converted 19

R. J. GRAYER and

20

J. B. HARBORNE

ways, first according to their taxonomic distribution, and second according to their chemical structures. This was done to see whether certain plant families or genera specialize in the accumulation of certain types of constitutive compounds as they are known to do for induced constituents (e.g. sesquiterpenoid phytoalexins in Solanaceae; isoflavonoid phytoalexins in Leguminosae [9]), and to see whether any structure-activity relationships are apparent. Taxonomic distribution

2 COCMS

OH

Ii

*

3 R=a-OH 4 R-f%OIi

&4 ‘/

P \ ‘m

R’

k2

5 R-iPr,R’-R*-0 6 R-iFr,R’=OH,R*=H 7 R=OE,R’=R2=H

in papers to which of these subclasses a given preinfectional substance belongs. Furthermore, the survey is restricted to antifungal metabolites of low molecular weight, although it has recently become apparent that the production of antifungal macromolecules such as proteins may also play an important role in the defence systems of higher plants against pathogens. CONSTITUTIVE ANTIFUNGAL SUBSTANCES

Several useful short reviews on the occurrence of preformed antifungal compounds in relation to their role in plant resistance have appeared in the last two decades, notably those by Ingham Cl], Mansfield [7] and Hegnauer [8]. The present review covers the literature on the subject over the last 12 years. Antifungal compounds described during this period are classified here in two

Table 1 gives a listing of pre-infectional substances found since 1982 arranged according to their occurrence in plant families and species. The chemical class to which each compound belongs is also given, and additionally the plant organ from which it was isolated and the pathogenic fungus on which the antifungal tests were based. The format of this table is similar to that used by Mansfield [7]. From Table 1 it is apparent that the antifungal compounds found in the taxa surveyed in the last decade belong to a very wide range of chemical classes, and that even closely related species produce their own specific antifungal substances. Thus, although the antifungal compounds newly isolated from Compositae are all phenolic, they belong to different chemical subclasses. Even in the two species of Helichrysum investigated two types of phenolic were recorded: phloroglucinol derivatives from H. decumbens [lo] and methylated flavonoids from H. nitens [ll]. In the Gramineae the range of constitutive antifungal substances reported is even wider, e.g. saponins in oats [12], an alkaloid in barley [13], fatty acids in rice [14,15], phenolics in Sorghum [16] and alkadienals in wheat [17]. In the Leguminosae there is also substantial variation, ranging from chalcones, flavans and a diphenylpropene in Bauhiniu mama [lS] to isoflavones in Lupinus albus [19,20] and saponins in Dolichos kilimandscharicus [21]. But most species listed in Table 1 in the Compositae, Gramineae and Leguminosae belong to different genera or tribes. On the other hand, species belonging to the same genus in Table 1 generally contain related antifungal substances, the two Helichrysum species mentioned above being an exception. Thus, Scutellaria uiolacea and S. woronowii (Labiatae) contain closely related antifungal neo-clerodane diterpenoids [22], Glycosmis cyanocarpa and G. mauritiana (Rutaceae) both produce antifungal sulphur-containing amides [23,24], and the epicuticular leaf wax of both Nicotiana tabacum and N. glutinosa (Solanaceae) contains antifungal diterpenoids [25,26]. Finally, cultivated rice (Oryza s&vu, Gramineae) produces a range of fatty acids as constitutive antifungal substances [15], whereas its wild relative, 0. o&in&, produces a biogenetically related compound, jasmonic acid (2) [27]. However, relatively little chemosystematic work on preformed antifungal compounds seems to have appeared in the last 12 years. An exception is perhaps the work by Picman [28] on the antifungal activity of sesquiterpene lactones found in Compositae, but the aim of this research was to investig-

Heartwood

Roots

Leaf surface Leaf surface

Leaf surface Fruit Leaves and twigs Tuber

Combretum ~pi~u~at~

Eupatorium riparitan

Helichrysum decumbens ~elichrys~ nitens

Wedelia bijlora Ecballium elaterium

Combretaceae

Compositae

Gramineae

Dipterocarpaceae Euphvrbiaceae

Cucurbitaceae Cupressaceae Dioscoreaceae

Resin

Humulus lupulus (hop)

Cannabidaceae

indica

Commiphora rostrata

Oryza oficinalis

(barley)

m&are

LMVeS

Papillae of cvleoptile inner epidermal cells

Leavef

Root

Aoena sativa (oat)

Hor~m

Bark

Stemonopom

Chamaecyparis pisifera Dioscorea batatas (Chinese yam)

(maw)

Burseraceae

Mangijkra

Anacardiaceae

organ

Peel and flesh of unripe fruits Stem bark

species

Plant family

Jasmonic acid (2)

Unidentified

Gramine (18)

Avenacins

Benxoquinone

Modified fatty acid

Phenylpropanvid

Indole alkaloid

Triterpenoid saponin

Phenanthrene

Flavonol Cucurbitacin Diterpene Oxygenated bibenzyl

2,6_Dimethvxybenxvquinone (45)

Prenylated phenol Methvxylated liavone and flavonvl

Dihydrostilbene Chromene

Stilbene trimer

(44);

Flavanone Chakvue Phen~threne

Alkanone

Alkylated phenol

Chemical class

Phloroglucinol derivatives (25-27) Chrysin dimethyl ether (30); gala&n trimethyl ether (31); baicaIin trimethyl ether (32k five more higbiy methoxylated flavonoids 7,3’Di-O-methylquercetin Cucurbitacin I Pisifcric acid 3-Hydroxy-S-methoxy~~~l 3,~~ihydrvxy-~methoxybi~~l (batatasin IV) (40); 6-hydrvxy-2,4,7-trimethvxyphenanthrene (batatasin I) (41k 6,7_dihydroxy-2,4-dimethoxyphenanthrene; 2,7dihydroxy+-dimethoxy ph~~~~ne Canaliculatol (39)

6-Isopentenylnaringenin (33); xanthvhumol(35) 4,7-Dihydroxy-2,3,4t~methoxyphenanthr~e (63); 2,7-dihydroxy-3,4,6-trimethvxydihydrophenanthrene 4,4’-dihydroxy-3,%%methoxydihydrostilbene (42) Methyhipariochromene A

5-(12-cis-Heptadecenyl~resorcinol(22); 5pentadecylresvrcinol (23) 2Decanone; 2.undecanone; 2-dodecanvne

Compound(s)

Table 1. Constitutive antifungal compounds repvrted since 1982 which may play a role in plant resistance

69 45

10 Ii

93

67

27

Pyrkularia oryzae

13 Erysiphe graminis tsp. hordei

hordei

92

Cl&sporium cladosporioides Geumannomyces graminis var. tritici Rrysiphe penis f.sp.

12

86

Cladosp0rium cludospori0ides

Pyrkularia oryzae 35 Exsmined against 24 fungi 88

Rhizoctonia solani Rvtrytis ciwea

CIadOSpO?+iiwn cticun&mua

Colletotrichum gloeosporioides Cladosporium herbarum

89

73

50

58

Alternaria alternata Aspergillus and Penicillium species Trichophyton rubrum T. mentagrophytes P~~illi~ expansum

Reference

Microorganism studied

k & 8 t

Leaves and grains

Leaves

Sorghum cultivars

Triticumaestivum (wheat) Ribes nigrnm (redcurrant) Rosmarinuso#cinalis and other Labiatae Scutellariuoiolacea S. woronowii Euodio lunu-unke~a

Chisocheton pan~culatus

Mollugo pentaphylla

Muss (banana)

Molluginaceae

Musaceae

Roots

Dolichos kilim~scharicus Lupinusalbus

Unripe fruit peel

Aerial parts

Fruits

Roots

Leaf surface

wood

Dopamine (oxidation products)

Mollugenol A (16)

Luteone (37); wighteone (38); licoisoflavones A and S; parvisoflavone B 1,2-Dihydroxy-6a-acetoxyazadirone and three similar meli~ins

(2S~7,~-Dihydroxyflavan; (2S~3,~-dihydroxy-7-methoxyflavan; (2~7,~-~ihydrox~~-methox~avan~ Obtustyrene J-O-glucosides of hederagenin (12), bayogenin (13) and medicagenic acid (14) Luteone (37); wighteone (38)

ether; echinatin

Amine

Triterpenoid

~eliacin-ty~ penoid

Isoflavone

Isotlavone

nortriter-

Diphenylpropene Triterpenoid saponin

Flavan

Chalcone

Long-chain alcohol

1,2,4-T~hydroxyheptadec-16-yne; l~,~t~hydroxyh~tad~i6~n~ l-a~toxy-2,~dihydroxy-heptad~-l~yne Isoliqui~tigenin; isoliqui~tigeain~-ethyl

Peel of unripe fruit

Enol ester of hydroxycinnamic acid Neo-clerodane diterpenoid Phenylethanone

Flavanone

AlkadienaI

~ucoa~th~y~i~n

Fatty acid

Chemical class

Long-chain alcohol

Clerodin (10); jodrellin B (II)

2-(3,4-Rihydroxyphenyl) ethenyl esters of caffeic acid (24)

Sakuranetin

a-Triticene; /Witicene

Epoxy- and hydroxylinole~~ acids (e.g. 20 and 21) epoxy- and hydroxylinolenic acids Flavan-4-01s

Compound(s)

1-[2’,4’-Dihydroxy-6(3”-methyl-2”-butenyloxy~~(~-methyl~-butenyl)]phenylethanone and related compound (2% 29) Peel of unripe fruit cis, cis l-A~toxy-2-hydroxy~oxo-h~eico~-l2,15~~ene

Root bark

B~~h~ni~ manca

Persea a~ricana (avocado)

Leaf surface

Oryza sativa (rice)

Leaf glands of adaxial surface Leaves and callus culture-s Aerial parts

Organ

Species

Meliaceae

Leguminosae

Lauraceae

Labiatae

Grossulariaceae

Plant family

Table 1. Continued

64 22

Ciadosporiumherbarum Fusarium oxysporumtsp. lycopersici ~l~os~rium cladosporioides

~~v~l~ia verrucifonnis; Dreschferaoryzae; Alternariasolani ~l~osporium cucumerhtum Colletotrichummusae

Cladosporium cucumerinum ~elrnint~s~ri~ carbonurn Gladosporiumherbarum

Botrytiscinerea; Saprole0n~ asterophora and three other fungi

~o~leto~ichum gloeos~rio~es Cladosporium ~l~osporioides

3

48

44

46

20

19

21

51

66

17

16

Fusariummoniliforme; Curvularialunata ~l~osporium cucumerinum Botrytiscinerea

Reference 14,15,55

studied

Pyrlculariaoryzae

~icr~rga~

d B a

x $

F

B

Proanthocyanidjn Saponin Diaryiheptenone

Pin~b~n Tomatine 2-Ketoepimanool (9) (x-and &4,8,13-Duvatriene-1,3_diols Polymeric procyanidin Camellidins I and II Gingerenones A, B and C, isogingerenone B

Leaves

Roots

Leaves

Leaves

Leaves

Leaves

Leaf glands Green fruits

Epicuticuiar Ieaf wax Leaf surface

I%& shoot tissue

Leaf Rhizomes

Piper aduncum

Polygala ny~kens~s

Prunes yedoensis

Alibertia macrophylia

Glycosmis cyanocarpa

Glycosmis mauritiana

Populus deltoides Ly&opersi~on esculentum (tomato) ~~cotja~ giuti~sa

Piperaceae

Polygalaceae

Rosaeeae

Rubiaceae

Rutaceae

Rutaceae

Sahcaceae Solana~e

Theaceae Zingiberaeeae

Sterculiaceae

Diterpenoid

Stearic acid; ~Hydroxyd~e~noic acid; ~-hydroxytetrade~noic acid; ~~ydroxyhexade~oi~ acid 7-Ket~ehydroabieti~ acid (St; 7-hydroxyde~ydroabieti~ acid (6); lS-bydroxypodocarpic acid (7) Methyl 8-hydrox~~2-dimethyl-2H~hromene.~ carboxylate (46); 2,2-djmethyl-S~3-methyI-2-buteny~~2H~~omene-~ carboxylic acid (47) I;1-Dihydroxy-4-methoxyxanthone (48k ~,7-dihydroxy-3,~,6-t~methoxyxanthone (49) Benzylalcobot Coumarin la- and lfi-Hydroxydihydrocomin aglycones (3,4)

Needle surface

Nicotiana tabacum (tobacco) Theobroma cacao (cocoa) Camelha japonica Zingiber o@cinale (gin&

Astringin; rhaponticin

Bark

Picea sitchensis (sitka spruce) Pinus radiata

Illukumbin; methyli~lukumbins A and B

Sinharine (19f; methylsinha~e

Diterpenoid

Sulp~ur-~nt~njng amide Sulphur~ontaining amide FIavanone Steroidal afkafoid

Phenol Coumarin Non-glycosidic iridoid

Xanthone

Chromene

Oxidized diterpene acid

Lon~chain fatty acid

Stilbene

Naphth~uinone

Pinaceae

Naphthoxirenes

Root bark

Dihydrochalcone

Sesamum angolense

(36)

Pedaliaceae

3’-Formyl-2’,4’,6’-trihydroxy-dihydrochalc

Triterpenoid saponin

Twings and leaves

Sakurasosaponin (15)

Psidium acutangulum

Leaves

Myrtaceae

M yrsina~ae

26 78

Peronospora tabacina Crinipellis perniciosa Pestalotia longiseta Py~ular~ oryzae

25

Erysiphe cichoracearum

59

39,40

24

23

71 42

30

65

95

94

33

85

90

74

38

~l~os~riurn sphaerospermunq C. C~S~~~~ Aspergillus niger; Colletotrichum gioeos~rioidesCladosporium chuiosporioides Cladosporium cladosporioides Melampsora medusae Fusarium salani

Ciadosporium cucumerinum Cl~os~ri~ orbs

Peniciilium oxalicurn

Cladosporium ~~~in~ Rhizoetonia solani; Heimint~sporium teres ~l~ospor~~ ~~urn~in~ Paeolus ~hweinitzii~ Sparassis crispa Dothistro~ pini

3

V K

ff

OS

j f P 0 3 B

E T & c a

24

R. J. GRAYERand

ate the structure-activity relationships of the compounds rather than chemical interactions between plant taxa and their pathogenic fungi. For this reason this study has not been included in Table 1, but is discussed in the next section. Papers on the antifungal activity of plant constituents against fungi pathogenic to humans generally have not been included in the table either. On the whole our knowledge of the distribution of constitutive antifungal compounds in higher plants appear to be very fragmentary, and it would be well worth doing more chemotaxonomic screening in this respect. This can lead to interesting results, as has been revealed by a recent survey in our laboratory of the antifungal substances in the Rosaceae. A wide range of preformed constituents, especially of phenolic origin, was found to be involved in the protection of this plant family against fungal pathogens (T. Kokubun, unpublished results).

Chemical structures Table 1 shows the constitutive antifungal substances belonging to all major classes of secondary compounds: terpenoids (e.g. iridoids, sesquiterpenoids, saponins), nitrogen- and/or sulphur-containing constituents (e.g. alkaloids, amines, amides), aliphatics (especially long-chain alkanes and fatty acids) and aromatics (e.g. phenolics, flavonoids, stilbenes, bibenzyls, xanthones and benzoquinones). The importance of each of these compound groups towards plant defence against pathogenic fungi is discussed below. Terpenoids. Although monoterpenoids from essential oils are well known for their antimicrobial activities, only a few have been implicated in plant resistance to fungal pathogens, e.g. the strongly antifungal thujaplicins from the heartwood of Thuja and Cupressus species (Cupressaceae). These compounds have unusual 7-carbon ring structures [29]. Iridoids are a group of monoterpenoid lactones which usually occur as glycosides since their aglycones tend to be highly unstable [8]. However, antifungal activity of iridoids appears to be associated with the few stable unglycosylated structures known. For instance, four non-glycosidic iridoids were discovered recently in Alibertia macrophylla (Rubiaceae), two of which, lu- and l/l-hydroxydihydrocornin aglycones (3,4), showed fungitoxicity against a range of Cladosporium and Aspergillus species [30]. Other iridoids known to be antifungal are also non-glycosidic: isoplumericin, plumericin and plumieride from Plumeria species (Apocynaceae)

PI. In contrast to sesquiterpenoid phytoalexins which are characteristic of the plant families Solanaceae, Convolvulaceae and Malvaceae [9], few sesquiterpenoid constitutive antifungal compounds have been reported. On the other hand, many sesquiterpene lactones appear to be active. These compounds, which show a wide range of biological activities [29], are characteristic of the family Compositae [31,32]. Picman [28] screened 45 such compounds for their antifungal activity against Microsporum cookei, Trichophyton mentagrophytes and Fusar-

J. B. HARBORNE ium spp. No less than 62% of the sesquiterpene lactones

tested showed at least weak activity against the former two fungi, and nearly half of those inhibited them strongly. However, Fusarium was only inhibited weakly by 13% of the compounds tested. The antifungal activity of the different skeletal classes of sesquiterpene lactones was also compared, and the eudesmanolides came out as the group showing the highest proportion of strongly active lactones, whereas the germacranolides had the highest proportion of inactive compounds. The natural role of constitutive sesquiterpenoid lactones in the resistance of species of the Compositae against their plant pathogens does not seem to have been investigated, however, but from the results of the above test and the fact that several composite species have recently been found to produce sesquiterpene lactone phytoalexins (see below), this may be an important one. Many diterpenoids show antifungal activity; some of these are phytoalexins (e.g. the momilactones and oryzalexins of rice plants, Table 2) whereas others are preformed. The latter may be involved in the resistance of several conifers against their pathogenic fungi. For instance, the oxidized diterpenoid resin acids 7-ketodehydroabietic acid (5), 7-hydroxydehydroabietic acid (6) and 15-hydroxypodocarpic acid (7) from the needle surface of Pinus rudiuta (Pinaceae) appeared to be highly fungistatic to the pine pathogen Dothistroma pini. The compounds inhibited both spore germination and mycelial growth [33]. The diterpene pisiferic acid (8) from leaves and twigs of Chamaecyparis pi$era (Cupressaceae) [34] showed antifungal activity against the rice pathogen Pyricularia oryzae [35], but whether the compound also exhibits antifungal activity against pathogens of Chamaecyparis itself has not yet been investigated. A norditerpene dilactone, 2c+hydroxynagilactone F, was isolated as an anti-yeast principle from root bark of Podocarpus nagi (Podocarpaceae); perhaps the compound is active as well against Podocarpus pathogens [36]. Antifungal diterpenes have also been found as constitutive antifungal compounds in species of Angiospermae. The diterpene 2ketoepimanool (= 13(S)-hydroxylabda-8(20),14-dien-2one, 9) was isolated from epicuticular leaf waxes of Nicotiana glutinosa (Solanaceae), a tobacco species immune to powdery mildew. When applied externally to leaf surfaces of susceptible tobacco plants, 9 strongly inhibited mildew development. The compound was not, however, detected in three resistant varieties of cultivated tobacco, N. tabacum [25]. In the latter species the major cuticular leaf diterpenoids, r- and /3-4,8,13_duvatriene1,3-diols, appear to play a role in the resistance to blue mould [37], and removal of these constituents from the surface of tobacco leaves increases their susceptibility to Peronospora tabacina [26]. Two neo-clerodane diterpenoids from Scutelhnia (Labiatae), clerodin (10) and jodrellin B (ll), reduced growth of Fusarium oxysporum f.sp. lycopersici and other fungi, and inhibited spore germination. Thus, neo-clerodane diterpenoids, some of which are known to show insect-antifeedant activity, may contribute not only to the defence of the plants against insects, but also to that against fungal pathogens [22].

25

Antifungal compounds from higher plants

enin (12), bayogenin (13) and medicagenic acid (14) [21], and that of Rapanea as sakurasosaponin (15) [38], which also occurs in the roots of Primula sieboldi (Primulaceae). Related saponins from Rapanea lacking an epoxy group between C-13 and C-28 are not antifungal. The triterpenoid saponins camellidin I and II from the leaves of Camellia japonica (Theaceae) display antifungal activity towards Pestalotia long&eta [39]; the sugar moieties in

5Tff -I

\\

’

H

8

fl

OH

0

I

H

H

9

12 R~=EI,R~=~~OH 13 RI = OH, R2 = CF120H 14 R* =OH,R2-COOH

OcoiPr

11 16

An important source of constitutive antifungal triterpenoids are the saponins. This is another group of substances showing a wide range of biological activities. For instance, fungicidal triterpenoid saponins which also have molluscicidal activity have been isolated from the roots of Dolichos kilimandscharicus (Leguminosae) and the leaves of the African evergreen tree Rapanea melanophloeos (Myrsinaceae). The saponins from Dolichos were identified as the 3-0-/l-D-glucopyranosides of hederag-

26

R. J. GRAYERand J. B. HARBORNE

these saponins are tetrasaccharides [40]. Saponins appear to be an exception among antifungal compounds in general in that their antifungal activity is usually correlated with the sugar moiety glycosylated to the 3-hydroxyl group of the triterpenoid (and thus a polar part of the molecule), whereas most other antifungal constituents tend to be strongly lipophilic and inactive in glycosidic form. For instance, by removing one sugar from the avenacins, triterpenoid glycosides from oat (Arena sat&, Gramineae) [12], the antifungal activity is reduced (see ref. [ 11). Furthermore, structure-activity studies of saponins based on hederagenin and its derivatives revealed that compounds with rhamnose as terminal sugar, as in tlhederin from ivy (Hedera helix, Araliaceae), showed highest antifungal activity [41]. Earlier reports of antifungal triterpenoid saponins include aescin from the horsechestnut, Aesculus hippocastanum (Hippocastanaceae) and yiamoloside B from Phytolacca octandra (Phytolaccaceae) [29]. Oat not only produces antifungal triterpenoid saponins (see above) but steroidal ones as well, avenacosides [29]. Digitalis (Scrophulariaceae) is another genus from which antifungal steroidal saponins have been isolated [29]. Closely related steroidal glycoalkaloids with antifungal activity are present in the Solanaceae, e.g. tomatine in tomato, Lycopersicon esculentum [42], and cl-solanine and r-chaconine in potato, Solanum tuberosum [43]. Tomatine is thought to be the cause of the restricted development of Botrytis cinerea in green tomato fruits [42]. An antifungal triterpenoid aglycone, mollugenol A (16) was isolated from Mollugo pentaphylla (Molluginaceae). The closely related mollugenol B (17) lacked activity, however [44]. Further triterpenoid-based antifungal compounds are cucurbitacin I from Echallium elaterium (Cucurbitaceae) and four meliacins from Chisocheton paniculatus (Meliaceae). When applied to cucumber fruits or cabbage leaves prior to inoculation with Botrytis cinerea, cucurbitacin I prevented fungus infection of the tissues. The protective effect was not due to the induction of lignification, although localized lignification did take place, but was thought to be caused by the inhibition by cucurbitacin I of lactase formation by Botrytis [453. The meliacin-type triterpenoids showed inhibitory activity against the lemon-grass pathogenic fungus Curvuluria verruciformis, the rice pathogen Drecshlera oryzae, and the tomato pathogen Alternaria solani

Cl$-N

\ /

0

o”Jc NWS\

I’

H

19

5?=21

Ho

71’ dn

22 R-(CE2)11-QI=~-(Ca,b-CR3 23 R = @2)14-3

1461.

Nitrogen-containing compounds. There are many reports of alkaloids showing activity against human fungal pathogens, e.g. the isoquinoline alkaloid jatrorrhizine which occurs in Berberidaceae, Ranunculaceae and Magnoliaceae, a range of peptide alkaloids from Rhamnaceae, the quinolizidine alkaloid dictamnine from many species of Rutaceae, and the pyrrolizidine alkaloid juliflorine from Prosopis julifora (Leguminosae) [29]. Whether these compounds also play a role in the defence of those plants against potentially pathogenic fungi has not been investigated, but this is possible. For instance, the indole alkaloid gramine (18) which occurs in various Gramineae including barley (Hordeum) and the quinolizidine alkaloids sparteine, lupanine and 13-tigloyloxylupanine which

24

occur in Leguminosae species, were found to inhibit the germination of conidia of the barley pathogen Erysiphe graminis f.sp. hordei and also the further development to appressoria [ 131. Furthermore, the isoquinoline alkaloid berberine protects the roots of Makonia trifoliata and M. swaseyi (Berberidaceae) against the root rot pathogen Phymatotrichum omnivorum [47]. The antifungal activit-

Antifungal compounds from higher plants

ies of steroidal glycoalkaloids, such as tomatine from tomato and u-solanine from potato have already been discussed in the section on terpenoids, since the compounds are closely related to steroidal saponins. Thus, alkaloids, often thought to have evolved in plants as a defence mechanism against insects, may be broad-based defence substances which also act against plant pathogenic micro-organisms. Amines are another group of N-containing compounds which include representatives showing antifungal activity. For instance, in banana skins (Musa spp., Musaceae) high levels of dopamine are found which are active against the pathogen Colletotrichum musae [48]. Furthermore, the polyamines spermidine and spermine, which have a universal occurrence in plants, inhibit spore germination of Penicillium species [29], and presence of hordatines A and B and their glucosides in barley seedlings (Hordeum uulgare, Gramineae) makes young barley shoots resistant to a range of pathogenic fungi [49]. Sulphur-containing amides such as sinharine (19) showing antifungal activity against Cladosporium cladosporioides have been found in two species of Glycosmis (Rutaceae) [23,24]. Further groups of nitrogen- and sulphur-containing plant compounds which exhibit antifungal properties are glucosinolates (= mustard oil glycosides), which occur characteristically in the Cruciferae and some related families [31,32], and cyanogenic glycosides, which have a very wide, though sporadic, distribution in plants [S]. Both glucosinolates and cyanogenie glycosides are post-inhibitins sensu Ingham [l] in that they occur in the plant in an inactive form, but are transformed into the active isothiocyanates and hydrogen cyanide, respectively, after plant damage or infection. The main function of glucosinolates and cyanogenic glycosides in plants is supposed to be the prevention of herbivory, but since the isothiocyanates and HCN are not only toxic to insects and other animals but to microorganisms as well, they may additionally protect the plants in which they occur against certain fungi. Aliphatic compounds. The simple alkanones 2-decanone, 2-undecanone and 2-dodecanone, present in the volatile resin exudate from the stem bark of Commiphora rostrata (Burseraceae), have been reported to have considerable antifungal activity against a number of Aspergillus and Penicillium species at 5000 ppm [SO]. However, this activity may be too low for these compounds to play a role in the plant’s defence, unless their concentration in the exudate is very high. Antifungal long-chain alcohols, some of which are acetylenic, have been found as preformed substances in the peel of immature avocado fruits (Persea americana, Lauraceae). They are thought to be involved in the latency of the fungal disease anthracnose in unripe avocados [Sl, 521. Many other acetylenes, e.g. safynol and falcarindiol, are constitutive antifungal compounds in some species of Compositae and Umbelliferae, and phytoalexins in others [29], whereas the furanoacetylene wyerone acid, produced in broad bean leaves (Viciafaba, Leguminosae) and which is often considered to be a phytoalexin, may be a post-inhibitin instead [l].

21

Long-chain fatty acids, especially those having 18 carbon atoms, are emerging as a major group of antifungal plant compounds. Some of these are produced as phytoalexins, e.g. 9,12,13-trihydroxy-(E)-octadecenoic acid, which is induced in tubers of taro (Colocasia antiquorum, Araceae) after infection with an incompatible strain of Ceratocystis Jimbriatn [53], but many are preformed, although they often increase in concentration after fungal infection (inhibitins sensu Ingham [ 11). Four epicuticular fatty acids (see Table 1) have been isolated from the needles of Pinus radiata (Pinaceae), which are highly fungistatic to the pathogen Dothistroma pini [33]. The compounds inhibited both spore germination and mycelial growth in vitro, and results of experiments in which the epicuticular constituents had been removed with acetone suggested that these substances could be pre-infectional factors contributing to the resistance of mature P. radiuta trees. Resistance of the grass Phleum pratense against leafspot disease (caused by Cladosporium phlei) present in plants infected by the endophyte Epichloe typhina, is thought to be caused by four fungitoxic fatty acids accumulating in P. pratense infected by the endophyte [54]. Whether these compounds should be called phytoalexins or post-inhibitins is not clear. Ten C,, unsaturated fatty acids, five of which contain an epoxy-group and five a hydroxyl group (e.g. 20 and 21), and which show antifungal activity against the rice blast fungus, Pyricularia oryzae, were found in the leaves of two resistant varieties of rice, Oryza satiua (Gramineae) [14]. No activity was found in uninfected plants of a susceptible cultivar, but the hydroxylated acids only accumulated in blast-inoculated plants of this variety [lS]. At the same time, the activity of fatty acid hydroperoxidases increased, so that perhaps these hydroxylated acids were formed from the corresponding epoxy fatty acids, which may have been present in low concentrations in uninfected plants (e.g. as inhibitins). Resistance against P. oryzae could be induced in the susceptible plants by feeding them with epoxy fatty acids [SS]. It has also been discovered that four of the antifungal fatty acids found in rice, the 13-hydroperoxides and 13hydroxides of both linoleic and linolenic acids, increase rapidly in concentration after inoculating press injured spots of rice leaves with P. oryzae, and that the highest concentrations are reached within 24 hr after inoculation. Evidence was found that these fatty acids may act as endogenous elicitors of the diterpenoid phytoalexins which rice leaves start producing 24 hr or more after inoculation [56]. Some additional C,, diene and triene fatty acids, which were found in Miscanthus sinensis (Gramineae), also showed antifungal activity against P. oryzae [57]. Jasmonic acid (2) isolated from a wild species of rice, Oryza oficinalis, as a constitutive antifungal compound [27] shows a range of other biological activities such as inhibition of seed germination, promotion of senescence in leaves, and tuber induction in potato. It is interesting that this compound is biosynthesized from the same C-18 fatty acid precursor, linoleic acid, as the preformed antifungal fatty acids found in cultivated rice plants.

28

R. J. GRAYER and J. B. HARBORNE

Some further long-chain antifungal substances have mixed origins in that they contain phenolic rings, e.g. the heptadecenyl- (22) and pentadecyl-resorcinol (23) from mango (Mangiferu indica, Anacardiaceae) [SS], and the gingerenones from ginger (Zingiher ojficinalis, Zingiberaceae) which are diarylheptenones [59]. Aromatic compounds. A large proportion of aromatic plant substances shows antibacterial and often also antifungal activities. They include simple and alkylated phenols, phenolic acids, phenylpropanoids, coumarins, flavonoids, isoflavonoids, stilbenoids, quinones and xanthones. Quite frequently the same aromatic compound is a phytoalexin in one species and a constitutive antifungal constituent in another; this applies especially to isoflavonoids and stilbenoids [see ref. 29). However, phenolics do not necessarily have to show antifungal activity to be involved in plant resistance; since phenolic hydroxyl groups have a high affinity to proteins they may act as inhibitors of fungal enzymes such as cutinases, which are necessary to infect a plant [60]. Many phenolic acids have been reported as constitutive antifungal compounds, e.g. benzoic, protocatechuic and gentisic acids 129, 611. Gallic acid is inhibitory to both pathogenic and saprophytic fungi, so that leaf litter derived from plants containing gallic acid decomposes rather slowly. Only some Penicillium species, in which polyphenol oxidase activity is very low or absent, are uninhibited by this phenol and can break down the leaf litter. The inhibition of fungi by gallic acid seems to be due to accumulation of oxidation products such as quinones which are formed in the polyphenol oxidase catalysed reactions [62]. Antifungal phenylpropanoid or hydroxycinnamic acids include p-coumaric, ferulic, caffeic, sinapic and chlorogenic acids [29]. All these compounds have a widespread distribution in plants and often accumulate after fungal infection [63]. The aromatic rather than the carboxyl group seems to be needed for activity, since these acids are still antifungal in esterified form, e.g. orobanchoside, a caffeic acid ester from species of broomrape (Orobanche, Orobanchaceae) [29]. Furthermore, enol esters formed by condensation of dopaldehyde with caffeic acid (e.g. 24) found in the foliage and cell cultures of species belonging to several genera of the Labiatae (e.g. Rosmarinus, Salvia) are potent fungicides towards Cladosporium herbarum. Colony formation of this fungus was inhibited by very low concentrations of these compounds [64]. Additionally, aromatics without carboxyl groups, both simple and alkylated derivatives, have been reported as antifungal components [29]. Examples are benzyl alcohol which accumulates in mechanically damaged leaves of the cherry Prunus yedoensis (Rosaceae) and which inhibits the growth of Cladosporium herbarum [65], isoprenylated phloroglucinol derivatives (25-27) found on the surface of Helichrysum decumbens (Compositae) which inhibit growth of the same fungus [lo], and fungicidal isoprenylated phenylethanones (28,29) from the root bark of Euodia luna-ankenda (Lauraceae) 1661. The antifungal diarylheptenones found in ginger roots and alkylated resorcinols from mango fruits and rice

25 R=H 26 R=Me 27 R-Et

?H

!?I-

no’

‘I

o--R

28 R=isopreoyl 29 R=genmyl

30 R1-R~=OCHs,R3=R’==H 31 R1=R2=R3=OCE3rR’=H 32 RI-Rz=R’-0CE3,R3=H

33 Rl=H,R2=bopr,R3=H 34 R1=C!E3,R2=&R3=bopr

roots have already been discussed in the section on aliphatic compounds. Finally, papillae of a mildew-resistant isoline of barley (Hordeum uulgare, Gramineae) were found to contain a light-absorbing component which was absent from the susceptible isoline. Inhibition of the formation of this component with chlorotetracycline made the mildew resistance disappear. Autofluorescence, UV absorbance and staining suggested that the light-absorbing compound was rich in phenylpropanoids [67]. Coumarins, which have a phenylpropanoid nucleus, are another group of aromatic substances rich in antifungal representatives. Examples include coumarin itself,

29

Antifungal compounds from higher plants

esculetin, herniarin, scopoletin and umbelliferone, which all have a wide distribution in higher plants [29,68]. Apart from benzyl alcohol (see above), coumarin was also found to accumulate in mechanically damaged leaves of Prunus yedoensis, where it inhibited growth of Cladosporium herbarum [65].

Many flavonoids and especially isoflavonoids have been reported to play a role in plant protection agaifist pathogens, both as preformed antifungal compounds and phytoalexins. Flavonoid classes most often associated with antifungal activity are flavanones and flavans, but additionally lipophilic flavones and flavonols, certain biflavones, chalcones and dihydrochalcones are known to be active. For example, Tom&Barberan et al. [ 1l] found eight methylated flavones and flavonols in leaf surface extracts of Helichrysum nitens (Compositae), e.g. chrysin dimethyl ether (30), and galangin and baicalin trimethyl ethers (31,32), which showed antifungal activity against Cladosporium cucumerinum. The leaf surface of another composite, Wedelin &flora, contains another antifungal methylated flavonol, quercetin 7,3’-0-diiethyl ether [69]. Antifungal flavones are found in rosaceous trees, e.g. chrysin as a glycoside in wood of Malus fusca and fruit and bark of Malus sieboldii [T. Kokubun, unpublished results], and tectochrysin (as the 5glucoside) in Prunus cerusus bark [70]. As to flavanones, pinocembrin, secreted by leaf glands of Populus deltoides (Salicaceae) is active against some pathogens of this tree, but not against others [71]. Naringenin is an antifungal constituent of the heartwood of many trees belonging to the family Rosaceae, e.g. Amelanchier o&is, Prunus lusitanica [T. Kokubun, unpublished results], and Prunus domestica [72]. The 7-methyl ether of naringenin, sakuranetin (1) also occurs as a constitutive antifungal agent in the heartwood of various species of Prunus, and in glands on the adaxial surface of most varieties of blackcurrant, Ribes nigrum (Grossulariaceae) as mentioned earlier. Conidia of Botrytis cinerea are inhibited from germination on this side of the leaves, but not on the abaxial surface which does not contain sakuranetin [3]. The hard resins of hop, Humulus lupulus (Cannabidaceae) contain the flavanones 6-isopentenyl naringenin (33) and isoxanthohumol (34), and the chalcone xanthohumol(35). Compounds 33 and 35 show a high antifungal activity against Trichophyton mentagrophytes and T. rubrum, but isoxanthohumol, though possessing the same substitution pattern as xanthohumol, is 60 times less active [73]. Three chalcones from the wood of Bauhiniu manta (Leguminosae), isoliquiritigenin, its 2’-methyl ether and echinatin, show antifungal activity to five different fungi [18]. An unusual dihydrochalcone (36) containing an aldehyde group, isolated from the twigs and leaves of Psi&urn acutangulum (Myrtaceae), demonstrated antifungal activity against Rhizoctonia solani and Helminthosporium teres [74]. There are opposing views as to whether dihydrochalcones such as phloretin and its glycoside phloridzin, which occur in apple foliage, play a role in plant defence against pathogenic fungi [see ref. 751. Apparently these compounds are not themselves antifungal, but are converted into the corresponding 0-quinones which are fungicidal [l]. It is also

35

36

37 R-OH 38 R-H

39

controversial whether flavan-3-01s play such a role. There appeared to be no positive correlation between resistance of apples to apple scab (caused by Venturia inaequalis) and preformed flavan-3-01s [76]. On the other hand, the epicatechin concentration in avocado peel contributes to the resistance of avocado to anthracnose, because this

30

R. J. GRAYER and J. B. HARBORNE

flavan-3-01 inhibits the lipogenase activity which is involved in breaking down the antifungal long-chain alcohols present in avocado skin [77]. Jambunathan et al. Cl63 found a positive correlation between flavan-4-01 levels and resistance of sorghum against mould; in mouldresistant cultivars the levels of these compounds were two-three fold higher in the grains, and similar results were found in the leaves. A flavanol polymer, procyanidin, found in the flush shoot tissue of cocoa (Theobroma cacao, Sterculiaceae) inhibited the germination of basidiospores of the witches’ broom pathogen Crinipellis perniciosa [78]. Structure-activity studies of the range of procyanidins and monomeric flavan-3-01s revealed a strong trend of increasing antifungal potency with increased molecular weight of the procyanidins tested. The non-ionic detergent Tween-20 inhibited the antifungal effects of procyanidin, suggesting that non-covalent complexes with fungal macromolecules may be responsible for these effects [793. Some fungi have themselves taken protective measures against condensed tannins such as procyanidin. For instance, Colletotrichum graminicola produces its spores in a water-soluble mucilage, a glycoprotein fraction of which has an exceptionally high affinity for binding to purified condensed tannins, and so protects spores from inhibition of germination by polyphenols [80]. Although flavans are occasionally produced by higher plants as phytoalexins, some occur constitutively, for example (2S)-7,4’-dihydroxyflavan, (2S)-3’,4’-dihydroxy7-methoxyflavan and (2S)-7,4’-dihydroxy-3’-methoxyflavan, which are found in the wood of Bauhinia manta (Leguminosae), and which show high antifungal activities against a number of fungi [18]. Among aromatic compounds, isoflavonoids are one of the major groups of antifungal constituents. Again, a large proportion of these are formed as phytoalexins, mainly in species of Leguminosae, but many also occur as preformed substances, especially in leguminous and rosaceous trees. For instance, genistein, present as the 5glucoside, is a constitutive antifungal constituent in Prunus cerasus bark [70], and so is biochanin A, also present as a glucoside, in the wood of Prunus lusitanica and Cotoneaster henryana [T. Kokubun, unpublished results]. In the leaves of Lupinus species (Leguminosae), preformed antifungal isoflavones appear to replace the isoflavonoid phytoalexins which occur characteristically in many papilionate legumes. For instance, luteone (37) and wighteone (38), isolated from the leaf surface of L. albus, are fungitoxic to Helminthosporium carbonum [19]. Constitutive antifungal isoflavonoids are also found in other organs of L. albus, e.g. roots, stems and fruits. The activities of these compounds were assessed against Cladosporium herbarum; luteone and licoisoflavone A showed highest fungitoxicity, followed by wighteone, parvisoflavone B, licoisoflavone B and lupisoflavone [20]. Equally rich in antifungal constituents as the isoflavonoids are the biogenetically related stilbenoids. These C&-C6 structures comprise stilbenes, phenanthrenes and bibenzyls [Sl]. Hydroxylated stilbenes, occurring as constitutive substances in the heartwood of pine species

and other trees, have often been associated with decay and disease resistance in these plants [82]. Schultz et al. [83,84] assessed the role of stilbenoids in the natural durability of wood. They measured fungicidal activities of a number of (E)4-hydroxylated stilbenes and related bibenzyls, and found that stilbenes with 3’-substitution were all active against two brown-rot fungi, GZoeophyllum tribeurn and Poria placenta. Most bibenzyls had moderate activity against these fungi, but no structural requirement was apparent. Additionally, three bibenzyls showed activity against white-rot, Coriolus versicolor, but no stilbene was found to be active against this pathogen. There appeared to be no synergism between the various stilbenes and bibenzyls tested. In tree bark, stilbenes may be present as glycosides, e.g. astringin (5,3’,4’-trihydroxystilbene-3/3-D-glucoside) and rhaponticin (5,3’-dihydroxy-4’-methoxystilbene-38_Dglucoside) which are present in the bark of sitka spruce (Picea sitchensis, Pinaceae). Although the glucosides are antifungal themselves, they decrease after fungal challenge, whereas the corresponding aglycones, which have a much higher activity, accumulate [85]. Stilbene oligomers may also be present in trees as antifungal agents, e.g. the resveratrol trimer canaliculatol (39) which was isolated from Stemonoporus canaliculatus. This species belongs to the Dipterocarpaceae, a family of hardwood trees. The trimer showed activity against Cludosporium cladosporioides [86].

Although stilbenes are active against many fungi, some pathogens are less affected because of their ability to produce hydroxystilbene-degrading enzymes. For instance, Botrytis cinerea produces a stilbene oxidase which oxidizes both pterostilbene and resveratrol to nontoxic products 1871. Chinese yam, Dioscorea batatas (Dioscoreaceae) produces a range of antifungal bibenzyls (= dihydrostilbenes) and phenanthrenes, some of which are constitutive, whereas others are induced after microbial infection. Examples of preformed antifungal constituents in D. batatas are 3,2’-dihydroxy-5-methoxybibenzyl or batatasin IV (40), and 6-hydroxy-2,4,7_trimethoxyphenanthrene (= batatasin I) (41) [88]. Similar antifungal stilbenoids are found in the heartwood of Combretum apiculatum (Combretaceae), e.g. 4,4’dihydroxy-3,5-dimethoxydihydrostilbene (4% 4,7dihydroxy-2,3,6_trimethoxyphenanthrene (43), and 2,7dihydroxy-3,4,6_trimethoxydihydrophenanthrene (44). When 20 pg was spotted on a TLC plate these compounds showed a total inhibition of the growth of Penicillium expansum [89].

Some quinones are known to inhibit mycelial growth of fungi or are even fungicidal. Examples are two naphthoquinonoid naphthoxirene derivatives and their glucosides from Sesamum angolense (Pedaliaceae) [90] and the benzoquinones juglone present in a range of plants, e.g. pecan (Carya illinoensis, Juglandaceae) [91] and 2,6-dimethoxybenzoquinone (45) from Croton laccijkus (Euphorbiaceae). The latter constituent displays antifungal activity against Cladosporium cladosporioides ~921.

Antifungal compounds from higher plants

\ -/ -o--h-

Ho

OMe

47 3

rT?k

G

‘IO’

OH

42

31

Ho

48

- \-ohb

ohle

0

Me0

‘oOMe

45

Chromenes are a further class of aromatics containing antifungal representatives. Methylripariochromene A (6acetyl-7,8-dimethoxy-2,Zdimethylchromene), a root constituent of Eupatoriwm ripariwm (Compositae), displayed antifunga1 activity against five out of seven fungal species tested, especially against the tropical pathogen Colletotrichum gloeosporioides [93]. From the small tree Piper aduncum (Piperaceae) two chromenes were isolated, 46 and 41, which showed inhibition against Penicillium oxalicurn [94].

PHYTO 37:1-D

Finally, two xanthones out of four isolated from the roots of Polygala nyikensis (Polygalaceae), 1,7dihydroxy-4-methoxyxanthone (48) and 1,7-dihydroxy3,5,6-trimethoxyxanthone (49), exhibited an antifungal activity against the plant pathogenic fungus Cladosporium cucumerinum [95]. PHYTOALEXINS

The only earlier major review of plant phytoalexins is the monograph edited by Bailey and Mansfield 193 and published in 1982. More recent reviews of some aspects of phytoalexin research include those of Brooks and Watson [96], Gottstein and Gross [97] and Harborne [98,99]. The present review covers the literature since 1982. New phytoalexins are listed in Table 2, according to plant family, in alphabetical order. Stress compounds per se are generally excluded from this listing, unless there is also good evidence that they can be formed as genuine phytoalexins. Consideration will be given to the natural distribution of these new phytoalexins and to any taxonomic regularities that are present in these distribution patterns. The chemical structures uncovered during the period under

Leaf Leaf Leaf Leaf Leaf Leaf Tuber Leaf Leaf

Leaf Leaf Leaf

Bras&a juncea

Bras&a napus

Camelina sativa

Euphorbiaceae Gramineae

Dioscoreaceae

Convolvui~eae Costaceae Cruciferae

Compositae

Dioscorea batatas D. bulb~era 0. du~ntor~ D. rotundata Hevea br~i~iensis Avena sativa Festuca versuta Oryza sativa

Leaf Cortex

Mela~r~um jirmum C~cldiphylfum ja~o~i~urn Carthamus ti~torius Cichorium ~nty&us Coleostephu~ myconis Hetianthus annuw Lactuca sativa Taraxacum oficinale lpomoea batatas Costusspeciosus Brassica ~ampestris

BulbiI Tuber Tuber Leaf Leaf Leaf Leaf

Leaf

cell culture Leaf

Cephalacereus set&s Diantbus curyophyllus

Cactaceae Caryo~hylIa~ae

CercidiphyIlaceae

Bulb

A&urn cepa

Alhaceae

Safynol; dehydrosafynol Cichorabxin (78) Mycosinol(81) Scopoletin; Ayapin Costuno~ide (77); ~ettu~in A (80) Lettucenin A (80) ~squiter~nes Al and A2 Glyceoliins II and III Spirobra~inin (61); cycl~br~s~~n (59); oxymethoxybrassinin (62); methoxybrassinin (58); brassinin (57); d~oxybras. sinin (70); bra&canals A-C (63-65) Cycfobrassinin su~pboxide (60); brassilexin (67) Methaxybrassinin (58); cyclobrassinin (59) Camalexin (68); metboxy~malexin (69) Spirobrassinin (61); methoxybrass~in (58); bras&in (57); oxymethoxybrassini~ (62) D~hydropinosy~vin ~methyIbatatasi~ IV Dihydroresveratro~ Batatasin IV, dihydropinosylvin Sco~~etin Avenalum~n I (76), II and If1 Resveratrol Sakuranetin (1) MomiIactones A and 8; oryzalexins A-E, oryzalexin S

~-~exylcyclo~nta-1,3~~one (53); 5-~ylcycio~nta-l,3-dione (54) 4,5-~ethylenediox~6”bydroxyanrone (5@) Dianthalexins (3) (e.g. 73); dianthramides (27) (e.g. 74) N-p-Hydroxybenzoyl-S-hydroxyadhfanilic acid Ma~olo~

Table 2. Phytoalexins reported in pfants since 1982

Bibenzyi Bibenzyl Bibcnzyl Bibenzyt Coumarin Anthranilic acid Stiibene Plavanone

Indoie

Indole

Indole

Acetylenic Sesquiterpene lactone A~tylenic Coumarin Sesquiterpene factone ~squiter~ne lactone Sesquiterpene Pterocarpan Indole

Biphenyl

Aurone Anthranihc acid

Cyclic dione

88 II9 119 120 121 I22 123 4 124, 125, 126,127

118

117

116

115

106 107 108 109 110 111 112 113,114

105

103 104

102 103

Verbena-e

Umbelliferae

Rubiaceae Rutaceae ~opbula~~ae Tiliaeeae Ulmaeeae

Mahaceae Papaveraceae Pinaceae Rosaceae

Liliaceae

L.eguminosae

Benzofuran Biphenyl Renzofuran Biphenyi Renzofuran Biphenyl Acetophenone Anthraquinone Coumarin Phenylpropanoid ~uite~e Diterpene Sesquiterpene Coumarin

Eriobofuran Au~upa~n; ~-methoxyaucupa~n a~ton~ur~ 2’- and ~-Methoxyaucupa~ a-, /I- and y-Pyrufuran (8587) Rhaphiolepsin; 4’.methoxyaucuparin ~,~“~hydroxy~-me~oxya~tophenone Purpurin l-methyl ether, etc. Se∈ scoparone Acteoside; 8alactosylact~side 7-Hydroxy~lamen~e Mansonon~ A-P 7-Hydroxycalamenene Psoralen; bergapten, etc.

Leaf Leaf Leaf Seed Bulb Leaf

Leaf Sapwood Sapwood Leaf Sapwood Leaf Root Bark; cell culture Root bark Root Sapwood Sapwood Sapwood Leaf Wound tissue

Sapwood

(88)

~~furan

Diphysolone; kievitone; ferrerein Nissicarpin; fruticarpin; nissolicarpin Furan~ihydrok~mpf~ol Dalbergioidin, kievitone; phaseollidin Yurenohde (55) Resveratrol Hemigossypol San8uinarine (51) Renzoic acid A~upa~n (St); r-and ~-me~oxyaucupa~n (83,8@ c+ and B-Cotonefuran

Leaf Leaf

Cell culture Needle Sapwood

Pterocarpan Pterocarpan

Ison~ra~teno~ demethylm~i~a~in, Desmocarpin; kievitone, etc.

Leaf Leaf Leaf

Triticum aestivum Arachis hypogaea Cassiaobt~~o~~ ~alo~go~~ mucunoides Desmodium ganget~~ Diphysarobin~ides Nisso~~~ticosa Shnteria vesrita Vigna spp. Lilium maximowczii Veratrum~a~ipor~ ~ossypiumspp. Papaver br~teatum Pinus radiata AroGa arbut$oEa;C~e~~&s cathayensif;C. japonica Cotoneasterlactea; C. acutifoliw,C. divaricata Eriobotryajaponica Malus ~urnt~a Mespilusgermanica Photinia&bra Pyrus commnnis ~hap~o~epis ~beilata Sanguisor~ minor Cinchona ledger&urn Citrus limon Rehmanniagiutinosa Rlia x europaea Ulmusamericana U. glabra Apiumgraveolens; Petroselinumcrispum Avicenniamarina etc.

Anthra~lic acid Pterocarpan Chromone

Leaf

Sorghum bicolor

Naphthoiuranone

Isoflavanone Pteroearpan Dihydroflavonol Isoflavonoid ~~~ioxin-2-one stilbene Sesquiterpene Alkaloid Phenolic acid Biphenyl

Anthocyaniclm Aath~~~n

Stilbene

Piceacannol(52) Lut~~idin APige~~d~ 5~ffe~lara~mosi~, luteotinidin HDIBOA &rcoside Mediearpin 56

Leaf

slrccharumo~cinarum

161

151 152,153 154,155 156 157 158 1.59 160

147 145 145 148 149 150

145,146

t36 137 138 139 140 141 142 143 144 145

134 135

131 132 133

128 129 130

34

R. J. GRAYERand J. B. HARBORNE

review will be outlined. Chemical differences from constitutive secondary metabolites will be emphasized. Tissue variation in phytoalexin response, uncovered in recent years in certain plants, will also be mentioned. Where data are available, relative fungitoxicities will additionally be considered. Much work has been carried out on the process of elicitor recognition and signal transduction in the phytoalexin response, but there is still little agreement on the precise structure of the natural elicitor agent. Little will be included here on elicitation, since the subject has been well reviewed elsewhere [see e.g. ref. 100-J. Taxonomic distribution New phytoalexins reported since 1982 have been found in ca 60 species representing 24 plant families (Table 2) [101--1611. Families which have received especial attention for the first time include Compositae, Cruciferae, Dioscoreaceae, Gramineae and Rosaceae. Much work has continued on families where the phytoalexin response is well characterized, e.g. the Leguminosae and Solanaceae. Positive phytoalexin identifications were recorded by Bailey and Mansfield [9] in 15 plant families. The present data extend this to twice this number, to 31 plant families. At the species level, it is more difficult to estimate the extent of phytoalexin coverage. The only family which has been surveyed extensively is that of the Leguminosae, where an excess of 600 species has been examined [ 1621, the great majority of which have yielded one or more phytoalexins on fungal inoculation. Otherwise, relatively few species have been examined in the remaining families. Recent research at Reading [145] on phytoalexin induction in the Rosaceae has indicated a relatively low frequency in this family, with no more than 15% of species forming phytoalexins. Here the failure to give a positive response is correlated to some extent with the presence of constitutive antifungal constituents in the tissues. Present evidence suggests that Rosaceae is unusual in giving such a poor phytoalexin response. At least, experience in several other families besides the Leguminosae, e.g. Compositae, Solanaceae, Gramineae and Umbelliferae, indicate that usually most species tested respond positively. It is reasonable to assume at the present time that the phytoalexin response is relatively universal within the flowering plants and that any new family to be examined is likely to give a positive reaction. Furthermore, examination of further species within plant families already studied is likely to yield phytoalexins of related structure to those already characterized in the family. The taxonomic aspect, already noted earlier [163], is an important feature of the phytoalexins produced in any given plant. Ingham, who has pioneered the use of phytoalexin induction as a taxonomic tool in the family Leguminosae [162], has recently extended his surveys to the tribe Phaseoleae, which includes a number of economically important food or fodder crops [164]. Seventy-six species, representing 37 genera, each produce about six compounds. A total of 30 structures, isoflavones, isoflavanones, pterocarpans or isoflavans, were characterized,

so chemically the responses were typical for the family. The survey revealed distinct trends in phytoalexin production, with compounds with prenyl substitution occurring in some of the more advanced systematic groupings. In a more limited survey of the phytoalexin response in 11 species of Vigna (also Phaseoleae), Seneviratne and Harborne [139] found a generally similar response in all taxa. However, while the seed, hypocotyl or epicotyl produced three compounds, the root only gave two (dalbergioidin and kievitone). Seven other isoflavonoids may be produced in Vigna roots following stress C16.51.Some difficulties were experienced by Seneviratne and Harborne in getting a positive response in the common leaf diffusate experiments with Vigna; use of other tissues besides leaves was therefore recommended for these plants [139]. Chemical structures

Some of the new phytoalexins reported in Table 2 are already known as phytoalexins in either related or unrelated plant groups. Stilbenes, such as resveratrol, have been recognized earlier as phytoalexins produced by Arachis and Trijolium (Leguminosae), Broussonetia (Moraceae) and Vitis (Vitaceae) [9]. New sources reported here include Gramineae (Festuca, Saccharum) and Liliaceae (Veratrum). The ability of no less than five unrelated families to produce, at least occasionally, stilbene phytoalexins suggests that this biosynthetic pathway may be more widespread than is at present envisaged. It is interesting to note that the enzyme for stilbene production, i.e. stilbene synthase, has been successfully transferred from one plant producing stilbene phytoalexins (Vitis oinifera) to one that does not (Nicotiana tabacum) and that the resulting transformed plant makes resveratrol in addition to its normal sesquiterpenoid phytoalexins expected in a member of the Solanaceae [166]. Coumarins, either hydroxy- or furanocoumarins, are well-known phytoalexins in several families, including Umbelliferae. They are newly reported as phytoalexins in Compositae (Helianthus), Euphorbiaceae (Heoea), and Rutaceae (Citrus). By contrast, isoflavonoids are generally confined as phytoalexins to members of the Leguminosae, where a wealth of different structures have been encountered [162]. It is noteworthy, therefore, that such a class of phytoalexins has recently been encountered in a completely unrelated plant source. Thus, glyceollin II and III, two isoprenyl substituted pterocarpans of Glycine max, have been identified as the phytoalexins of Costus speciosus leaf, a monocotyledonous plant in the family Costaceae [112]. Several classes of secondary metabolite not previously noted for their antifungal properties have now been uncovered as phytoalexins. Aurones, for example, are a class of yellow flavonoid pigments, mainly occurring constitutively in the flowers of a number of Heliantheae (e.g. Coreopsis) in the Compositae. An aurone has been elicited in cell cultures of the cactus Cephalocerus senilis and identified as the 4,5-methylenedioxy-6-hydroxy derivative (50) [102]. The first alkaloidal phytoalexin san-

Antifungal compounds from higher plants

35 R

0

Lc

0

53 R = (CH&Me 54 R = (CH&Me

e

I’

I’

’

Oi-0

’

O)

%Y

51

guinarine (Sl), an orange compound, has been obtained by elicitation of Papaoer bracteatum cell suspension cultures. Its synthesis is stimulated by combined fungal elicitation and hormonal deprivation. It is not present in the intact plant as a constitutive alkaloid although widespread elsewhere in Papaveraceae. It proved to be significantly fungitoxic at 1 x 10e5 M to three of four fungal pathogens tested [143]. The first anthraquinones to be encountered as phytoalexins have been identified in Cinchona Iedgeriana bark. A mixture of several structures is present, including purpurin l-methyl ether [ 1531. Similar compounds are elicited by the fungus Phytophthora cinnamomi in cultured cells of the same plant [152]. Finally, mention should be made of anthocyanidin phytoalexins, recognized for the first time as such in two economically important grasses, sorghum and sugarcane. Two 3-desoxy pigments, apigeninidin and luteolinidin, orange-red in colour, were first recognized, e.g. in mesocotyl tissue of sorghum Cl303 in response to infection by both a pathogen and a nonpathogen. More recently, an apigeninidin 5-caffeoylglu-

A0 55

coside has also been detected in inoculated tissues [ 1671. The sorghum phytoalexins are synthesized in subcellular inclusions within the host epidermal cells, and only in the first cells that come under fungal attack [ 1671. In sugarcane, luteolinidin and one of its glycosides were detected, but the activity of these compounds against the sugarcane red rot disease, CoUetotrichumfalcatum, was minimal. A more active compound, the stilbene piceatannol(52), has subsequently been recognized as a phytoalexin of this plant [128]. Turning now to phytoalexins of novel structural type that have recently been encountered in nature, two of the simplest are the cyclic diones (53) and (54) produced in infected bulbs of the onion Allium cepa [loll. Although onion and other Allium species are rich in sulphur compounds some of which are known to be antimicrobial, it is noteworthy that the phytoalexins in the plant are not sulphur-based. Two other structurally unusual phytoalexins are yurinelide (55), a benzodioxin-a-one, from the monocot Lilium maximowczii [ 1401, and the chromone (56) from Cassia obtusifolia (Leguminosae) [133]. The

R. J. GRAYERand 1. B. HARBORNE

36

latter is an unexpected structure from a family where isoflavonoid phytoalexins are so regularly encountered. The most original series of new phytoalexins encountered in recent years are the indole-based sulphur compounds of the family Cruciferae. Analysis of the phytoalexin response in only a few well-known species of crucifers (Table 2) has yielded a profusion of at least 16 structures (57-72). These are all clearly derived from a common indole-based precursor and almost all have one

57 R-H 58 R==OMe

or more sulphur substituents. One possible partial biosynthetic sequence, suggested from in oibo chemical experiments, is the formation of brassilexin (67) from cyclobrassinin (59), via its sulphoxide [168]. These indole-based phytoalexins appear to have significant antifungal activity and are formed in sufficient quantity to act as a barrier to infection. Thus, 486 g of Brassica campestris infected tissues yielded 39 mg methoxybrassinin (5J3), 8 mg of brassinin (57) and 20 mg of cyclobrassinin (59),

65

59 R=SMe 60 R=SOMe

67

61 68 R=H 69 R=OMe

&Me 62

63

OJC S

H

64

X

b

dm. 72

Antifungal compounds from higher plants while these compounds demonstrated antifungal activity at concentrations of 100 ppm [113, 1141. The crucifer phytoalexins are related to the constitutive glucosinolates or mustard oil glycosides, which characterize the family as bound toxins. In particular, the glucosinolate glucobrassicin or indol-3-ylmethyl glucosinolate, which is of common occurrence, is an obvious structural analogue. It is interesting from the point of view of defence mechanisms in this family, that recent research has shown that indole-based glucosinolates may specifically increase in concentration in crucifer plants which are subject to insect herbivory [169]. There is thus a parallel between phytoalexin induction and induced chemical increases in response to herbivory in this one family. Yet another series of novel phytoalexins have been described in recent years from the carnation Dianthus

73

0‘I

37

caryophyllus (Caryophyllaceae). As many as 30 compounds have been isolated from infected carnation tissues [103]. Three of these are dianthalexins (e.g. 73), while the rest are anthranilamides (e.g. 74), in which various benzoic acids are substituted on the amino group of an anthranilic acid moiety. The amides can be formed readily in vitro by hydrolysis of the dianthalexins and the question has been raised as to whether the amides may not be artifacts of the isolation process. However, careful analysis of infected carnation tissue has confirmed that both alexin and amide are present in the phytoalexin mixture that accumulates. One novel feature of the carnation system is the presence in infected plants of an ‘anti-phytoalexin’. This is the compound dianthramine (75), which accumulates in susceptible varieties. Pretreatment of rooted cuttings with salicylic acids causes a switch from phytoalexin synthesis to dianthramine accumulation, there being a common precursor involved. This switch reduces the plant’s ability to resist fungal invasion [103]. Remarkably enough, anthranilic acid amides and related benzoxazinones have been recognized for some time as phytoalexins in a completely unrelated plant, the oat Avena sativa (Gramineae) [170]. Here, the major compounds that accumulate are avenalumins I-III, in which an anthranilic acid moiety is substituted by cinnamic rather than by benzoic acids. The benzoxazinone structure for avenalumin 1(76) has been questioned recently by Crombie and Mistry [ 1221. From synthetic experiments, these authors provide evidence that the natural phytoalexin of oat leaves is the ring-opened amide rather than the benzoxazinone. The Compositae is another plant family which has been recently surveyed for phytoalexins, and sesquiterpene lactones, acetylenics and coumarins have been encountered (Table 2). Here, the phytoalexins are generally closely related to constitutive secondary metabolites known in this large family. Thus, sesquiterpene lactones are characteristic toxins and three have been recognized as phytoalexins in the dandelion, lettuce and chicory. Two have expectable structures: costunolide (77) from lettuce which occurs elsewhere as a constitutive lactone; and cichoralexin (78) from chicory which is closely related to lactucin (79), a constitutive lactone of the same plant. The only really novel structure is lettucenin A (So) from lettuce, since this is a highly unsaturated lactone uniquely formed as part of the phytoalexin response. It is synthesized in inoculated lettuce leaves in very low amounts (0.00084%), but fortunately can be detected as a trace constituent from its green-yellow fluorescence in UV light [109]. As with the first two lactones mentioned above, the coumarins and acetylenic phytoalexins reported in the Compositae have expectable structures. For example, the spiroketal enol ether mycosinol(81) from infected leaves and stems of Coleostephus myconis occurs naturally in roots of the related Compositae, Santolina oblongifolia [107]. Mycosinol is formed as a phytoalexin in Coleostephus along with the (Z)-isomer, but it is not clear whether this isomer is a true phytoalexin, since it can be formed from the @)-form by photochemical action [107].

R. J. GRAYERand J. B. HARBORNE

R H

OH

.,H

\

\

0

77

0

@3 H

H’

4

-__

83 Rl=H,R*=Me 84 Rt=Me.R*=H

OH

85

G

CHO

0

86 R=H 87 R=Me

80

81

Tissue variability in phytoalexin response

At one time it was assumed that the phytoalexin response of a plant would be similar whether leaf, stem, hypocotyl, root or seed was the inoculated organ. Experience in the Leguminosae suggested that this was so, with only minor differences in phytoalexin profile according to the tissue examined [9]. Recent research on the plants of the Rosaceae, carried out at Reading [145], suggests that at least with members of this family the response can be

88

quite different according to the tissue being infected. This has been apparent from the earlier literature on rosaceous phytoalexins, since the fruit and sapwood of the apple tree, Malus pumila, produce benzoic acid and the biphenyls aucuparin and 2’-methoxyaucuparin, respectively. Recent research on fruits of Malus spp. failed to show any further examples of henzoic acid production, and preformed phenolics, especially phloridzin, appear to be more important as defence against fungal infection here.

Antifungal compounds from higher plants

The sapwoods of the variety of Maloideae have responded positively, confirming the generalization that biphenyl phytoalexins are produced in these tissues. In fact, two classes of phytoalexin may be formed (Table 2). Thus, biphenyls such as aucuparin (82) or related structures (83,84), are produced in sapwood of Chaenomeles, Malus and Rhaphiolepis, while benzofurans like a-pyrufuran are formed in Cotoneaster and Pyrus (85-87). Although biphenyls and benzofurans are biosynthetically related, no plant has so far been found to produce both classes of phytoalexin simultaneously. Biphenyl or benzofuran phytoalexins can also be formed in inoculated leaf tissue, e.g. in Rhaphiolepis umbellata and Eriobotrya japonica, but in general the response is not so easily demonstrated as with sapwood. The most striking example of phytoalexin restriction to plant tissue is that of the plant Sanguisorba minor, the salad burnet, a perennial herbaceous member of the Rosaceae. Here the only tissue that would respond positively to inoculation with Botrytis cinerea was the root. The compound produced, 2’,6’-dihydroxy-4’-methoxyacetophenone, is a novel phytoalexin for the family, although it does occur constitutively elsewhere, e.g. in the bark of Prunus domestica [151]. This simple acetophenone (88) proved to have a similar fungitoxicity to the biphenyls and benzofurans produced in the sapwood of woody rosaceous members [151]. As a phytoalexin, it is not only unique to root tissue but also to this particular plant, since attempts to induce in it roots of several related plants, e.g. Geum rivale, Acaena sanguisorba, were unsuccessful. DlSCUSSION

Some 250 new antifungal metabolites have been characterized in plants since 1982. About half of these are constitutive constituents (Table l), whereas the remainder are induced as phytoalexins (Table 2). They are all typically secondary metabolites, mainly being of terpenoid or phenolic biosynthetic origin. There is no sharp chemical division between constitutive and induced antifungal agents. However, fatty acid derivatives, reported in rice and pine, represent a relatively new class of constitutive antifungal activity in plants. Likewise, a variety of phytoalexins with novel structures have been uncovered. The sulphur-containing indole phytoalexins of the Cruciferae are a remarkable new group of such phytoalexins. It is also clear that the complexity of the phytoalexin reaction in a plant can far exceed that of any constitutive barrier to infection. The formation of as many as 30 phytoalexins in the fungally infected carnation plant provides a striking example of this. The fungitoxicity of the newly reported metabolites, where measured, is generally of the same activity as those reported earlier. The toxicity of phytoalexins was reviewed in 1982 and it was generally concluded that they were multi-site toxicants against fungi. Little new data have emerged in the last decade. Structure-activity relationships have been explored among the stilbenoids [84] and the isoflavonoids [ 171,172] without yielding much

39

new information. Synthesis of some 3-phenylcoumarin analogues of the isoflavonoid phytoalexins produced compounds with less fungitoxicity than the natural agents [173]. Methods of inducing phytoalexins in plant tissues have been extended, and a variety of elicitors, including the fungal glucan Polytran L [174] are now available for experimental purposes. Elicitation in cell cultures is used frequently [102,152]. It is clear that the simple drop diffusate technique, used with leaf tissue, is not as widely applicable as originally assumed. Although it works well with legume plants, there are occasional difficulties even with members of this family [ 1391. The technique failed to give a positive response when applied to the Rosaceae, but this was partly because the family is well endowed with constitutive bound toxins [145]. Difficulties with the drop diffusate technique have also been reported with leaves of tropical plant species [175], and phytoalexins could only be induced in leaves of Gramineae by employing other methods [R. Grayer, unpublished results]. Thus, several different inducing techniques, preferably using more than one tissue, are recommended for phytoalexin studies. At one time, it appeared that within a given family, a particular class of phytoalexin was produced, so that legumes provided isoflavonoids, solanaceous plants sesquiterpenoids, etc. Recent research has now shown that several different classes of phytoalexin may be produced within the same family. This is true of the Compositae, Gramineae and Rosaceae (Table 2). In the grass family, no less than five classes of phytoalexin have been detected already. There are even flavanone and diterpenoid phytoalexins formed in one plant, Oryza satiua. As mentioned before, the range of constitutive antifungal compounds in this family is equally wide. The taxonomic aspects of phytoalexin induction and production of constitutive antifungal compounds in plants are therefore quite intriguing and further investigations of antifungal agents in families such as the Gramineae are likely to be very rewarding. Acknowledgements-The

authors are grateful to Mr T. Kokubun for helpful suggestions and allowing us to use his unpublished results, and to Mr C. Grayer for help in the preparation of this article. REFERENCES

1. Ingham, J. L. (1973) Phytopath. Z. 78, 314. 2. Schmidt, M. (1933) Planta (Berlin) 20, 407. 3. Atkinson, P. and Blakeman, J. P. (1982) New Phytologist 92, 93. 4. Kodama, O., Miyakawa, J., Akatsuka, T. and Kiyosawa, S. (1992) Phytochemistry 31, 3807. 5. Cartwright, D. W., Langcake, P., Pryce, R. J., Leworthy, D. P. and Ride, J. P. (1981) Phytochemistry 20, 535. 6. Kato, T., Kabuto, C., Sasaki, N., Tsunagawa, M., Aizawa, H., Fujita, K., Kato, Y., Kitahara, Y. and Takahashi, N. (1973) Tetrahedron Letters 3861.

40

R. J. GRAYERand J. B. HARBORNE

7. Mansfield, J. W. (1983) in Biochemical Plant Pathology (Callow, J. A., ed.), p. 237. Wiley, Chichester. 8. Hegnauer, R. (1986) Chemotaxonomie der Pfunzen, Vol. 7. Birkhauser, Basle. 9. Bailey, J. A. and Mansfield, J. W. (eds) (1982) Phytoalexins. Blackie, Glasgow. 10. Tomas-Lorente, F., Iniesta-Sanmartin, E., TomasBarberan, F. A., Trowitzsch-Kienast, W. and Wray, V. (1989) Phytochemistry 28, 1613. 11. Tom&-Barber&n, F. A., Msonthi, J. D. and Hostettmann, K. (1988) Phytochemistry 27, 753. 12. Crombie, L., Crombie, W. M. L. and Whiting, D. A. (1984) J. Chem. Sot., Chem. Commun. 244 and 246. 13. Wippich, C. and Wink, M. (1985) Experientia 41, 1477. 14. Kato,

35. Kobayashi, K., Nishino, C., Tomita, H. and Fukushima, M. (1987) Phytochemistry 26, 3175. 36. Kubo, I., Himejima, M. and Ying, B.-P. (1991) Phytochemistry 30, 1467. 37. Cruickshank, I. A. M., Perrin, D. R. and Mandryk, M. (1977) Phytopath. Z. 90, 243. 38. Ohtani, K., Mavi, S. and Hostettmann, K. (1993) Phytochemistry 33, 83. 39. Nagata, T., Tsushida, T., Hamaya, E., Enoki, N., Manabe, S. and Nishino, C. (1985) Agric. Biol.

Chem. 49, 1181. 40. Nishino, C., Manabe, S., Enoki, N., Nagata, T., Tsushida, T. and Hamaya, E. (1986) J. Chem. Sot., Chem. Commun. 720. 41. Takechi, M. and Tanaka, Y. (1990) Phytochemistry

T., Yamaguchi, Y., Uyehara, T. and Yokoyama, T. (1983) Tetrahedron Letters 24, 4715. 15. Kato, T., Yamaguchi, Y., Namai, T. and Hirukawa, T. (1993) Biosc. Biotechn. Biochem. 57, 283. 16. Jambunathan, R., Butler, L. G., Bandsyopadkyay, R. and Mughogho, K. (1986) J. Agric. Food Chem.

29, 451. 42. Defago, G. and Kern, H. (1988) Physiol. Plant Path. 22, 29. 43. Allen, E. H. and Kuc, J. (1968) Phytopathology 58, 776. 44. Hamburger, M., Dudan, G., Ramachandran Nair,

34, 425. 17. Spendley,

A. G., Jayaprakasam, R. and Hostettmann, K. (1989) Phytochemistry 28, 1767. 45. Bar-Nun, N. and Meyer, A. M. (1990) Phyto-

P. J., Bird, P. M., Ride, J. P. and ’ Leworthy, D. P. (1982) Phytochemistry 21, 2043. 18. Achenbach, H., Stocker, M. and Constenla, M. A. (1988) Phytochemistry 27, 1835. 19. Ingham, J. L., Tahara, S. and Harborne, J. B. (1983) 2. Naturforsch. 38~. 194. 20. Tahara, S., Ingham, J. L., Nakahara, S., Mizutani, J. and Harborne, J. B. (1984) Phytochemistry 23,1889. 21. Marston, A., Gafner, F., Dossagi, S. F. and Hostettmann, K. (1988) Phytochemistry 27, 1325. 22. Cole, M. D., Bridge, P. D., Dellar, J. E., Fellows, L. E., Cornish, M. C. and Anderson, J. C. (1991) Phytochemistry 30, 1125. 23. Greger, H., Hofer, O., Kahlig, H. and Wurz, G. (1992) Tetrahedron 48, 1209. 24. Greger, H., Zechner, G., Hofer, O., Hadacek, F. and Wurz, G. (1993) Phytochemistry 34, 175. 25. Cohen, Y., Eyal, H., Goldschmidt, Z. and Sklarz, B. (1983) Physiol. Plant Path. 22, 143. 26. Reuveni, M., Tuzun, S., Cole, J. S., Siegel, R., Nesmith, W. C. and Kuc, J. (1987) Physiol. Mol. PIant Path. 30, 441. 27. Neto, G. C., Kono, Y., Hyakutake, H., Watanabe, M., Susuki, Y. and Sakurai, A. (1991) Agric. Biol. Chem. 55, 3097. 28. Picman, A. K. (1984) Biochem. Syst. Ecol. 12, 13. 29. Harborne, J. B. and Baxter, H. (eds) (1993) Phytochemical Dictionary. Taylor and Francis, London.

30. Young, M. C. M., Braga, M. R., Dietrich, S. M. C., Gottlieb, H. E., Trevisan, L. M. V. and da S. Bolzani, V. (1992) Phytochemistry 31, 3433. 3 1. Hegnauer, R. (1964) Chemotaxonomie der PJunzen, Vol. 3. Birkhauser, Basle. 32. Hegnauer, R. (1989) Chemotaxonomie der PJlanzen, Vol. 8. BirkhHuser, Basle. 33. Franich, R. A., Gadgil, P. D. and Shain, L. (1983) Physiol. Plant Path. 23, 183. 34. Fukui, H., Koshimizu, K. and Egawa, H. (1978) Agric. Biol. Chem. 42, 1419.

chemistry 29, 787. 46. Bordoloi, M., Saikia,

B., Mathur, R. K. and Goswami, B. N. (1993) Phytochemistry 34, 583. 47. Greathouse, G. A. and Watkins, G. H. (1938) Am. J. Botany 25, 743. 48. Muirhead, J. F. and Deverall, B. J. (1984) Aust. J. Botany 32, 575. 49. Stoessl, A. and Unwin, C. H. (1969) Can. J. Botany 48,465. 50. McDowell, P. G., Lwande, W., Deans, S. G. and Waterman, P. G. (1988) Phytochemistry 27, 2519. 51. Prusky, D., Keen, N. T. and Eaks, I. (1983) Physiol. Plant Path. 22, 189. 52. Adikaram, N. K. B., Ewing, D. F., Karunaratne, A. M. and Wijeratne, E. M. K. (1992) Phytochemisry 31, 93. 53. Masui, H., Kondo, T. and Kojima, M. (1989) Phytochemistry 28, 2613. 54. Koshino, H., Yoshihara, T., Sakamura, S., Shimanuki, T., Sato, T. and Tajimi, A. (1989) Agric. Biol. Chem. 53, 2527. 55. Namai, T., Kato, T., Yamaguchi, Y. and Hirukawa, T. (1993) Biosc. Biotechn. Biochem. 57, 611.

56. Li, W. X., Kodama,

0. and Akatsuka, T. (1991)

Agric. Biol. Chem. 55, 1041.

57. Mori, A., Enoki, N., Shinozuka, K., Nishino, C. and Fukushima, M. (1987) Agric. Biol. Chem. 51, 3403. 58. Cojocaru, M., Droby, S., Glotter, E., Goldman, A., Gottlieb, H. E., Jacoby, B. and Prusky, D. (1986) Phytochemistry 25, 1093. 59. Endo, K., Kanno, E. and Oshima, Y. (1990) Phytochemistry 29, 797. 60. Prusky, D. and Keen, N. T. (1993) Plant Disease 77, 114. 61. Walker, J. C. and Stahmann, M. A. (1955) A. Rev. Plant Phys. 6, 351. 62. Dix, N. J. (1979) Trans. Br. Mycol. Sot. 73, 329.

Antifungal compounds from higher plants 63. Kuc, J., Henze, R. E., Ullstrup. A. J. and Quackenbush, F. W. (1956) J. .Am. Chem. Sec. 78, 3123. 64. Banthorpe, D. V., Bilyard, H. J. and Brown, G. D. (1989) Phytoehemis~ry 28, 2109. 65. Ito, T. and Kumazawa, K. (1992) B&c. Biotechn. B&hem, 56,16X 66. Kumar, V., Karunaratne, V., Sanath, M. R., Meegalle, K. and MacLeod, J. K. (1990) Phytochemistry 29, 243. 67. Aist, J. R., Gold, R. E., Bayles, C. J., Morrison, G. H., Chandra, S. and Israel, H. W. (1988) Physiol. Mol. Plant Path. 33, 17. 68. Murray, R. D., Mend&z, J. and Brown, S. A, (1982) The Natural Coumarins. Occurrence, Chemistry and Bi~hemistry. Wiley and Sons, Chichester. 69. Miles, D. H., Chittawong, V., Hedin, P. A. and Kokpol, U. (1993) Phytochemistry 32, 1427. 70. Geibel, M., Geiger, H. and Treutter, D. (1990) Phytochemistry 29, 1351. 71. Shain, L. and Miller, J. B. (1982) Phytopathology 72, 877. 72. Parmar, V. S., Vardhan, A., Nagarajan, G. R. and Jain, R. (1992) Phytochemistry 31, 2185. 73. Mizobuchi, S. and Sato, Y. (1984) Agric. Biol. Chem. 48,277l. 74. Miles, 1). H., Rosa de Medeiros, J. M., Chittawong, V., Hedin, P. A., Swithenbank, C. and Lidert, 2. (1991) Phytochemistry 30, 1131. 75. Hunter, M. D. and Hull, L. A. (1993) Phytoc~~s~y 34,12X 76. Sierotzki, H. and Gessler, C. (1993) Physiol. Mol. Plant Path. 42, 291. 77. Karni, L., Prusky, D., Kobiler, I., Bar-Shim, E. and Kobiler, D. (1989) Physiol. Mol. Plant Path. 35,367, 78. Brownlee, H. E., McEuen, A. R., Hedger, J. and Scott, I. M. (1990) Physiol. Mol. Plant Path. 36, 39. 79. Brownlee, H. E., Hedger, J. and Scott, I. M. (1992) Physiol. Mol. Plant Path. 40, 227. 80. Nicholson, R. L., Butler, L. G. and Asquith, T. N. (1986) Phytopathology 76, 1315. 81. Gorham, J. (1980) Progress in Phytochemistry 6,203. 82. Hart, J. H. (1981) A. Rev. P~ytopat~ 19, 437. 83. Schultz, T. P., Hubbard, T. F., Jin, L., Fisher, T. H. and Nicholas, D. D. (1990) Phyt~hemistry 29,1501. 84. Schultz, T. P., Cheng, Q., Boldin, W. D., Hubbard, T. F., Jin, L., Fisher, T. H. and Nicholas, D. D. (1991) Phytochemistry 30,2939. 85. Woodward, S. and Pearce, R. B. (1988) Physiol. Mol. Plant Path. 33, 127. 86. Bokel, N., Diyasena, M. N. C., Gunatilaka, A. A. L., Kraus, W. and Sotheeswaran, S. (1988) Phytochemistry 27, 377. 87. Pezet, R., Pont, V. and Hoang-Van, K. (1991) Physiol. Mol. Plant Path. 39,441. 88. Takasugi, M., Kawashima, S., Monde, K., Katsui, N., Masamune, T. and Shirata, A. (1987) Phytochemistry 26, 371. 89. Malan, E. and Swinny, E. (1993) Phytochemistry 34, 1139. 90. Potterat, O., Stoeckli-Evans, H., Msonthi, J. D. and Hostettmann, K. (1987) Helv. Chim. Acta 70, 155.

41

91. Hedin, P. A., Collum, D. H., Langham, V. E. and Graves, C. H. (1980) J. Agric. Food Chem. 28,340. 92. Ratnayake Bandara, B. M. and Wimalasiri, W. R. (1988) Phytochemistry 27, 225. 93. Ratnayake Bandara, B. M., Hewage, C. M,, Karunaratne, V., Wannigama, P. and Adikaram, N. K. B. (1992) Ph~ochemistry 31, 1983. 94. Orjala, J., Erdelmeier, C. A. J., Wright, A. D., Rali, T. and Sticher, 0. (1993) Phyt~~mistry 34, 813. 95. Marston, A., Hamburger, M., Sordat-Diserens, I., Msonthi, J. D. and Hostettmann, K. (1993) Phytochemistry 33, 809. 96. Brooks, C. J. W. and Watson, D. G. (1985) Natural Product Reports 2, 427, 97. Gottstein, D. and Gross, D. (1992) Trees 6, 55. 98. Harbome, J. B. (1986) in Natural Resistance of Plants to Pests (Green, M. B. and Hedin, P. A., eds), pp. 22-35. American Chem. Sot., Washin~on, D. C. 99. Harborne, J. B. (1993) Natural Product Reports 10, 327. 100. Scheel, D, and Parker, J. E. (1990) Z. Nafurfirsch. 49,564. 101. Tverskoy, L., Dmitriev, A., Kozlovsky, A. and Gradzinsky, D. (1991) Phytoe~m~try 30,799. 102. Pare, P. W., Dmitrieva, N. and Mabry, T. J. (1991) Phytochemistry 30, 1133. 103. Niemann, G. J. (1993) Phytochemistry 34, 319. 104. Takasugi, M. and Katui, N. (1986) Phytochemistry 25, 2751. 105. Tietjen, K. G. and Matern, U. (1984) Arch. Biochem. Biophys. 229, 136. 106. Monde, K., Oya, T., Shirata, A. and Takasugi, M. (1990) Phytochemistry 29,3449. 107. Marshall, P. S., Harbome, J. B. and King, G. S. (1987) Phytochemistry 26, 2493. 108. Tal, B. and Robeson, D. J. (1986) Phytochemistry 25, 77. 109. Takasugi, M., Okinaka, S., Katsui, N., Masamune, E., Shirata, A. and Ohuchi, M. (1985) J. Chem. Sot., Chem. Commun. 621. 110, Tahara, S., Hanawa, F., Harada, Y. and Mizutani, J. (1988) Agric. Bid. Chem. 52, 2947, 111. Ito, I., Kato, N. and Uritani, I. (1984) Agric. Biol. Chem. 48, 1.59. 112. Kumar, S., Shukla, R. S., Singh, K. P., Paxton, J. D. and Husain, A. (1984) Phytopatho~ogy 74, 1349. 113. Takasugi, M., Katsui, N. and Shirata, A. (1986) J. Chem. Sot. Chem. Commun. 1077. 114. Monde, K., Katsui, N., Shirata, A. and Takasugi, M. (1990) Chemical Letters 209. 115. Devys, M., Barbier, M., Lo&let, I., Rouxel, T., Sarguignet, A., Kollmann, A. and Bousquet, I. F. (1988) Tetrahedron Letters 29, 6447. 116. Dahiya, J. S. and Rimmur, S. R. (1988) Phytochemistry 27,3105. 117. Browne, L. M., Conn, K. L., Ayer, W. A. and Tewari, I. P. (1991) Tetrahedron 47,3909. 118. Takasugi, M., Monde, K., Katsui, N. and Shirata, A. (1987) Chemical Letters 1631. 119. Adesanya, S. A., Ogundana, S. K. and Roberts, M. F. (1989) Phyr~hemistry 28, 773.

42

R. J. GRAYER and J. B. HARBORNE

120. Fagboun, D. E., Ogundana, S. K., Adesanya, S. A. and Roberts, M. F. (1987) Phytochemistry 26, 3187. 121. Giesemann, A., Biehl, B. and Lieberei, R. (1986) J. Phycopath. 17, 373. 122. Crombie, L. and Mistry, J. (1990) Tetrahedron Letters 31, 2647. 123. Powell, R. G., TePacke,

M. R., Plattner,

White, J. F. and Clement,

R. D., S. L, (1994) Phyto-

chemistry 35, 335. 124. Kodama,

O., Suzuki, suka, T. (1988) Agric. 125. Sekido, H., Endo, T., suka, T., Kono, Y.

T., Miyakawa, J. and AkatBiol. Chem. 52,2469.

Suga, R., Kodama, O., Akatand Takeuchi, S. (1986) f.

Pesticide Sci. 11, 369. 126. Kato, H., Kodama, 0. and Akatsuka, T. (1993) Phytochemistry 33, 79. 127. Tamegami, S., Mitani, M., Kodama, 0. and Akatsuka, T. (1993) Tetrahedron 49, 2025. 128. Brinker, A. M. and Seigfer, D. S. (1991) Phytochemistry 30, 3229. 129. Godshall, M. A. and Lonergan, T. A. (1987) Physiol. Mol. Plant Path. 30, 299. 130. Nicholson, R. L., Kollinara, S. S., Vincent, S. R,, Lyons, P. C. and Gomez, G. C. (1987) Proc. Nat. Acad. Sci. U.S.A. 84, 5520. 131. Biicker, C. and Grambow, H. J. (1990) Z. Naturjbrsch. 4Sc, 11.51.

132. Strange, R. N., Ingham, J. L., Cole, D. L,, Cavil], M. E., Edwards, C., Cooksey, C. J. and Garratt, R. J. (1985) 2. Naturforsch. &, 313. 133. Sharon, A., Ghirlando, R. and Gressel, 5. (1992) Plant Physio~. 92, 303. 134. Ingham, J. L. and Tahara, S. (1985) Z. Naturforsch. 4Oc, 482. 135. Ingham, J. L. and Dewick, P. M, (1984) Z. Naturforsch. 39c, 531. 136. Ingham, J. L. and Tahara, S. (1983) Z. ~atur~rsch. 3%c, 899. 137. Ingham, J. L. and Markham, K. R. (1984) Z. Naturforsch. 3!k, 13. 138. Xngham, J. L., Tahara, S. and Dziedzic, S. 2. (1986) J. Nat. Prod. 49, 631. 139. Seneviratne, G. I. and Harborne, J. B. (1992) Biothem. Syst. Ecol. 20,459. 140. Monde, K., Kishimoto, M. and Takasugi, M. (1992) Tetrahedron Letters 33, 5395. 141. Hanawa, F., Tahara, S. and Mizutani, J. (1992) Phytochemistry 31, 3005. 142. Bell, A. A., Mace, M. E, and Stipanovic, R. D. (1986) ACS Symp. Ser. 2%, 36. 143. Cline, S. D. and Coscia, C. J. (1988) Plant Physiol. 86, 161.

144. Franich, R. A., Carson, M. J. and Carson, S. D. (1986) Physiol. Mol. Plant Path. 28, 267. 145. Kokubun, T. and Harborne, J. 3. (1994) Z, Natur@sch. (in press). 146. Burden, R. S., Kemp, M. S., Wiltshire, C. W. and Owen, J. D. (1984) J. Chem. Sot. Perkin Trans. I 1445. 147. Watanabe,

K., Ishiguri, Y., Nonaka, F. and Morita, A. (1982) Agric. Biol. Chem. 46, 567.

148. Widyastuti, S. M., Nonaka, F., Watanabe, K., Sako, N. and Tanaka, K. (1992) Ann. Phytopath. Sot. Japan 58, 228. 149. Kemp, M. S. and Burden, R. S. (1984) J. Chem. Sot. Perkin Trans. I 1441. 150. Watanabe, K., Widyastuti, S. M. and Nonaka, F. (1990) Agric. Biol. Chem. 54, 1861.

151. Kokubun, T., Harbome, J. B. and Eagles, J. (1994) Phytochemistry 35, 33 1. 152. Wijnsma, R., Go, J. T., van Weerden, I. N., Harkes, P. A. A., Verpoorte, R. and Svendsen, A. B. (1985) Plant Cell Rep. 4, 241. 153. Wijnsma, R., van Weerden, 1. N. and Verpoorte, R. (1986) Planta Med. 52, 21 t. 154. Vernenghi, A., Ramiandrasoa, F., Chuilon, S. and Ravise, H. (1987) Fruits (Paris) 42, 103. 155. Afek, V. and Sztejnberg, A. (1988) Phytopathology 78, 1678. 156. Shoyama, Y., Matsumato, M. and Nishioka, 1. {t987) Phytochemistry 26, 983. 157. Burden, R. S. and Kemp, M. S. (1983) Phytochemistry 22, 1039.

158. Dumas, M. T., Strunz, G. M., Hubbes, M. and Jeng, R. S. (1983) Experientia 39, 1089. 159. Burden, R. S. and Kemp, M. S. (1984) Phytochemistry 23, 383.

160. Knogge, W., Kombrink, E., Schmelzer, E. and Hahlbrock, K. (1987) Planta 171, 279. 161. Sutton, D. C., Gillan, F. T. and Susie, M. (1985) Phytochemistry 24, 2877. t62. Ingham. J. L. (198 1) Advances in Legume Systematics 1, 599. 163. Harbome, J. B, and Turner, B. L. (1984) Plant Chemosystematics. Academic Press, London. 164. Ingham, J. L. (1990) Biochem. Syst. Ecol. 18,329, 165. Abe, N., Sato, H. and Sakamura, S. (1987) Agric. B&l. Chem. 51, 349. 166. Hain, R., Reif, H. J., Krause, E., Langebartels, R.,

Kind], H., Vornau, B., Wiese, W., Schmetzer, E. and Schreier, P. H. (1993) Nature 361, 153. 167. Snyder, B. A. and Nicholson, R. L. (1990) Science 248, 1637. 168. Devys, M. and Barbier, M. (1992) Z. Naturfirsch. 47c, 318. 169. Koritsas, V. M., Lewis, J. A. and Fenwick, G. R. (1989) Experientia 49, 493.

170. Mayama, S., Hayashi, S., Yamamoto, R., Tani, T., Ueno, T. and Fukami, H. (1982) Physiol. P/ant Path. 20, 305. 171. Stiissel, P. (1985) Phys~o~.Plant Path. t6, 269. 172. Arnofdi, A. and Merlini, L. (199s) J. Agr. Food Chem. 38, 834. 173. Arnoldi, A., Farina, G., Galli, R., Merlini, L. and Parrino, M. G. (1986) J. Agric. Food Chem. 34, 185. 174, Bhandal, I. S. and Paxton, J. D. (1991) J. Agric. Food

Chem. 39,2156. 17.5. Braga, M. R., Young, C. M., Ponto, J. V. A., Dietrich, S. M. C., Emerenciano, V. de P. and Gottlieb. 0. R. (1986) Biochem. Syst. Ecol. 14, 507.