terpenos a hongos.pdf

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 COMPOUNDS FROM HIGHER PLANTS, 1982-1993

RENBE J. GRAYER and JEFFREY B. HARBORNE

Department of Botany, University of Reading, Whiteknights, Reading RG6 2AS, U.K.

(Received 30 March 1994)

IN HONOUR OF PROFESSOR ROBERT HEGNAUER’S SEVENTY-FIFTH BIRTHDAY

Key Word Index-Flowering plants; antifungal agents; constitutive compounds; phytoalexins; secondary 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

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

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 com- partment. Damage or infection of the plant brings to- gether 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. More- over, 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 dis- tinguished between preformed antifungal compounds and phytoalexins, but the former are not further sub- divided into prohibitins, inhibitins and post-inhibitins, since it is not always possible to infer from the data given

19

20 R. J. GRAYER and J. B. HARBORNE

2

COCMS OH Ii

*

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

&4 ‘/ P R’

\ ‘m

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 pre- infectional 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 Heg- nauer [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

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 Solana- ceae; isoflavonoid phytoalexins in Leguminosae [9]), and to see whether any structure-activity relationships are apparent.

Taxonomic distribution

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 Heli- chrysum 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 (Ruta- ceae) 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-

Tab

le

1. C

onst

itutiv

e an

tifun

gal

com

poun

ds

repv

rted

si

nce

1982

whi

ch m

ay p

lay

a ro

le i

n pl

ant

resi

stan

ce

Plan

t fa

mily

sp

ecie

s or

gan

Com

poun

d(s)

C

hem

ical

cla

ss

Mic

roor

gani

sm

stud

ied

Ref

eren

ce

Ana

card

iace

ae

Bur

sera

ceae

Can

nabi

dace

ae

Com

bret

acea

e

Ma

ng

ijkra

in

dica

Pe

el a

nd f

lesh

of

5-(1

2-ci

s-H

epta

dece

nyl~

reso

rcin

ol(2

2);

(maw

) un

ripe

fru

its

5pen

tade

cylr

esvr

cino

l (2

3)

Com

mip

hora

ros

trat

a St

em b

ark

2Dec

anon

e;

2.un

deca

none

; 2-

dode

canv

ne

Hum

ulus

lup

ulus

(ho

p)

Res

in

Com

bret

um

~p

i~u

~a

t~

Hea

rtw

ood

Com

posi

tae

Eup

ator

ium

rip

arit

an

Roo

ts

Hel

ichr

ysum

dec

umbe

ns

Lea

f su

rfac

e ~

elic

hrys

~

nite

ns

Lea

f su

rfac

e

Cuc

urbi

tace

ae

Cup

ress

acea

e D

iosc

orea

ceae

Wed

elia

bij

lora

E

cbal

lium

ela

teri

um

Cha

mae

cypa

ris

pisi

fera

D

iosc

orea

bat

atas

(C

hine

se y

am)

Leaf

sur

face

Fr

uit

Lea

ves

and

twig

s T

uber

Dip

tero

carp

a-

ceae

E

uphv

rbia

ceae

Ste

mon

opom

B

ark

Gra

min

eae

Aoe

na sa

tiva

(oat

) R

oot

Hor

~m

m

&ar

e (b

arle

y)

Lea

vef

Papi

llae

of

cvle

optil

e in

ner

epid

erm

al

cells

O

ryza

ofi

cina

lis

LMV

eS

Alk

ylat

ed p

heno

l A

lter

nari

a al

tern

ata

58

Alk

anon

e

6-Is

open

teny

lnar

inge

nin

(33)

; xa

nthv

hum

ol(3

5)

4,7-

Dih

ydro

xy-2

,3,4

t~m

etho

xyph

enan

thr~

e (6

3);

2,7-

dihy

drox

y-3,

4,6-

trim

ethv

xydi

hydr

ophe

nant

hren

e (4

4);

4,4’

-dih

ydro

xy-3

,%%

met

hoxy

dihy

dros

tilbe

ne

(42)

M

ethy

hipa

rioc

hrom

ene

A

Flav

anon

e C

hakv

ue

Phen

~thr

ene

Asp

ergi

llus

and

50

P

enic

illi

um s

peci

es

Tri

chop

hyto

n ru

brum

73

T

. m

enta

grop

hyte

s P

~~

illi

~

expa

nsum

89

Dih

ydro

stilb

ene

Chr

omen

e

Phlo

rogl

ucin

ol

deri

vativ

es

(25-

27)

Chr

ysin

di

met

hyl

ethe

r (3

0);

gala

&n

trim

ethy

l et

her

(31)

; ba

icaI

in t

rim

ethy

l et

her

(32k

fi

ve m

ore

higb

iy m

etho

xyla

ted

flav

onoi

ds

7,3’

Di-

O-m

ethy

lque

rcet

in

Cuc

urbi

taci

n I

Pisi

fcri

c ac

id

3-H

ydro

xy-S

-met

hoxy

~~~l

3,

~~ih

ydrv

xy-~

met

hoxy

bi~~

l (b

atat

asin

IV

) (4

0);

6-hy

drvx

y-2,

4,7-

trim

ethv

xyph

enan

thre

ne

(bat

atas

in

I) (

41k

6,7_

dihy

drox

y-2,

4-di

met

hoxy

- ph

enan

thre

ne;

2,7d

ihyd

roxy

+-di

met

hoxy

ph

~~~~

ne

Can

alic

ulat

ol

(39)

Pren

ylat

ed

phen

ol

Met

hvxy

late

d lia

vone

an

d fl

avon

vl

Col

leto

tric

hum

93

gl

oeos

pori

oide

s C

lado

spor

ium

he

rbar

um

10

CIa

dOSp

O?+

iiwn

Ii

ctic

un&

mua

Flav

onol

R

hizo

cton

ia s

olan

i 69

C

ucur

bita

cin

Rvt

rytis

ciw

ea

45

Dite

rpen

e P

yrku

lari

a or

yzae

35

O

xyge

nate

d bi

benz

yl

Exs

min

ed

agai

nst

24 f

ungi

88

Phen

anth

rene

2,6_

Dim

ethv

xybe

nxvq

uino

ne

(45)

Ave

naci

ns

Gra

min

e (1

8)

Uni

dent

ifie

d

Stilb

ene

trim

er

Ben

xoqu

inon

e

Tri

terp

enoi

d sa

poni

n

Indo

le a

lkal

oid

Phen

ylpr

opan

vid

Cla

dosp

0riu

m

clud

ospo

ri0i

des

Cl&

spor

ium

cl

ados

pori

oide

s

Geu

man

nom

yces

gr

amin

is v

ar.

triti

ci

Rry

siph

e pe

nis

f.sp.

ho

rdei

E

rysi

phe

gram

inis

tsp

. ho

rdei

86

92

12

13

67

Jasm

onic

ac

id (

2)

Mod

ifie

d fa

tty a

cid

Pyr

kula

ria

oryz

ae

27

k &

8 t

Plan

t fa

mily

Sp

ecie

s O

rgan

C

ompo

und(

s)

Tab

le

1. C

ontin

ued

Che

mic

al c

lass

B

~icr

~rga

~ st

udie

d R

efer

ence

Ory

za s

ativ

a (r

ice)

Sorg

hum

culti

vars

Tri

ticu

m ae

stiv

um

(whe

at)

Gro

ssul

aria

ceae

R

ibes

nig

rnm

(r

edcu

rran

t)

Lab

iata

e R

osm

arin

us o#

cina

lis

and

othe

r L

abia

tae

Scut

ella

riu o

iola

cea

S. w

oron

owii

L

aura

ceae

E

uodi

o lu

nu-u

nke~

a

Per

sea

a~ri

cana

(a

voca

do)

Leg

umin

osae

B

~~

h~ni

~ man

ca

Dol

icho

s ki

lim

~sc

hari

cus

Lup

inus

albu

s

Mel

iace

ae

Chi

soch

eton

pa

n~cu

latu

s

Mol

lugi

nace

ae

Mol

lugo

pen

taph

ylla

Mus

acea

e M

uss

(ban

ana)

Lea

f su

rfac

e

Lea

ves

and

grai

ns

Lea

ves

Lea

f gl

ands

of

adax

ial

surf

ace

Lea

ves

and

callu

s cu

lture

-s

Aer

ial

part

s

Roo

t ba

rk

Peel

of

unri

pe f

ruit

Peel

of

unri

pe f

ruit

woo

d

Roo

ts

Lea

f su

rfac

e

Roo

ts

Frui

ts

Aer

ial

part

s M

ollu

geno

l A

(16

)

Unr

ipe

frui

t pe

el

Dop

amin

e (o

xida

tion

prod

ucts

)

Epo

xy-

and

hydr

oxyl

inol

e~~

acid

s (e

.g. 2

0 an

d 21

) ep

oxy-

and

hyd

roxy

linol

enic

ac

ids

Flav

an-4

-01s

a-T

ritic

ene;

/W

itice

ne

Saku

rane

tin

2-(3

,4-R

ihyd

roxy

phen

yl)

ethe

nyl

este

rs o

f ca

ffei

c ac

id (

24)

Cle

rodi

n (1

0); j

odre

llin

B (

II)

1-[2

’,4’

-Dih

ydro

xy-6

(3”-

met

hyl-

2”-b

uten

ylox

y~~(

~-m

ethy

l-

~-bu

teny

l)]p

heny

leth

anon

e an

d re

late

d co

mpo

und

(2%

29)

cis,

cis

l-A

~tox

y-2-

hydr

oxy~

oxo-

h~ei

co~-

l2,1

5~~e

ne

1,2,

4-T

~hyd

roxy

hept

adec

-16-

yne;

l~

,~t~

hydr

oxyh

~tad

~i6~

n~

l-a~

toxy

-2,~

dihy

drox

y-he

ptad

~-l~

yne

Isol

iqui

~tig

enin

; is

oliq

ui~t

igea

in~-

ethy

l et

her;

ech

inat

in

(2S~

7,~-

Dih

ydro

xyfl

avan

; (2

S~3,

~-di

hydr

oxy-

7-m

etho

xyfl

avan

; (2

~7,~

-~ih

ydro

x~~-

met

hox~

avan

~ O

btus

tyre

ne

J-O

-glu

cosi

des

of h

eder

agen

in

(12)

, bay

ogen

in

(13)

and

med

icag

enic

ac

id (

14)

Lut

eone

(3

7); w

ight

eone

(3

8)

Lut

eone

(3

7); w

ight

eone

(3

8); l

icoi

sofl

avon

es

A a

nd S

; pa

rvis

ofla

vone

B

1,

2-D

ihyd

roxy

-6a-

acet

oxya

zadi

rone

an

d th

ree

sim

ilar

mel

i~in

s

Fatty

ac

id

~uco

a~th

~y~i

~n

Alk

adie

naI

Flav

anon

e

Eno

l es

ter

of h

ydro

xy-

cinn

amic

ac

id

Neo

-cle

roda

ne

dite

rpen

- oi

d Ph

enyl

etha

none

Lon

g-ch

ain

alco

hol

Lon

g-ch

ain

alco

hol

Cha

lcon

e

Flav

an

Dip

heny

lpro

pene

T

rite

rpen

oid

sapo

nin

Isot

lavo

ne

Isof

lavo

ne

~elia

cin-

ty~

nort

rite

r-

peno

id

Tri

terp

enoi

d

Am

ine

Pyr

lcul

aria

oryz

ae

Fusa

rium

mon

ilifo

rme;

C

urvu

lari

a lun

ata

~l~

ospo

rium

cu

cum

erin

um

Bot

ryti

s cin

erea

Cia

dosp

oriu

m he

rbar

um

Fusa

rium

oxy

spor

um ts

p.

lyco

pers

ici

~l~

os~

rium

cl

ado-

sp

orio

ides

~o~

leto

~ic

hum

gl

oeos

~ri

o~es

C

lado

spor

ium

~

l~os

pori

oide

s

Bot

ryti

s cin

erea

; Sa

prol

e0n~

ast

erop

hora

an

d th

ree

othe

r fu

ngi

Cla

dosp

oriu

m

cucu

mer

inum

~

elrn

int~

s~ri

~

carb

onur

n G

lado

spor

ium

herb

arum

~~

v~l~

ia

verr

ucif

onni

s;

Dre

schf

era o

ryza

e;

Alt

erna

ria s

olan

i ~

l~os

pori

um

cucu

mer

htum

C

olle

totr

ichu

m m

usae

14,1

5,55

16

17

3 64

22

F

66

x $

51

d B

a

21

19

20

46

44

48

M y

rsin

a~ae

Myr

tace

ae

Peda

liace

ae

Pina

ceae

Psi

dium

acu

tang

ulum

Sesa

mum

ang

olen

se

Pic

ea s

itch

ensi

s (s

itka

spru

ce)

Pin

us r

adia

ta

Leaves

Tw

ings

and

lea

ves

Roo

t ba

rk

Bar

k

Nee

dle

surf

ace

Pipe

race

ae

Pip

er a

dunc

um

Lea

ves

Poly

gala

ceae

P

olyg

ala

ny~

kens

~s

Roo

ts

Ros

aeea

e P

rune

s ye

doen

sis

Lea

ves

Rub

iace

ae

Alib

erti

a m

acro

phyl

ia

Lea

ves

Rut

acea

e

Rut

acea

e

Sahc

acea

e So

lana

~e

Gly

cosm

is c

yano

carp

a L

eave

s

Gly

cosm

is m

auri

tian

a L

eave

s

Pop

ulus

del

toid

es

Ly&

oper

si~

on

escu

lent

um (t

omat

o)

~~

cotj

a~

giut

i~sa

Lea

f gl

ands

G

reen

fr

uits

Epi

cutic

uiar

Ie

af

wax

L

eaf

surf

ace

Ster

culia

ceae

The

acea

e Z

ingi

bera

eeae

Nic

otia

na

taba

cum

(t

obac

co)

The

obro

ma

caca

o (c

ocoa

) C

amel

ha ja

poni

ca

Zin

gibe

r o@

cina

le

(gin

&

I%&

sh

oot

tissu

e

Lea

f R

hizo

mes

Saku

raso

sapo

nin

(15)

3’-F

orm

yl-2

’,4’

,6’-

trih

ydro

xy-d

ihyd

roch

alc

(36)

Nap

htho

xire

nes

Ast

ring

in;

rhap

ontic

in

Stea

ric

acid

; ~H

ydro

xyd~

e~no

ic

acid

; ~-

hydr

oxyt

etra

de~n

oic

acid

; ~~

ydro

xyhe

xade

~oi~

ac

id

7-K

et~e

hydr

oabi

eti~

ac

id (

St;

7-hy

drox

yde~

ydro

abie

ti~

acid

(6)

; lS-

bydr

oxyp

odoc

arpi

c ac

id (

7)

Met

hyl

8-hy

drox

~~2-

dim

ethy

l-2H

~hro

men

e.~

carb

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ate the structure-activity relationships of the compounds ium spp. No less than 62% of the sesquiterpene lactones rather than chemical interactions between plant taxa and tested showed at least weak activity against the former two their pathogenic fungi. For this reason this study has not fungi, and nearly half of those inhibited them strongly. been included in Table 1, but is discussed in the next However, Fusarium was only inhibited weakly by 13% of section. Papers on the antifungal activity of plant consti- the compounds tested. The antifungal activity of the tuents against fungi pathogenic to humans generally have different skeletal classes of sesquiterpene lactones was not been included in the table either. On the whole our also compared, and the eudesmanolides came out as the knowledge of the distribution of constitutive antifungal group showing the highest proportion of strongly active compounds in higher plants appear to be very frag- lactones, whereas the germacranolides had the highest mentary, and it would be well worth doing more chemo- proportion of inactive compounds. The natural role of taxonomic screening in this respect. This can lead to constitutive sesquiterpenoid lactones in the resistance of interesting results, as has been revealed by a recent survey species of the Compositae against their plant pathogens in our laboratory of the antifungal substances in the does not seem to have been investigated, however, but Rosaceae. A wide range of preformed constituents, espe- from the results of the above test and the fact that several cially of phenolic origin, was found to be involved in the composite species have recently been found to produce protection of this plant family against fungal pathogens sesquiterpene lactone phytoalexins (see below), this may (T. Kokubun, unpublished results). be an important one.

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 (Cupressa- ceae). 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, plumer- icin and plumieride from Plumeria species (Apocynaceae)

PI. In contrast to sesquiterpenoid phytoalexins which are

characteristic of the plant families Solanaceae, Convol- vulaceae 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 Micro- sporum cookei, Trichophyton mentagrophytes and Fusar-

Many diterpenoids show antifungal activity; some of these are phytoalexins (e.g. the momilactones and oryzal- exins 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-ketodehy- droabietic 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 di- lactone, 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 diter- penes have also been found as constitutive antifungal compounds in species of Angiospermae. The diterpene 2- ketoepimanool (= 13(S)-hydroxylabda-8(20),14-dien-2- one, 9) was isolated from epicuticular leaf waxes of Nicotiana glutinosa (Solanaceae), a tobacco species im- mune 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_duvatriene- 1,3-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 jodrel- lin 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].

Antifungal compounds from higher plants 25

5Tff -I ’

\ \ H

8

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

OH 0 fl I H

H

9

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11

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 melanop- hloeos (Myrsinaceae). The saponins from Dolichos were identified as the 3-0-/l-D-glucopyranosides of hederag-

12 R~=EI,R~=~~OH

13 RI = OH, R2 = CF120H

14 R* =OH,R2-COOH

16


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 tl- hederin from ivy (Hedera helix, Araliaceae), showed highest antifungal activity [41]. Earlier reports of antifungal triterpenoid saponins include aescin from the horse- chestnut, Aesculus hippocastanum (Hippocastanaceae) and yiamoloside B from Phytolacca octandra (Phytolac- caceae) [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 Solana- ceae, 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 Chiso- cheton paniculatus (Meliaceae). When applied to cucum- ber 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 Drec- shlera oryzae, and the tomato pathogen Alternaria solani

1461. Nitrogen-containing compounds. There are many re-

ports of alkaloids showing activity against human fungal pathogens, e.g. the isoquinoline alkaloid jatrorrhizine which occurs in Berberidaceae, Ranunculaceae and Mag- noliaceae, 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

Cl$-N / \

0

o”Jc I’ NWS\ H

19

5?=- 21

Ho

7 1’ dn

22 R-(CE2)11-QI=~-(Ca,b-CR3

23 R = @2)14-3

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-


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]. Fur- thermore, 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 cyano- genie 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 hydro- gen 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 micro- organisms as well, they may additionally protect the plants in which they occur against certain fungi.

Aliphatic compounds. The simple alkanones 2-decan- one, 2-undecanone and 2-dodecanone, present in the volatile resin exudate from the stem bark of Commiphora rostrata (Burseraceae), have been reported to have con- siderable antifungal activity against a number of Asper- gillus 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 avoca- dos [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].

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 hydroper- oxidases 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 13- hydroxides 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.


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, Zingibera- ceae) 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 broom- rape (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 Clados- porium 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’ o--R !?I-

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 chlorotetracyc- line made the mildew resistance disappear. Autofluore- scence, 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,


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 Cladospor- ium 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. [ 1 l] 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, secre- ted 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 Rosa- ceae, e.g. Amelanchier o&is, Prunus lusitanica [T. Koku- bun, 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 Botry- tis 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-isopen- tenyl 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 oppo- sing views as to whether dihydrochalcones such as phlor- etin 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


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 mould- resistant 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 basi- diospores 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 com- plexes 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 glyco- protein fraction of which has an exceptionally high affinity for binding to purified condensed tannins, and so protects spores from inhibition of germination by poly- phenols [80].

Although flavans are occasionally produced by higher plants as phytoalexins, some occur constitutively, for example (2S)-7,4’-dihydroxyflavan, (2S)-3’,4’-dihydroxy- 7-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 5- glucoside, 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 Cla- dosporium herbarum; luteone and licoisoflavone A showed highest fungitoxicity, followed by wighteone, parviso- flavone 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 moder- ate activity against these fungi, but no structural require- ment 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_D- glucoside) which are present in the bark of sitka spruce (Picea sitchensis, Pinaceae). Although the glucosides are antifungal themselves, they decrease after fungal chal- lenge, whereas the corresponding aglycones, which have a much higher activity, accumulate [85]. Stilbene oligo- mers 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_trimethoxyphen- anthrene (= 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% 4,7- dihydroxy-2,3,6_trimethoxyphenanthrene (43), and 2,7- dihydroxy-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 naph- thoquinonoid 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.


o--h- \ - - / Ho OMe OH

42

rT?k \-ohb - ohle

0

Me0

‘o- OMe

45

Chromenes are a further class of aromatics containing antifungal representatives. Methylripariochromene A (6- acetyl-7,8-dimethoxy-2,Zdimethylchromene), a root constituent of Eupatoriwm ripariwm (Compositae), dis- played 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].

47

3

G ‘IO’ Ho

48

Finally, two xanthones out of four isolated from the roots of Polygala nyikensis (Polygalaceae), 1,7- dihydroxy-4-methoxyxanthone (48) and 1,7-dihydroxy- 3,5,6-trimethoxyxanthone (49), exhibited an antifungal activity against the plant pathogenic fungus Cladospor- ium 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 Wat- son [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 regu- larities that are present in these distribution patterns. The chemical structures uncovered during the period under

PHYTO 37:1-D

Tab

le

2. P

hyto

alex

ins

repo

rted

in

pfa

nts

sinc

e 19

82

Alh

acea

e

Cac

tace

ae

Car

yo~h

ylIa

~ae

Cer

cidi

phyI

lace

ae

Com

posi

tae

Con

volv

ui~e

ae

Cos

tace

ae

Cru

cife

rae

Dio

scor

eace

ae

Eup

horb

iace

ae

Gra

min

eae

A&

urn

cepa

Cep

hala

cere

us s

et&

s D

iant

bus

cury

ophy

llus

Mel

a~r~

um ji

rmum

C

~cl

diph

ylfu

m

ja~

o~i~

urn

Car

tham

us t

i~to

rius

C

icho

rium

~nt

y&us

C

oleo

step

hu~

myc

onis

H

etia

nthu

s an

nuw

L

actu

ca s

ativ

a T

arax

acum

ofic

inal

e lp

omoe

a ba

tata

s C

ostu

s spe

cios

us

Bra

ssic

a ~

ampe

stri

s

Bra

s&a

junc

ea

Bra

s&a

napu

s

Cam

elin

a sa

tiva

Dio

scor

ea b

atat

as

D. b

ulb~

era

0. d

u~nt

or~

D

. rot

unda

ta

Hev

ea b

r~i~

iens

is

Ave

na s

ativ

a Fe

stuc

a ve

rsut

a O

ryza

sat

iva

Bul

b

cell

cultu

re

Leaf

Lea

f C

orte

x

Lea

f L

eaf

Lea

f L

eaf

Lea

f L

eaf

Tub

er

Lea

f L

eaf

Leaf

Lea

f

Lea

f

Lea

f

Bul

biI

Tub

er

Tub

er

Lea

f L

eaf

Lea

f L

eaf

~-~e

xylc

yclo

~nta

-1,3

~~on

e (5

3);

5-~y

lcyc

io~n

ta-l

,3-d

ione

(5

4)

4,5-

~eth

ylen

edio

x~6”

bydr

oxya

nron

e (5

@)

Dia

ntha

lexi

ns

(3) (

e.g.

73)

; di

anth

ram

ides

(2

7) (e

.g. 7

4)

N-p

-Hyd

roxy

benz

oyl-

S-hy

drox

yadh

fani

lic

acid

M

a~ol

o~

Safy

nol;

dehy

dros

afyn

ol

Cic

hora

bxin

(7

8)

Myc

osin

ol(8

1)

Scop

olet

in;

Aya

pin

Cos

tuno

~ide

(77

); ~

ettu

~in

A (

80)

Let

tuce

nin

A (

80)

~squ

iter~

nes

Al

and

A2

Gly

ceol

iins

II a

nd I

II

Spir

obra

~ini

n (6

1); c

ycl~

br~s

~~n

(59)

; ox

ymet

hoxy

bras

sini

n (6

2);

met

hoxy

bras

sini

n (5

8); b

rass

inin

(5

7); d

~oxy

bras

. si

nin

(70)

; bra

&ca

nals

A

-C

(63-

65)

Cyc

fobr

assi

nin

su~p

boxi

de (

60);

br

assi

lexi

n (6

7)

Met

haxy

bras

sini

n (5

8);

cycl

obra

ssin

in

(59)

C

amal

exin

(6

8);

met

boxy

~mal

exin

(6

9)

Spir

obra

ssin

in

(61)

; met

hoxy

bras

s~in

(5

8);

bras

&in

(5

7); o

xym

etho

xybr

assi

ni~

(62)

D

~hyd

ropi

nosy

~vin

~m

ethy

Ibat

atas

i~

IV

Dih

ydro

resv

erat

ro~

Bat

atas

in

IV,

dihy

drop

inos

ylvi

n Sc

o~~e

tin

Ave

nalu

m~n

I (

76),

II a

nd I

f1

Res

vera

trol

Sa

kura

netin

(1

) M

omiI

acto

nes

A a

nd 8

; or

yzal

exin

s A

-E,

oryz

alex

in

S

Cyc

lic d

ione

Aur

one

Ant

hran

ihc

acid

Bip

heny

l

Ace

tyle

nic

Sesq

uite

rpen

e la

cton

e A

~tyl

enic

C

oum

arin

Se

squi

terp

ene

fact

one

~squ

iter~

ne

lact

one

Sesq

uite

rpen

e Pt

eroc

arpa

n In

dole

Indo

le

Indo

le

116

Indo

ie

Bib

enzy

i B

iben

zyl

Bib

cnzy

l B

iben

zyt

Cou

mar

in

Ant

hran

ilic

acid

St

iiben

e Pl

avan

one

102

103

103

104

105

106

107

108

109

110

111

112

113,

114

115

117

118

88

II9

119

120

121

I22

123 4

124,

125

, 12

6,12

7

L.e

gum

inos

ae

Lili

acea

e

Mah

acea

e Pa

pave

race

ae

Pina

ceae

R

osac

eae

Rub

iace

ae

Rut

acea

e ~o

pbul

a~~a

e T

iliae

eae

Ulm

aeea

e

Um

belli

fera

e

Ver

bena

-e

slrc

char

um o~

cina

rum

Sorg

hum

bic

olor

Tri

ticum

aes

tivum

A

rach

is h

ypog

aea

Cas

sia o

bt~

~o~

~

~al

o~go

~~

m

ucun

oide

s D

esm

odiu

m

gang

et~

~

Dip

hysa

robi

n~id

es

Nis

so~~

~tic

osa

Shnt

eria

ves

rita

V

igna

spp

. L

ilium

max

imow

czii

Ver

atru

m ~

a~ip

or~

~

ossy

pium

spp.

P

apav

er b

r~te

atum

Pi

nus

radi

ata

Aro

Ga

arbu

t$oE

a; C

~e~

~&

s ca

thay

ensi

f; C. j

apon

ica

Cot

onea

ster

lact

ea;

C. a

cuti

foli

w, C

. div

aric

ata

Eri

obot

rya j

apon

ica

Mal

us ~

urnt

~a

Mes

pilu

s ger

man

ica

Pho

tini

a &br

a P

yrus

com

mnn

is

~ha

p~o~

epis

~

beil

ata

Sang

uiso

r~ m

inor

C

inch

ona

ledg

er&

urn

Cit

rus l

imon

R

ehm

anni

a giu

tinos

a R

lia

x eu

ropa

ea

Ulm

us am

eric

ana

U. g

labr

a A

pium

grav

eole

ns;

Pet

rose

linu

m cr

ispu

m

Avi

cenn

ia m

arin

a

Leaf

Leaf

Leaf

Le

af

Leaf

Lea

f L

eaf

Leaf

Le

af

Lea

f Se

ed

Bul

b L

eaf

Cel

l cu

lture

N

eedl

e Sa

pwoo

d

Sapw

ood

Lea

f Sa

pwoo

d Sa

pwoo

d L

eaf

Sapw

ood

Lea

f

Roo

t B

ark;

cel

l cu

lture

R

oot

bark

R

oot

Sapw

ood

Sapw

ood

Sapw

ood

Lea

f

Wou

nd

tissu

e

Pice

acan

nol(

52)

Lut

~~id

in

APi

ge~~

d~

5~ff

e~la

ra~m

osi~

, lu

teot

inid

in

HD

IBO

A

&rc

osid

e M

edie

arpi

n 56

Ison

~ra~

teno

~ de

met

hylm

~i~a

~in,

et

c.

Des

moc

arpi

n;

kiev

itone

, et

c.

Dip

hyso

lone

; ki

evito

ne;

ferr

erei

n N

issi

carp

in;

frut

icar

pin;

ni

ssol

icar

pin

Fura

n~ih

ydro

k~m

pf~o

l D

albe

rgio

idin

, ki

evito

ne;

phas

eolli

din

Yur

enoh

de

(55)

R

esve

ratr

ol

Hem

igos

sypo

l Sa

n8ui

nari

ne (5

1)

Ren

zoic

aci

d A

~upa

~n

(St)

; r-

and

~-m

e~ox

yauc

upa~

n (8

3,8@

c+

and

B-C

oton

efur

an

Eri

obof

uran

A

u~up

a~n;

~-m

etho

xyau

cupa

~n

a~to

n~ur

~ 2’

- and

~-M

etho

xyau

cupa

~ a-

, /I

- an

d y-

Pyru

fura

n (8

587)

R

haph

iole

psin

; 4’

.met

hoxy

aucu

pari

n

~,~“

~hyd

roxy

~-m

e~ox

ya~t

ophe

none

(8

8)

Purp

urin

l-

met

hyl

ethe

r, e

tc.

Se&

in;

scop

aron

e A

cteo

side

; 8a

lact

osyl

act~

side

7-

Hyd

roxy

~lam

en~e

M

anso

non~

A

-P

7-H

ydro

xyca

lam

enen

e Ps

oral

en;

berg

apte

n,

etc.

Stilb

ene

Ant

hocy

anic

lm

Aat

h~~~

n

128

129

130

Ant

hra~

lic

acid

13

1 Pt

eroc

arpa

n 13

2 C

hrom

one

133

Pter

ocar

pan

Pter

ocar

pan

134

135

Isof

lava

none

t3

6 Pt

eroe

arpa

n 13

7 D

ihyd

rofl

avon

ol

138

Isof

lavo

noid

13

9 ~~

~iox

in-2

-one

14

0 st

ilben

e 14

1 Se

squi

terp

ene

142

Alk

aloi

d 14

3 Ph

enol

ic

acid

14

4 B

iphe

nyl

145

~~fu

ran

145,

146

Ben

zofu

ran

Bip

heny

l R

enzo

fura

n B

iphe

nyi

Ren

zofu

ran

Bip

heny

l

147

145

145

148

149

150

Ace

toph

enon

e 15

1 A

nthr

aqui

none

15

2,15

3 C

oum

arin

15

4,15

5 Ph

enyl

prop

anoi

d 15

6 ~u

ite~e

15

7 D

iterp

ene

158

Sesq

uite

rpen

e 1.

59

Cou

mar

in

160

Nap

htho

iura

none

16

1


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 at- tention 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 Solana- ceae. 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 Legumin- osae, e.g. Compositae, Solanaceae, Gramineae and Um- belliferae, 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. Further- more, 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, isoflav- anones, 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), Senevir- atne 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 Senev- iratne 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 (Mor- aceae) 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 trans- ferred 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 Legum- inosae, 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 de- rivative (50) [102]. The first alkaloidal phytoalexin san-


R

Lc 0 0

I’ O) e I’ ’ Oi-0 ’ %Y

51

53 R = (CH&Me

54 R = (CH&Me

A0 55

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 signi- ficantly 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 pur- purin 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 non- pathogen. More recently, an apigeninidin 5-caffeoylglu-

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 chromo- ne (56) from Cassia obtusifolia (Leguminosae) [133]. The

36 R. J. GRAYER and 1. B. HARBORNE

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 cruci- fers (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

59 R=SMe 60 R=SOMe

61

&Me 62

63

OJC S

H

64 X b

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 campes- tris infected tissues yielded 39 mg methoxybrassinin (5J3), 8 mg of brassinin (57) and 20 mg of cyclobrassinin (59),

65

67

68 R=H 69 R=OMe

dm. 72


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 charac- terize 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

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 Coleos- tephus 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. GRAYER and J. B. HARBORNE

H .,H R \

\

0

77

0 H

@3 H’ 4 -__

G CHO

0

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. Experi- ence 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

OH

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

OH

85

86 R=H 87 R=Me

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.


The sapwoods of the variety of Maloideae have re- sponded 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-pyru- furan 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’-meth- oxyacetophenone, is a novel phytoalexin for the family, although it does occur constitutively elsewhere, e.g. in the bark of Prunus domestica [151]. This simple acetophen- one (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 terpen- oid 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 Cruci- ferae 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

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 employ- ing 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 intri- guing 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.

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