JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND

217

Maataloustieteellinen Aikakauskirja
Vol. 59: 217—230. 1987

Critical aspects of phytoalexins in potato

STURE BRISHAMMAR

Department ofPlant and Forest Protection, The Swedish University ofAgricultural Sciences,
P.O. Box 7044, S-750 07 UPPSALA, Sweden

Abstract. Phytoalexins in potato are sesquiterpenoidsubstances produced in response to
infections and are believed to help plants resist attack by pathogens. However, these compounds
appear in response to compatible as well as incompatible interactions and only accumulate
in the tubers. The amounts of phytoalexins produced depend on the physiological condition
of the tubers. Young tubers don’t gel easily infected with Phylophlhora infestanseven though
they synthesize extremely small amounts of phytoalexins. Furthermore, confusion as to the
identity of specific races and the propensity for a given race to produce different effects in
the same type of host makes it extremely difficult to predict host-parasite interactions with
any acceptable degree of accuracy. It is doubtful that the production of phytoalexins in response
to artificial inoculations is representative of that occurring in natural infections. Markedly dif-
ferent types of pathogens induce synthesis of same substances in the host cells. It therefore
seems most probable that all the phytoalexins are synthesized in response to stimulation by
an endogenous elicitor. Little knowledge is available regarding the biosynthesis of these
sesquiterpenes, and many previous determinations have presumably been erroneous. When
potato tubers were inoculated with the late blight fungus, secondarily appearing bacteria were
not retarded, despite the presence of phytoalexins. There is no generally accepted hypothesis
describing the mechanism by which phytoalexins inhibit pathogens and no distinction has been
made between the effects on necrotrophs and biotrophs. Adequate bioassays capable of
measuring the effects of inhibition have yet to be developed, thus far, no convincing inhibitory
effects have been reported. During purification of the phytoalexins there is a high risk for artifact
forming, implying that specific compounds cannot be detected with certainty. Moreover, pre-
sent analytical methods must be improved before we can determine how phytoalexins act in
vivo. Probably, phytoalexins are synthesized at a stage in the infection too late to be able to
restrict its expansion with the tissues of the host. Phytoalexins are restricted to the attacked
parts of the tubers and there is no evidence indicating that these compounds pose any health
risks when present in potatoes used fqr consumption.

Index words: potato, tuber, phytoalexins, sesquiterpenes, elicitation, bioassays, purification, detection

Introduction ginning of World War 11, plant pathologists
showed a notable interest in induced resistance
in plants. They presumed that plants had anFrom the turn of this century until the be

https://www.c-info.fi/en/info/?token=JBisRK4vzsbSX6k8.OKUd0L0mq6SXyVPrFn31bg.VVg_7AumsnuzSHQsY3uS_TwTvvANjhRFgMf8HSIfmwPbt0KYw6-IrsrTHt_2uWWCjF8wYArKseeElGidpgpyxZPA0LBBHmA5S26m4P70PTo41zx2SCtQSb1qnGqHAZL73im4OxstosCK5liPMzyH70UHHPAk4oomuVkCizC-


immunological system similar to that of higher
animals. Although most researchers consid-
ered the occurrence of circulating antibodies
to be highly improbable, many of them were
looking for proteins that, like antibodies, are
capable of binding pathogens (Chester 1933).
Meanwhile, other scientists regarded the tan-
nins as the antibodies of plants (Meyer
1939).

Against this background we will consider the
postulate formulated by Muller and Boroer
in 1940, based on their experiments on locally
induced resistance in potato tubers following
inoculations with Phytophthora infestans
(Muller and Börger 1940). After inocula-
tions with avirulent races on tuber cross-sec-
tions exposed by slicing, an ’effect of immu-
nization’ was obtained i.e., the preventively
treated surfaces resisted attack by virulent
races. It was thereby assumed that defensive
substances so-called ’phytoalexins’ were
produced in response to the first infection.

More than one and a half decades later,
when beans were examined instead of pota-
to tubers, Muller (1956) regarded the phy-
toalexins as antibiotics synthesized when host
plant tissues respond to pathogen infections
by forming local lesions. His view had some-
what altered, since he now proposed that
phytoalexins even take part in defending
against the primarily infecting fungus.

Tomiyama et al. (1968) isolated the ses-
quiterpene rishitin from potato tubers after
inoculation with P. infestans, and subsequent-
ly, this substance and some other closely re-
lated sesquiterpenes have generally been con-
sidered to be synonymous with the hypothetical
phytoalexins in potato, postulated by Muller
and Boroer (1940). Gradually, however, def-
initions of phytoalexins became increasingly
generalized until a wide variety of substances
with real or assumed inhibitory effects against
microorganisms were regarded as phytoalexins.

Due to this confusion, attempts were made
by a large number of researchers to agree on
a stringent definition. One was consequently
formulated as follows: ’Phytoalexins are low
molecular weight antimicrobial compounds

that are both synthesized by and accumulated
in plants after their exposure to microorga-
nisms’ (Paxton 1981).

Some scientists consider the production of
phytoalexins as a consequence, not a cause,
of plant resistance to infection (Kiraly et al.
1972), but current opinions basically support
the view that phytoalexins are part of the
resistance mechanism of plants against patho-
gens and that they act through direct contact
with these pathogens. Still, it has yet to be
unequivocally proved that substances with the
above-mentioned qualities actually exist.

Experimental material

In general, a resistance reaction resulting
in a hypersensitive response has been asso-
ciated with the generation of phytoalexins.
After studying the experimental material in
its entirety, however, 1 can find no basis
for this assumption. For example, very few
proper investigations of phytoalexin produc-
tion in leaves have been performed (Kud
1982, Rohwer et al. 1987) even though infec-
tions of P. infestans are mostly initiated in
the haulm.

There is an obvious difference between
green parts of the plant and storage tissues
e.g. potato tubers. The green cells of leaves
have photosynthetically produced energy at
their disposal, while the more isolated tubers
are dependent on the degradation of starch
stores for energy. Thus there is a pronounced
difference in the availability of energy between
green leaves and tubers, and this difference
can increase under stress conditions. In green,
photosynthesizing cells all metabolic pathways
seem to be continuously active; therefore, the
turnover of sesquiterpenes is presumably also
more steady, implying that no real accumula-
tion of such substances will occur.

The physiological state of the tubers is of
great importance in determining how much of
and how fast the various phytoalexins are
synthesized and transformed once the tubers
become infected. Tuber condition is in turn

218



dependent on environmental variables e.g.
soil quality, water availability and fertiliza-
tion. Storage conditions e.g. temperature,
humidity and light will also affect tuber phys-
iology.

The period between harvest and infection
must also be considered. It is well-known that
freshly harvested tubers produce little or no
phytoalexins when inoculated; nevertheless,
they are more resistant than physiologically
aged tubers (Bhatia and Young 1985). More-
over, the amounts of phytoalexins obtained
with compatible and incompatible races of
P. infestans do not differ when young tubers
are infected (Brishammar et al. 1987).

The method used for inoculation also af-
fects the way in which the metabolism is
activated. An attack on the periderm dif-
fers in character from an attack on the pith.
Tuber varieties may differ in their degree of
sensitivity and resistance within their various
tissue regions. In cv ’Grata’, for example, the
skin is relatively resistant to fungal attack
while the pulp of the tuber is very sensitive.
In contrast, other varieties generally show less
pronounced differences in this respect.

Consequently, interactions may differ, de-
pending on whether the fungus is inoculated
through a bored hole in the tuber (and with
the core, reintroduced afterwards) (Horikawa
et al. 1976), or spread on the cut surface of
a tuber cross section (Tomiyama et al. 1968).

Boring and especially sectioning are
drastic disturbances, causing severe reactions
in the affected tissues. Hydrolyzing enzymes
are released from the cracked lysosomes
(Wilson 1973), and large amounts of carbon
dioxide evaporate from the exposed tuber
surfaces (Kahl 1974). The reconstruction of
destroyed units involves a considerable re-
synthesis of individual molecules as well as
complex cellular structures. These energy-
demanding activities require an accelerated
rate of starch degradation, simultaneously
with an activation of glycolysis and the pentose
shunt (Kahl 1974). Furthermore, the citric
acid cycle is switched on, thereby leading to
the opening of synthetic routes for the forma-

tion of fatty acids and terpenoids (Kahl
1974).

Cutting, for instance, involves the activa-
tion of tuber tissues in a way that presumably
does not occur during spontaneous, natural
infections, in which the production of ses-
quiterpenes is delayed compared with produc-
tion following laboratory inoculations preceded
by a sectioning procedure.

The pulp consists of a variety of tissues
(Reeve et al. 1969) that each react at a char-
acteristic rate when a tuber is cut. Thus the
peripheral tissues react faster than the interior
ones i.e., maximum respiration is attained
in the cortex cells after one day while it takes
2 days to reach a maximum in the pith cells
(Kahl 1974). A sample of infected material
taken as a 1-mm slice below the inoculated
surface across the tuber therefore comprises
a series of divergent tissue cells that are out
of phase with each other regarding the bio-
synthesis of sesquiterpenes.

For these reasons it is doubtful that labo-
ratory experiments can simulate the condi-
tions associated with natural infections while
simultaneously obtaining enough homoge-
neous sample material to undertake a chemical
analysis of phytoalexins using techniques
available at present. Likewise, it is probable
that the cutting of potato tubers and similar
manipulations alter the characteristics of the
surface exposed to pathogens during infec-
tions. Consequently, under artificial condi-
tions the interactions between host and com-
patible and incompatible races of pathogens
such as P. infestans can be expected to differ
from interactions occurring in natural infec-
tions.

The pathogen

Races of P. infestans used for inoculating
tubers to induce production of phytoalexins
were initially classified based on the symptoms
induced on the leaves of test plants, repre-
senting an assortment of potato varieties
(Black et al. 1953). Since leaf and tuber tis-
sues can react differently, an incompatible

219



reaction on a leaf does not ensure that the same
type of response will occur in corresponding
tubers. Consequently, race determinations
should be carried out on tubers as well as
leaves.

It is also generally taken for granted that
in inoculation assays, test plants exhibit an
all-or-nothing response, irrespective of the
size of the plants. However, uncertain ’transi-
tional’ symptoms probably occur, in which
case they would complicate the readings and
race determinations. It is also conceivable that
a given variety of potato could react different-
ly between locations and tests. Any latent viral
infection in a test plant will affect the reading,
and the risk for such infections can never be
excluded. Thus an absolute race determination
is probably not possible with this methodology.

Races of late blight fungus also tend to
change with time. Thus it is conceivable that
their features differ to some extent during
the course of an experimental period. As
a consequence, isolates should be submitted
for recurrent testing to confirm their racial
identity.

P. infestans can induce various levels of
phytoalexins in the same tuber variety, de-
pending on the pathogen race involved (Bris-
hammar et al. 1987). On the other hand, a
specific isolate can vary in its effects from oc-
casion to occasion depending on its physio-
logical condition e.g., its ability to absorb
amino acids. To explain differential resistance
to various fungal races, it might be more
rewarding to determine differences in their
essential-nutrient needs and in their ability to
utilize such nutrients instead of using the con-
ventional approach of defining compatible
and incompatible races (Pauli, and Cassel-
ton 1984).

Finally, it should be added that laboratory
inoculations with pure races may differ in
character from spontaneous infections, in
which several races probably occur simulta-
neously. In natural infections, it is also likely
that the infecting fungus is accompanied by
contaminating microorganisms such as bac-
teriä.

Turnover of sesquiterpenes in potato tubers

When the sesquiterpene rishitin was first
isolated in infected tubers (Tomiyama et al.
1968) and since it was thought to have anti-
fungal activity (Tomiyama et al. 1968)
it was therefore not surprising that rishitin
was proposed as the hypothetical phytoalexin
of potato (Muller and Börger 1940). That
rishitin, in particular, was designated as a
phytoalexin may simply have been a con-
sequence of the analytical technique used,
which emphasized sesquiterpenes. If other
categories of substances had been under ana-
lytical consideration, other types of com-
pounds would most certainly also have been
regarded as phytoalexins of potato.

The most recently accepted definition of
phytoalexins (Paxton 1981) states that such
substances must accumulate at infection sites;
such accumulations are deemed necessary for
detecting these compound with present-day
analytical methods. Still, the question remains
as to whether an accumulation of phytoalexins
occurs specifically in response to infections or
if other factors (not directly related to an in-
fection) can induce accumulations. It must
also be proved that the tentatively determined
phytoalexins actually take part in the plant’s
defence against pathogens.

In which tissues and to what extent are phy-
toalexins present in uninfected cells? Rishitin
and phytuberin could not be detected in ex-
tracts of potato leaves that had been inoculated
with incompatible or compatible races of
P. infestans (Kuc 1972, Rohwer et al. 1987).
In sprouts, an accumulation of rishitin only
occurred in connection with compatible in-
teractions between the sprouts and P. infestans
(Yarns et al. 1971).

Sesquiterpenoid substances similar to or
even exactly resembling the phytoalexins in
potato tubers have been detected in the leaves
of other members of the Solanaceae (e.g.
tobacco and tomato), but it is uncertain as to
whether these compounds were synthesized in
response to infections (Kud 1972, Takagi et
ai. 1979).

220



Various amounts of rishitin and closely
related sesquiterpenes have also been found
in healthy tubers of certain potato varieties
(Kuc et al. 1976, Schöber 1978, Brishammar
et al. 1987), indicating that not all phytoalexin
production is dependent on infections (Kuc
et al. 1976).

According to Muller and Börger (1940)
phytoalexins should be end-products, but a
number of researchers (Murai et al. 1977,
Ward 1977) do not regard the formation of
rishitin as the last step in the proposed bio-
synthetic sequence. Murai et al. (1977) sug-
gested that in cells with undisturbed metabo-
lism, rishitin is transformed into two new
compounds (rishitin-M-1 and rishitin-M-2),
which are then further transformed into water-
soluble forms. There are, however, no data
to support hypotheses describing the final
catabolic sequence, and degradation into
C5 -compounds has never been observed.

Added rishitin can probably be metabolized
by cells in which all metabolic pathways are
activated as in photosynthesizing cells or
tuber cells with an activated citric acid cycle
(Kahl 1974). Thus cut tubers that have been
permitted to age should be able to metabolize
rishitin in contrast to freshly sectioned tubers
(Ishiguri et al. 1978).

Thus the biosynthetic pathway responsible
for the production of rishitin and related
sesquiterpenes may actually operate in a variety
of healthy tissues in both leaves and tubers.
If true, then such a pathway should be pos-
sible to detect if the sensitivity of current
analytical techniques can be improved.

The accumulation of rishitin and associated
metabolites in connection with infection may
either indicate that a certain function of the
cell needs an extra stimulus to counteract the
attacking pathogen or that the cells are in an
early stage of decline, leading to necrosis.

In contrast to higher animals, plants are
devoid of centralized excretory organs, con-
sequently, the individual cells have to eliminate
their own waste products to prevent them

from contacting the actively metabolizing cell
units (Sitte 1974). Limited amounts of ses-
quiterpenes can probably be detoxifiedby nor-
mal cells, but when present in great excess,
phytoalexins and other similar compounds
have to be transferred to adjacent, necrotic
cells, which thereby provide a waste storage
function.

In suspensions of potato tuber cells in-
fected with P. infestans the cells synthesize
phytoalexins, which then diffuse out into the
nutrient solution (Brindle et al. 1983). This
indicates that rishitin becomes an end product

possibly a waste product when the cells
are metabolically overloaded.

There are indications that rishitin can coun-
teract the growth stimulating effects induced by
additions of indole acetic acid (Tomiyama et
al. 1968). Rishitin can also inhibit the germina-
tion of Solanum pollen (Hodgin and Lyon

1979). These observations suggest that rishitin
and possibly other so-called phytoalexins
function primarily as cell-regulatory substances
rather than as pathogen inhibitors. It should
be noted that the sesquiterpenoid plant hor-
mone abscisic acid can accumulate in response
to wilting (Wain 1977). Consequently, the
anti-pathogenic activity of the phytoalexins is
perhaps only coincidental.

As is true for all sesquiterpenes, potato
phytoalexins are derived from farnesyl pyro-
phosphate (FPP) (Cordell 1976); moreover,
all of the phytoalexins seem to be synthesized
within the same part of the sesquiterpenoid
pathway. After FPP, rishitin and phytuberin
presumably have at least one precursor a
germacrene in common (Stoessl 1982). In
the sequence leading to rishitin, the various
steps are not yet known in detail. Still there
is good agreement that solavetivone, lubimin
and rishitin are synthesized in chronological
order, although there may be some other steps
in-between (Stoessl 1982).

Experience has shown that it is technically
difficult to conduct in vivo studies of ses-
quiterpenoid biosynthesis in higher plants

221



based on the incorporation of suspected pre-
cursors (Baker and Brooks 1976). Transport
barriers, compartmentation effects, rapid
catabolism of the desired products and con-
sumption of precursors by more competitive
biosynthetic pathways all contribute to the
reduced incorporation of assumed precursors
(Baker and Brooks 1976). Moreover, the
sesquiterpenes have very complex, three-
dimensional structures (Stoessl et al. 1976);
thus it is hard to obtain sufficient amounts of
uniform material.

In this treatment of phytoalexin biosynthe-
sis and accumulation the occurrence of ses-
quiterpene glycosides has been ignored. How-
ever, such a compound has been demonstrated
in potato tubers infected with Phoma exigua
var foveata, and its aglycone is closely related
to solavetivone (Malmberg and Theander
1980). Also four similar sesquiterpene glyco-
sides have been isolated from healthy tobac-
co leaves after flue-curing (Anderson et al.
1977).

In studies of biosynthesis using a radioactive
precursor ( 14C-labelled mevalonate), a sub-
stantial degree of radioactivity was incorpo-
rated into terpenoid glycosides (Francis and
O’Connel 1969, Banthorpe et al. 1972).
Thus sesquiterpene glycosides probably occur
more frequently within the ’phytoalexin-path-
way’ than previously believed. The glycosides
are more hydrophilic than the phytoalexins;
thus, the former may have eluded discovery,
since when isolating phytoalexins, all extrac-
tions have been performed with hydrophobic
solvents. Furthermore, the glycosidic linkages
may be enzymatically broken by the action
of glycosidases, which are released during
homogenization.

The fact that the sesquiterpene glycoside
detected in potato tubers (Malmberg and
Theander 1980) has an aglycone that is close-
ly akin to solavetivone suggests that the glyco-
side occurs at a comparatively early stage
during the biosynthesis and therefore may not
be in a state of decline. Instead, the glycosides
may actually be the functional forms of the
sesquiterpenoid phytoalexins.

The course of infection and the production
of phytoalexins

The phytoalexins are regarded to have
broad-spectrum activity and may be syn-
thesized in response to infections by a num-
ber of pathogens. In the course of a late blight
infection phytoalexin production apparently
does not occur during spore germination,
germ tube growth, or development of the
appressorium. Not until the infection peg has
penetrated the tissues, does an elicitor act
upon the host cell membrane. The fungus then
develops a haustorium, which is an indenta-
tion of the host cell’s plasmalemma. At that
time P. infestans is at a biotrophic stage. If
the infected cell and adjacent cells subsequent-
ly begin to produce phytoalexins, and if these
compounds really have anti-pathogenic prop-
erties, then they will either have to act directly
against the fungus at very low concentrations
outside the host cell, or act indirectly, regu-
lating host cells in such a way as to render
them less accessible to the attacking fungus.

There is no evidence to date that phyto-
alexins accumulate before the onset of necro-
sis. It should also be stressed that pathogens
that develop haustoria are not very susceptible
to direct-acting anti-microbial substances
(Schönbeck and Schlösser 1976).

Changes over time during the infection by
an incompatible race of P. infestans have been
followed using microscopical examinations
(Sato et al. 1971, Jones et al. 1975 a, Jones
et al. 1975 b). The primarily infected host cell
generally dies after 3—4 h, while the incipient
formation of rishitin was first detected 10—
11 h after inoculation, in cut tubers that had
been allowed to age for 15 h before being in-
fected (Sato et al. 1971).

Cell death and accumulation of phytoalexins
occur later in association with compatible inter-
actions than in association with incompatible
ones. As previously mentioned, phytoalexin
production should start off slower in natural
infections than in artificial infections, since
spontaneous infections are not preceded by

222



the metabolic activation resulting from the
cutting of tubers.

In pathogens inducing phytoalexin synthesis
in host cells the course of infection will vary,
depending on pathogen type. Therefore it is
doubtful that inhibitory mechanisms are the
same in different types of interactions. Still,
we do not know if there are any differences
in the stage at which and mechanisms by
which phytoalexins inhibit a hemi-biotroph
such as P. infeslans and a necrotroph such
as Phoma exigua. If inhibition occurs dur-
ing the necrotrophic stage of a pathogen with-
in the host plant, then direct contact can
occur between the fungus and the phyto-
alexins, and it should be possible to clearly
register the effect in bioassays in vitro. If
this scenario is valid, then phytoalexins are
probably not involved in defending against
biotrophs, since necrosis occurs prior to the
synthesis of these substances, and the biotrophs
will die in dead or dying cells because of their
complete dependence on the intact metabolism
of the host cells. If, on the other hand, phy-
toalexins contribute to the inhibition of path-
ogen development when the host cells are still
intact and phytoalexin concentrations are very
low, then they would most likely act as cell
activity regulators, inducing disease resistant
mechanisms in the cells. Unfortunately, it is
doubtful that present analytical techniques are
sensitive enough to reveal differences between
infected and healthy cells, since only minute
amounts are present.

Elicitation

Higher and lower fungi and bacteria (Lyon
et al. 1975) apparently elicit formation of the
same kinds of phytoalexins in infections of
potato tubers and other plant species. Phenolic
phytoalexins also have been detected in plants
after attacks by necrotizing viruses (Klarman
and Hammerschlag 1972), which can elicit
synthesis of sesquiterpenes in tobacco leaves
in the same way (Bailey et al. 1976). Thus it
appears as if widely differing categories of
pathogens can induce synthesis of the same

types of compounds. Is there one type of
elicitor common to all pathogens or do a
variety of elicitors exist that each use a dif-
ferent mechanism to induce the same type of
host cell biosynthesis? It seems most likely that
in all cases an endogenous elicitor is released
from the wall of the host cell (Hargreaves
and Bailey 1978) in response to pathogen
attack irrespective of pathogen type. The
endogenous elicitor may be a saccharide that
reacts with a specific receptor, probably a
lectin (Garas and Kuc 1981), located in the
plasma membrane of the tuber cell. It is al-
ready known that small amounts of rishitin
are present in healthy tubers after cutting. The
cutting procedure sets lysosomes free in the
tissues, and their enzymes may degrade con-
stituents of the cell wall (Ray 1972), making
the endogenous elicitor available.

The sesquiterpenoid phytoalexins accumu-
late after infections, in association with both
compatible and incompatible interactions,
but the accumulation occurs faster and to a
greater extent in the latter case (KuC 1982).
Even then, potato tubers treated with sonicated
mycelium of P. infeslans develop necroses
and accumulations of phytoalexins, but the
responses are exactly the same regardless
as to whether virulent or avirulent races are
used (Yarns et al. 1971). Two fatty acids,
eicosapentanoic and arachidonic acid, detected
in the mycelium of P. infestans, have been
proved to induce synthesis of phytoalexins in
potato tubers (Bostock et al. 1981). Their ef-
fects, however, may be nonspecific, and they
probably act by interfering with the lipoid
parts of the host cell membrane, thereby im-
pairing their function (Lode and Pedersen
1970). Since these two fatty acids are also
found in the cell wall of the late blight fungus,
it is also possible that treatment with sonicated
mycelium elicits nonspecific reactions as well.
Sonicates and cell wall preparations of other
fungi or heat-treated bacteria, however, do
not induce accumulations of phytoalexins
(KuC et al. 1984), suggesting that these orga-
nisms are devoid of the two fatty acids. If this
is the case, then the substances of current in-

223



terest are probably not of universal occurrence;
consequently, they should not serve as specific
elicitors. Instead, the evidence suggests that
in cases where attacks by necrotizing viruses
are accompanied by the formation of phyto-
alexins, an endogenous elicitor is involved
(Klarman and Hammerschlag 1972), Bailey

et al. 1976).
It can therefore be assumed that all patho-

gens penetrating the host cell wall cause the
endogenous elicitor to be released. Compatible
races, however, may competitively inhibit
bonding between the endogenous elicitor and
the specific receptor by emitting glycans func-
tioning as suppressors (Doke et al. 1979).
This is in accordance with the theory that
resistance is normal while sensitivity is abnor-
mal (Ingram 1978).

To emit suppressors, a compatible race
must remain undisturbed during the infection
process. Additions of chloramphenicol or
streptomycin at the time of inoculation of
potato tubers with virulent races of P. infestans
consequently result in hypersensitive reac-
tions i.e., early necrosis formation and
accumulation of phytoalexins, two symptoms
of incompatible interactions (Kiraly et al.
1972). It is also conceivable that virulent races
can be influenced by other types of additions
associated with artificial infections, causing
the pattern of infection to deviate from that
characteristic of spontaneous, natural infec-
tions.

Bioassays

The new definition of phytoalexins (Pax-
ton 1981) does not set any concentration
limits above which activity is attributed to
non-phytoalexin-related effects. Nor does it
refer to how antimicrobial activity shall be
measured. Moreover, there is no generally
agreed upon procedure for estimating the
ability of phytoalexins to inhibit pathogens.

There are considerable difficulties involved
in the design of an in vitro bioassay to test for
antipathogenic activity, since the results may
not reflect the actual in vivo situation. Of the

many studies on phytoalexin occurrence only
a few deal with their antipathogenic activity,
and, on the whole, there are no firm concep-
tions to how and when inhibition occurs.

Phytoalexins must act during a stage when
their formation is in phase with the growth
of the attacking fungus. It is hardly relevant
to study inhibition of spore germination
(Hargreaves and Mansfield 1975, Doke et
al. 1979) or germ tube growth (Doke et al.
1979), since, to all appearances, the produc-
tion of phytoalexins does not begin until
the infection peg has penetrated the membrane
of the host cell. Furthermore, inhibitory ac-
tivity should be assayed with an actual path-
ogenic fungus such as P. infestans. Obvious-
ly, it is also necessary to test both compatible
and incompatible races. Thus little would
be gained by using a non-pathogenic fungus
such as Cladosporium sp. (Hargreaves and
Mansfield 1975).

It is very doubtful whether phytoalexins
such as rishitin function as inhibitors of spore
germination. For example, the established
endogenous inhibitor methyl-3,4-dimethoxy-
cis-cinnamate in the spores of the crown rust
fungus (Macko et ai. 1972) is about 107 times
more inhibitory than rishitin.

Furthermore, the inhibitory activity of
phytoalexins in in vitro bioassays is at least
one hundred times lower than that of the
fungicide metalaxyl when used against the
late blight fungus (Brishammar and Wid-
mark 1987). Furthermore, they are no better
at inhibiting pathogens than certain other
sesquiterpenes, that are quite irrelevant in
this connection (Brishammar and Widmark
1987). It should also be added that the in-
hibition achieved in these bioassays is not
complete. Accordingly there are no distinct
demarcation zones between the fungus and the
added phytoalexins; such zones are charac-
teristic of bioassays with certain bacterial
isolates and the fungus or with conventional
antibiotics against bacteria.

There are also disagreements in the litera-
ture concerning the inhibitory ability of the
various phytoalexins. Many researchers ob-

224



served inhibition with rishitin and to a
lesser extent with lubimin, but not with
solavetivone. However, other workers con-
sider solavetivone to be the most active com-
pound (StOssel and Hohl 1981, Brishammar
and Widmark 1987).

Estimates as to the amounts of specific
phytoalexins required to inhibit fungal growth
vary greatly. Generally, the necessary doses
are very high (Harris and Dennis 1976,
Smith 1982), although there are some excep-
tions (Ward et al. 1974). Lyon and Bayless

(1975) consider phytuberin to be ineffective
against bacteria, and according to Harris
and Dennis (1976) this phytoalexin is even
devoid of fungitoxic and fungistatic activity.

The variation in inhibitory activity between
studies may be due to differences in purity.
There is an obvious risk that sesquiterpenes
can be contaminated with fatty acids and
methyl esters of fatty acids, which may inhibit
growth of the fungus (Lindeberg and Linde-
berg 1974). Furthermore, it is probable that
the sensitive, three-dimensional structures of
the sesquiterpenes can be easily disturbed.
Therefore the configuration of any particular
phytoalexin may actually differ from one
preparation to another.

Some researchers consider phytoalexins
to be ineffective against primary pathogens
(Kiraly et al. 1972) while inhibiting subse-
quent secondary microorganisms (Van Der
Plank 1975). In experiments on interactions
between potato tubers and the late blight
fungus, however, the secondary bacterial
pathogens were not inhibited (Brishammar et
al. 1987).

Concrete suggestions as to the mechanisms
of the supposed inhibition by phytoalexins are
lacking apart from an interesting hypothesis
by StOssel and Home (1981). They proposed
that solavetivone acts as an inhibitor of glu-
canases, which are secreted by certain fungi,
including P. infestans. However, it is pre-
sumably not possible to detect this type of in-
hibitory activity on fungi in vitro on an agar
medium. In addition, it is doubtful that the
phytoalexin can reach inhibitory concentra-

tions in vivo quickly enough to catch the
fungus at its susceptible stage.

As inhibitors of pathogenic fungi the vari-
ous sesquiterpenoid phytoalexins seem to
be inferior to a number of phenolic sub-
stances (Malmberg et al. 1980). It is also
doubtful that hydrophobic compounds such
as sesquiterpenes come into direct contact with
a fungus restricted to a hydrophilic environ-
ment. Conceivably, glycosidic forms (Malm-
berg and Theander 1980), which are more
water-soluble, could have more inhibitory
activity.

Preparation, separation and detection

The quality of the sample material is of
particular importance when isolating ses-
quiterpenoids. It may also be difficult to
minimize heterogeneity in samples, which
generally consist of a mixture of dead cells,
cells in various degrees of decline and healthy
cells. Although the use of cut (infected) tubers
allows samples to be taken in very thin, even
layers, the sample slices comprise (as men-
tioned earlier) a variety of tissues whose bio-
syntheses are out of phase with each other.

In infections established deep in the tuber
pulp e.g. those occurring after artificial
inoculation or those associated with rots
it is difficult to exclusively isolate the tissues
of interest. Such problems can occur when,
for example, the concentrations of rishitin are
to be determined at various times after infec-
tion. In killed cells the amounts of rishitin
should remain constant, although some rishitin
is lost owing to problems involved in the ex-
traction of sesquiterpenoid substances from
wilted tissues. Furthermore, the extraction
procedure itself may induce decomposition or
other molecular changes, thereby decreasing
actual yields. If living cells producing relatively
large amounts of phytoalexins recover after
the stress induced by the forced synthesis, it
is probable that the rishitin produced will be
catabolized and decay. The markedly different
estimates of rishitin life expectancy within the

225



cells (Kuc 1972, Murai et al. 1977, Ward et
al. 1977, Brishammar et al. 1987) may stem
from variation in the relative proportions of
killed and living cells between samples. In this
respect the choice of tuber variety may play
an important role.

The formation and occurrence of phy-
toalexins seem to be markedly restricted to in-
fected areas. In association with compatible
reactions, however, the phytoalexins appear
deeper in the tubers, since accumulations are
formed in connection with the expansion of
the infection (Schöber 1980). On the other
hand, any reports of large amounts of ses-
quiterpenoid phytoalexins deep in tubersafter
incompatible interactions are certainly in-
correct and probably are the result of detec-
tion error. It should be noted that during gas
chromatography, the methyl esters of the fatty
acids mostly turn up in the same region as the
sesquiterpenes (Coxon et al. 1977).

The method of extraction and choice of
solvent may partly determine the types and
amounts of the sesquiterpenes recovered
(Stoessl et al. 1976). Moreover, if samples are
used in which cell sesquiterpene biosyntheses
are not synchronized, the variety of com-
pounds produced may be great. These sub-
stances may then be further modified by
secondary microorganisms.

During purification of sesquiterpenes, in
particular, there is a risk that the extraction
will give rise to artifacts (Kuc 1982, Wickbero
1983). Such compounds generally do not exist
in nature, either in living or in dead tissues.
Slight structural shifts in the three-dimensional
molecular pattern would probably render
a compound biologically inefficient. Con-
sequently, sesquiterpenes that are active in vivo
may show little or no activity after being
isolated from tissues (Wickberg 1983).

The type of methods used for identifying
and quantitating plant constituents can have
a definitive impact on the results. For in-
stance, it is unadvisable to exclusively base
sesquiterpene determinations on one system of
thin layer chromatography, even if reference
substances are used. Not even gas chromatog-

raphy can alone provide reliable qualitative and
quantitative results, especially when packed
columns are utilized. Capillary columns defi-
nitely permit better separation of substances;
nevertheless, confirmation of results by mass
spectrometry or other methods is necessary,
since more than one compound can have the
same retention time.

The general use of hydrophobic, organic
solvents for purification of sesquiterpenoid
phytoalexins has led to a de-emphasis on the
identification of water-soluble types, such
as sesquiterpene glycosides (Malmberg and
Theander 1980). Thus it is very likely that
more types of glycosides would be discovered
if a systematic search for such compounds was
undertaken. For instance, the FIS-toxin, which
is produced by Helminthosporium saccari,
has been shown to be a glycoside with a ses-
quiterpene as an aglycone to which four
hexoses are bound, forming the active unit
(Macko 1983).

Toxicity

Can phytoalexins in potato tubers cause
toxic reactions when the tubers are used as
food or forage? Since these sesquiterpenes
lack any appreciable fungitoxic effects, there
is little apparent risk for toxicity in either
humans or animals. Furthermore, phytoalexins
are generally restricted to the attacked parts
of the tubers, which are usually small unless
the infection expands.

Neither the substances of current interest
nor closely related ones are included in the in-
ternational index, ’Rotecs’ (1980), treating
toxic compounds. Moreover, no harmful ef-
fects were noted when mice were exposed
to phytuberin (Renwick 1972), and no em-
bryotoxic or teratogenic effects appeared when
pregnant mice were exposed to either rishitin or
phytuberin (Neudecker and Schöber 1984).
Mammals are probably also capable of syn-
thesizing glycosides of terpenes, which are
then excreted (Ishag 1984).

Certain injurious effects have been observed
in plants after exposure to sesquiterpenoid phy-

226



6

toalexins (Smith 1982). For instance, rishitin
can cause membrane injuries (Lyon 1980)
resembling those produced by some fatty acids
(Lode and Pedersen 1970). However, very
high concentrations of phytoalexins are usually
required to cause such adverse reactions, and
it is therefore uncertain as to whether they
actually occur in nature.

Conclusions

It is generally accepted that phytoalexins of
potato are sesquiterpenes. These substances
only accumulate in tubers in response to in-
fections. In fact, there is no comprehensive
picture regarding the turnover of these ses-
quiterpenes in the tuber and haulm. Thus no
information is available on the role of ses-
quiterpenoid glycosides in cells, although these
compounds may be important. The general
conception that phytoalexins in potato are ses-
quiterpenes may have developed by accident.
Phenolics, fatty acids, analogs of amino acids,
peptides, etc. could presumably have been
regarded as phytoalexins as well.

Phytoalexins are considered to be unspecific
inhibitors with a direct impact on pathogens.
However, no general mechanism of inhibi-
tion has been established, nor are there any
methods available for measuring the extent of
inhibition. Data has yet to be obtained on
in vivo concentrations of phytoalexins in
micro-sites harboring developing pathogens
(Deverall 1976), and the forms of the ses-
quiterpenes active on these sites remain un-
identified. It is also very doubtful that the
phytoalexins are produced in time to prevent
pathogen growth (Daly 1972) in fact the
pathogen and the phytoalexins may actually
never meet in vivol

It is conceivable that phytoalexin synthesis
is always induced by means of an endogenous
elicitor released from the cell wall of the host
when penetrated by microorganisms. Com-
patible interactions, however, may indicate
that the endogenous elicitor was not able
to reach its receptor, which may have been

occupied by so-called suppressors (Doke et
al. 1979) emitted by the pathogen.

Phytoalexin deposits are definitely restricted
to the infection sites and appear in connection
with cell death. Consequently, accumulations
of phytoalexins turn up earlier in association
with incompatible interactions than in associa-
tion with compatible ones.

It is difficult to determine whether the in-
hibitory ability of a metabolite in vitro actually
reflects its activity in vivo. Inhibitionobtained
with phytoalexins has been limited and may
also have been accidental (Stoessl 1980).
From an energetic point of view, it seems
’uneconomical’ for the plant to exert resistance
through directly operating inhibitors of this
kind. However, it is conceivable that phy-
toalexins or closely related metabolites could
serve as regulators, stimulating host cells
to defend themselves against pathogens by
preventing pathogen entrance through modi-
fications of the spatial patterns of the structural
molecules, by withdrawing essential nutrients
or by producing nutrient types e.g., amino
acids, that can be utilized by the host cells but
not by the pathogens (Faull and Casselton
1984).

No differences have yet been documented
in phytoalexin content between potato tubers
affected by compatible interactions and those
affected by incompatible reactions.

During the initial stages of pathogen de-
velopment, when the pathogens appear on the
superficial parts of the plants, pathogen
growth is probably retarded through contact
with individual inhibitory substances, most or
all of which originate from microorganisms
in the phyllo- and rhizospheres. Otherwise,
direct-acting anti-microbial substances should
not be assumed to exist until it is possible to
unambiguously demonstrate that host cells
really produce compounds capable of in-
hibiting pathogens in vivo at very low con-
centrations.

The designation of the term ’phytoalexin’,
in connection with a hypothesis (Muller and

227



Börger 1940), has led to a host of precon-
ceptions, that have introduced serious bias
into related studies on plant defense against
pathogens.

Acknowledgements. Thanks are due to Dr. David Tilles
for correcting the English. Financial support was obtained
from the Swedish Council for Forestry and Agricultural
Research.

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SELOSTUS

Perunan fyloaleksiinit: kriittinen tarkastelu

Sture Brishammar
Department of Plant and Forest Protection,
The Swedish University ofAgricultural Sciences,
P.O. Box 7044, S-750 07 Uppsala, Sweden

Perunan fytoaleksiinit ovat seskviterpecnejä, joitaker-
tyy mukuloihin infektioiden seurauksena. Niiden on ar-
veltu osittain säätelevän perunan taudinkestävyyttä, jos-
kin niitä muodostuu sekä alttiissa että kestävissä lajik-
keissa. Fytoaleksiinien määrä riippuu mukuloiden fysio-
logisesta tilasta, nuorissa mukuloissa niitä on hyvin vähän.
Koska monet patogeenit aiheuttavat samojen fytoalek-
siinien muodostumisen, on todennäköistä, että jokin so-
lunsisäinen elisitori laukaisee niiden tuotannon.

Sopivien testimenetelmien puuttuessa ei vielä tiedetä.

mihin fytoaleksiinien patogeenejä estävä vaikutus perus-
tuu. Fytoaleksiineja puhdistettaessa saattaa syntyä arte-
fakteja, jotkahaittaavat joidenkinyhdisteiden tunnistus-
ta. Nykyisiä analyysimenetelmiä onkin huomattavasti ke-
hitettävä, ennen kuin voidaan selvittää fytoaleksiinien vai-
kutusta in vivo. Todennäköisesti ne syntetoituvatkasvissa
liian myöhään pystyäkseen estämään infektion leviämis-
tä. Fytoaleksiinit eivät tiettävästi ole terveydelle vaaral-
lisia, ja niitä kertyy vain mukuloiden infektoituneisiin
osun.

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