Maataloustieteellinen Aikakauskirja
Vol. 59: 231—249, 1987

Induction of defence reactions in plants

HANS THORDAL-CHRISTENSEN, PER L. GREGERSEN, JAN B. ANDERSEN,
and VIGGO SMEDEGAARD-PETERSEN

Department of Plant Pathology, The Royal Veterinary and Agricultural University,
Thorvaldsensvej 40, 1871 Frederiksberg C, COPENHAGEN, Denmark

Abstract. Induced local resistance presumably involves the same mechanisms in the plants
asresistance elicited during normal plant-pathogen interactions. In many cases resistance elicitors
from pathogens have been found to be non-specific, i.e. unrelated to race-cultivar specificity.
Thus, existence of specific resistance suppressors has been suggested to make the virulent races
able to infect. In other cases specific resistance elicitors have been indicated toexist in avirulent
races, by which the race specific resistance may be accomplished.

At our Department resistance has been induced in the barley powdery mildew interac-
tion by use of double inoculation procedures. Both virulent and avirulent races of barley powdery
mildew can induce resistance, but avirulent races show an increased resistance induction ability
in relation to virulent races from 12 hours after inoculation. In barley plants wheat powdery
mildew induced more resistance than barley powdery mildew 1 to 8 hours after inoculation.
Induced resistance was mainly localized to the epidermal cells attacked by the inducer, but
an effect was also present in the surrounding epidermal cells.

The energetic consequences of resistance in barley to barley powdery mildew have been
found to be reflected in an increased respiratory rate at the time of infection attempt. Further,
these energy costs appeared to reduce grain yield by 7 Vo.

The experession of resistance in barley is thought to involve de novo synthesis mRNAs
and proteins, which makes it possible to apply gene technological methods to study induced
resistance. Research of this kind is in progress at our Department, which hopefully will give
information on the mechanisms of resistance triggering and resistance expression.

Index words: barley, powdery mildew, induced resistance

introduction

Induced resistance can be defined as ac-
tive defence based on physical and chemical
barriers elicited by preliminary inoculation
With pathogens or non-host pathogens, or by
application of metabolic products from such

organisms. It acts against subsequent infec-
tion by otherwise pathogenic organisms. In a
broader sense the term also includes resistance
induced by abiotic stimuli.

Distinction can be made between two func-
tionally different forms of induced resistance,
systemic and local. Induced systemic protec-

231

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tion against a pathogen can be elicited by
previous inoculation of the host with either
avirulent or virulent races of the same pathogen
or with non-host pathogens. The resistance-
inducing factor is translocated from the site
of induction to other, usually younger plant
parts, where it conditions the host tissue to
respond in a resistant fashion upon subse-
quent challenge by a pathogen. In contrast to
systemic protection, induced local resistance
is restricted to the site of inducer inoculation,
and until recently only avirulent races of a
pathogen and non-host pathogens have been
shown to act as inducers. This article will only
deal with aspects of induced local resistance,
and does not intend to cover induced systemic
resistance.

Presumably induced resistance, as it results
from double inoculation, involves the same
basic defence elements as known from ordinary
incompatible host-pathogen interactions or
from non-host resistance: recognition, rapid
elicitation or sensitation towards a resistant
stage, and subsequent accumulation of re-
sistance-related metabolites.One of the exiting
aspects of induced resistance is, however, that
it seems to constitute a suitable model for
detailed studies of the molecular basis of
disease resistance by the employment of DNA
techniques.

Induced, active resistance involves drastic
changes in the metabolic activity around the
site of attempted infection such as rapid cyto-
plasmatic movement, synthesis and deposit of
heterogeneous materials around the attempted
penetration sites, changes in respiration and
photosynthesis, and the accumulationof sec-
ondary fungitoxic metabolites. Research at
our Department has shown that such highly
energy-consuming defence reactions occur
at the expense of host energy resources and
therefore finally lead to reduction in plant
growth and yield.

Elicitation of defence reactions in plants

At the macroscopic level induced resistance
appears as the end-result of a complex process

involving extensive metabolic changes in the
plant tissue (see a later chapter about molecular
studies of resistance). It is, however, difficult
to establish the sequence of events in this
process following application of the inducer
and challenger agents. Here, we will consider
the efforts done to elucidate the first steps in
the inducing process, also called the deter-
minative phase as opposed to the subsequent
expressive phase (Keen 1982). The term elici-
tation used in this text covers the putative
decisive events taking place in these first steps
of interactions between hosts and pathogens.
The question to be raised is: What are the
primary events at the biochemical level re-
sponsible for the extensive changes involved
in induction of defence reactions in plants?

Elicitors of defence reactions

The term elicitor has been used for several
years to designate substances which signal the
plant to trigger defence responses. Especially
the term has been applied when dealing with
phytoalexin accumulation (Darvill and Al-
bersheim 1984). This somehow complicates
the matter because phytoalexin accumulation
in many cases has been regarded as essential to
resistance and this is not always the case. For
instance Rohwer et al. (1987) showed that
potato leaves are able to react with a strong
resistance response without accumulating
detectable amounts of phytoalexins.

Therefore, the work that has been done
with elicitors in different systems is in some
ways incommensurable because of the dif-
ferent methods applied, but still, it casts a
good light on the biochemical interactions
between hosts and pathogens at the initial
stages of infection.

The elicitors described in the literature
are either biotic, i.e. originating in plant or
pathogen, or of abiotic origin (Davis et al.
1986).

Elicitors offungal origin

Most of the extensive work trying to iso-

232



late substances of fungal origin which, when
applied in purified form, trigger defence re-
sponses in plants, has been done with soybean,
bean and potato.

In soybean it has been shown that fractions
isolated from the cell walls of Phytophthora
megasperma f.sp. glycinea (Pmg) provoke the
accumulation of phytoalexins when applied
to cotyledons of soybean (Darvill and Al-
bersheim 1984). One elicitor-active substance
from this cell wall fraction was purified by
Sharp et al. (1984) and characterized as a
/3-glucan. Apparently the elicitor activity of
the glucan is related to the specific arrange-
ment of branch points because other glucans,
similar in chemical composition but with other
arrangements of branch points, were inactive.

The elicitors from cell walls of Pmg are
race-non-specific, i.e. they work independent-
ly of the interaction between soybean cultivars
and races of Pmg as described by the gene-
for-gene model. Thus elicitors isolated from
avirulent races elicit as much phytoalexin ac-
cumulation as the ones isolated from virulent
races (Ayers et al. 1976).

In the interaction between potato and Phy-
tophthora infestans (Pi), Bostock et al. (1981)
showed that unsaturated fatty acids, espe-
cially eicosapentaenoic and arachidonic acid,
from the cell walls of Pi were able to elicit
a hypersensitive response, accompanied by
phytoalexin accumulation, in potato tuber
tissue. This elicitation was also non-specific,
i.e. unrelated to race-cultivar specificity. Fur-
thermore, it has been shown that the elicitor
activity of the fatty acids is enhanced by com-
bining them with glucans from the cell walls
of Pi (Preisig and Kuc 1985). The glucans
themselves are inactive as elicitors of the
hypersensitive response.

The non-specificity of elicitors requires
additional explanation concerning the bio-
chemical determination of race-specific in-
teractions. Such an explanation has been sug-
gested for the potato-/*/ interaction, where
race-cultivar specificity, defined by the R-genes
in potato cultivars, is ascribed to the existence
of fungal suppressors of the hypersensitive

reaction (Doke et al. 1979). The suppressors
are supposed to work by specifically hindering
the response to the non-specific elicitors in
compatible host-pathogen interactions (Doke
et al. 1987). The mechanisms of this suppres-
sion are totally unknown.

Another theory concerning race-cultivar
specificity postulates the existence of race-
specific elicitors as opposed to the non-specific
elicitors suggested for Pmg and Pi. Anderson
(1980), working with Colletotrichum linde-
muthianum (Cl) on bean, suggested the ex-
istence of such specific elicitors. She used three
races of Cl with differentvirulence characters
to show that the elicitor activity of extracellular
components obtained from liquid cultures
of the three races correlated well with their
virulence characters. This work was extended
by Tepper and Anderson (1986) who showed
that two cultivars of bean displayed differential
responses to extracellular components of Cl.
Although the results were not entirely con-
sistent with race-cultivar specificity, they con-
tribute to a more thorough understanding of
the events taking place in the interphase
between the invading pathogen and the host
tissue. Intuitively, extracellular/surface bound
substances are more likely to play a role here
than substances obtained from homogenates
of fungal cell walls.

Elicitors originating in plants

In several cases it has been shown that
plant tissues within the cell walls possess so-
called endogenous elicitors which, after being
released, are able to elicit the accumulation
of phytoalexins indicative of a defence reac-
tion (Darvill and Albersheim 1984).

Hargreaves and Bailey (1978) showed
that the contact with dead cells caused living
bean cells to produce phytoalexins, which was
ascribed to the release of an elicitor from
the dead cells. Hahn et al. (1981) showed
that a fraction from soybean <iell walls was ac-
tive as elicitor and that it contained oligosac-
charides rich in galacturonic acid. The ex-
istence of endogenous elicitors containing

233



234

galacturonic acids from the pectic fragments
of cell walls was also shown by Bruce and
West (1982) in castor bean. These authors
suggested that the endogenous elicitor could
be released upon infection with a pathogen be-
cause of a fungal endogalacturonase working
in the cell wall degradation process associated
with infection attempts.

The existence of endogenous elicitors is
in accordance with theories about cell wall
oligosaccharides being involved in many dif-
ferent physiological control processes as a
new kind of regulatory molecules (Fry 1986).
The hypothesis is that such cell wall oligosac-
charides under appropriate conditions can be
released from their built-in state, for instance
during degradation of the cell wall by invading
pathogens.

Elicitors and elicitation

Most of the experiments concerning elicitors,
which have been considered in this text, have
been carried out with elicitors applied to plant
tissue in a more or less purified form. This
gives rise to some problems when trying to
relate the results from the experiments to the
events actually taking place in the interface be-
tween pathogen and host tissue during the in-
fection process. It is difficult to infer anything
conclusive about the role of the isolated elici-
tors under in vivo conditions. There, they
necessarily will have to function in intimate
contact with many other compounds of both
plant and pathogen origin. One could get
the idea that elicitors isolated by more or
less harsh procedures could turn out to be
artefacts of these procedures, and that they
could be acting more like abiotic elicitors
rather than as biotic elicitors relevant to the
system under study. The results by Preisig

and Kuc (1985) showing that elicitor active
fatty acids interact with non-eliciting glucans
illustrates the complexity of systems com-
prising more than just one component. The
triggering of defence responses is probably
more complicated than what the action of one
single elicitor can account for.

How does elicitation function?
The working hypothesis about the function

of elicitors in triggering defence responses
implies a release of fungal elicitors from the
invading pathogen and a subsequent reaction
by theplant tissue resembling those obtained
when elicitors are applied in purified form
(Darvill and Albersheim 1984). To a certain
extent such a hypothesis is supported by the
observations that enzymes from plant tissues
are able to release elicitor-active substances
from fungal cell walls and perhaps thereby
promote the induction of defence responses
(Keen and Yoshikawa 1983).

After the release it is hypothesized that an
interaction takes place between the elicitor and
a receptor in the host tissue. Yoshikawa et al.
(1983) conclude to have found such a receptor
site on the membrane of soybean cells. This
conclusion is based on the observed specific
binding of the elicitor from Pmg to membrane
fragments from soybean. The receptor has not
been characterized any further and it is not
explained how the observed binding is able to
transfer a signal to the host cell ordering a
defence response to be triggered. However,
Yoshikawa et al. (1983) suggest that recep-
tors, like the one observed, may exhibit race-
specific properties and thereby account for the
race-cultivar interactions observed in many
host-pathogen systems.

Epperlein et al. (1986) suggest an action of
abiotic elicitors which gives a causal explana-
tion of elicitor action at the physiological level.
AgN0 3 and HgCl2 were used as elicitors and
the results suggested that the activity of these
agents was mediated by the generation of
OH in the treated tissue. The heavy metal
salts are able to initiate redox-reactions which
lead to the OH -generation. Once generated,
this reactive oxygen species is able to initiate
lipid peroxidation in the membranes which
can continue as an autoxidative chain reac-
tion leading to perturbation of membrane
function.

Epperlein et ai. (1986) put up the following
sequence of events as a model for the function



of elicitors: Free radical generation lipid
peroxidation diffusion of endogenous elic-
itor derepression of genes required for
phytoalexin synthesis. They argued that some
of the biotic elicitors mentionedearlier in this
text could act in a similar way. For instance,
eicosapentaenoic and arachidonic acids are
known to undergo spontaneous peroxidation
in the presence of 02 and they might thereby
start the listed events leading to phytoalexin
accumulation.

The theory by Epperlein et al. (1986) is
supported by the work of Doke (1983), who
observed a marked generation of reactive
oxygen species around sites where P. infestans
attempted infection into potato tuber tissue.
This reaction was related to the incompatible
interaction between host and pathogen. Chai
and Doke (1987) extended this work and
found a small race-non-specific elicitation of

generation in potato leaves at an early
time after inoculation with Pi. In the incompat-
ible interaction this was followed by a marked
increase in Oj generation, similar to that
found in potato tuber tissue, while in the com-
patible interaction the initial O 7 generation
faded away. The theory that reactive oxygen
species might initiate membrane damage is
also supported by other studies showing that
peroxidation of lipids in the membranes is in-
volved in early hypersensitive responses to
pathogens (Stelzig et al. 1983, Ocampo et al.
1986).

The model that appears from these observa-
tions suggests that elicitation of defence reac-
tions might depend on the initiation of an
autoxidation of lipid components in the host
plasmalemma followed by a derangement of
the membrane. From the deranged membrane
a signal could be transferred to the metabolic
and genetic apparatus of the cell, telling that
something is wrong and that a reaction has to
be put up. That the membrane functions as
a mediator of the signal could explain why
defence responses often are localized right
below the infection site in the shape of e.g.
papillae.

This model for elicitation of defence reac-
tions involving membrane derangement by
reactive free radicals does not give any sug-
gestions as to how race-specificity is working
in host-pathogen interactions. In some way
the pathogen in compatible interactions must
be able to prevent or suppress the development
of the defence response.

Under in vivo conditions a subtle balance
exists between pathogen and host when infec-
tion is attempted. Even in compatible interac-
tions many infection attempts run the risk of
failure. Among others, Andersen and Torp

(1986) have shown this for barley-powdery
mildew interaction. This could mean that just
a slight perturbation of the infection process
might tip over the subtle balance in favour of
the host. Race-specific elicitation of defence
reactions could be caused by »constitutive»
perturbation of the infection process, perhaps
working via interference with the coalescence
between the infection hypha and the host cell
wall. Raa et al. (1977) noted that such an
association apparently is essential for patho-
gens. Improper coalescence could from the
host cells point of view be taken as a rather
aggressive wounding and this could cause a
defence response to be induced. Raa et al.
(1977) noted that non-host pathogens ap-
parently are unable to attach firmly to plants
and this supports the idea that coalescence
with the host cell wall is essential for a suc-
cessful infection.

Unfortunately we do not know very much
about elicitation of defence reactions in mono-
cotyledons even though host-pathogen in-
teractions with monocotyledenous hosts have
been characterized rather well in genetic analy-
sis (El.ungboe 1982). The latter will, however,
be very helpful when trying to get into more
details concerning the elicitation events be-
cause hypothesized molecular mechanisms
can be compared to the results of genetic
analysis. The time course studies conducted
with induced resistance in the barley-powdery
mildew interaction (see the next part) may
also be useful tools in elucidating the initial
events taking place in resistant reacting tissue,

235



especially when combined with molecular in-
vestigations of the same interactions.

Induced resistance in barley-powdery
mildew interaction

Induced resistance in the barley powdery
mildew interaction has been studied extensively
during the last 15 years. In some experimental
procedures the inducer race remained on the
barley leaf surface after challenge inocula-
tion, and distinction between inducer and
challenger was made possible by testing only
avirulent and non-pathogenic races as inducer.
It was shown that avirulent (Andersen 1983,
Chaudhary et al. 1983, Chin et al. 1984)
and non-pathogenic (Kunoh et al. 1985) races
of powdery mildew were able to induce re-
sistance in barley. Andersen (1983) found
that 30 min. between inoculation with the
avirulent inducer and the virulent challenger
was sufficient to reduce the number of chal-
lenger colonies.

Ouchi et al. (1974 and 1976) introduced
removal of the inducer after the induction
period. This technique allowed the test of
virulent inducer races as well. Studies of the
resistance inducing ability of virulent and
avirulent barley powdery mildew races showed
that only avirulent races induced resistance in
24 and 48 hours (Ouchi et al. 1974). Further,
it was found that wheat powdery mildew was
able to induce resistance after an induction
period of 6 hours (Ouchi et al. 1976).

Cho and Smedegaard-Petersen(1986) and
Thordal-Christensen and Smedegaard-Pe-

tersen (in press) quantified the effect of in-
ducer race, induction period, and inducer den-
sity. Inoculations were made on the first leaves
of isogenic lines of the barley cultivar »Pallas».
The leaves were fixed in horizontal position
while still attached to the barley plants. Af-
ter the induction periods the inducers were
removed by rubbing the leaves with wet cotton
balls. Challenge inoculations were performed,
and the induced resistance was later assessed
as percentage reduction in number of colonies

developed by the virulent challenger race in
relation to a non-induced control.

The shortest induction period in which re-
sistance was induced by barley powdery mildew
was found to be 0.5 hours (Cho and Smede-
gaard-Petersen 1986) (Fig. 1) and 2 hours
(Thordal-Christensen and Smedegaard-Pe-

tersen (in press) (Fig. 2). In apparent dis-

Fig. 1. The influence of inducer-inoculum density on
the induction of resistance against subsequent
infection by Erysiphe graminis f.sp. hordei.
A, Near-isogenic line of the barley cultivar Pallas
with resistance gene Ml-a, inducer race H2l (avir-
ulent), challenger race A 6 (virulent). B, Near-
isogenic line of the barley cultivar Pallas with
the resistance gene Ml-al3, inducer race H2l
(virulent), challenger race H2l (virulent). Sym-
bols: • », inoculum density 20conidia/mm 2 ;

o o, inoculum density 200conidia/mm 2 . In-
ducer inocula were removed immidiately before
the challenge inoculation were made. (From Cho
and Smedegaard-Petersen 1986).

236



agreement with the results of Ouchi et al.
(1974), it was found that virulent and avirulent
races of barley powdery mildew showed the
same ability to induce resistance in barley in
induction periods of up to 10 hours (Figs. 1
and 2). Our results of induced resistance up
to 10 hours after inoculation are in agreement
with the findings that resistance elicitors are
often non-specific (Ayers et al. 1976, Bostock
et al. 1981, Rohwer et al. 1987).

However, when the induction period was
extended to 12—24 hours (Fig. 2) the resistance
induced by the avirulent race was increased
significantly in relation to that induced by the
virulent race. This difference, only present at
relatively low inducer densities, agrees with the
results of Ouchi et al. (1974), except that sig-
nificant resistance induction by the virulent
race in induction periods of 12—24 hours was
found (Fig. 2). The time of the establishment

Fig. 2. Comparison of the resistance induced in a near-isogenic line of the barley cultivar Pallas with the re-
sistance gene Ml-a. Resistance was induced by the virulent race A6, and the avirulent race Cl 5 of Erysiphe
graminis f.sp. hordei, and by the non-pathogenic race TB5 of E. graminis f.sp. trilici. The comparison was
made at four different inducer densities and in 13 different induction periods. Mean values of three replica-
tions. (From Thordal-Christensen and Smedegaard-Petersen in press, a).

237



238

of the difference, i.e. 10 to 12 hours after
inoculation, coincides with the time of specific
recognition suggested by Ellinoboe (1972).
The increase of induced resistance between 10
and 12 hours after inoculation may be caused
by an elicitor present only in the avirulent race.
Existence of specific elicitors has been indicated
by Anderson (1980), Keen and Legrand

(1980), De Wit et al. (1986), and Mayama

et al. (1986).
The non-host pathogen, wheatpowdery mil-

dew, was included in the studies of Thordal-
Christensen and Smedegaard-Petersen (in
press, a) (Fig. 2). It was found that wheat pow-
dery mildew induced more resistance than
barley powdery mildew in the first 6 to 8
hours after inducer inoculation. Disregarding
the inducer density of 6.5 conidia/mm2

, the
resistance induced by wheat powdery mil-
dew appeared to be insensitive to changes
in the induction period between 10 and 24
hours. This is similar to resistance induced
by virulent barley powdery mildew, while
resistance induced by avirulent barley powdery
mildew increased significantly between 10 and
12 hours of induction at 20 conidia/mm2

.

Ouchi et al. (1976) found that wheat powdery
mildew could induce resistance 6 hours after
inoculation, consistent with the results of
Thordal-Christensen and Smedegaard-Pe-

tersen (in press, a). Rohwer et al. (1987) has
in agreement with the rapid induction of
resistance by wheat powdery mildew found
that the non-host pathogen Phytophthora
megasperma f.sp. glycinea induced rishitin ac-
cumulation in potato tuber earlier than the
pathogen Phytophthora infestans.

Infection efficiency and induced resistance
by a virulent race

In preliminary experiments with single in-
oculation procedures it was found that the in-
fection efficiency of a virulent race was reduced
when the inoculum density was increased. This
effect and the effect of resistance induced by
a virulent race, assessed in a double inocula-
tion procedure, are considered to be caused

by the same phenomenon. Resistance appears
in both cases to be elicited by and acting
against the same virulent powdery mildew
race. Further, as resistance can be induced
0.5 hour after inoculation (Cho and Smede-
gaard-Petersen 1986), it may be functional
at the moment of cell wall penetration about
10hours later in the single inoculation proce-
dure, and hence reduce the infection efficiency.

To compare these effects an inoculum den-
sity gradient was constructed with a viru-
lent race of barley powdery mildew (Thor-
dal-Christensen and Smedegaard-Petersen

in press, a). At each inoculum density of the
gradient both the infection efficiency assessed
as number of haustoria per appressorium
(single inoculation procedure) and the induced
resistance (double inoculation procedure) were
tested. The infection efficiency was found to
be reduced from 97 % at 0.20 conidia/mm 2

to 11 %at 6.5 conidia/mm 2
, while the in-

duced resistance was increased from 23 °7o at
6.5 conidia/mm2 to 80 % at 200 conidia/mm2

.

Thus the inoculum density needed to cause
50 % induced resistance was 40 times higher
than the density at which 50 % reduction of
infection efficiency was found. This difference
may be explained by the fact that the infec-
tion efficiency is assessed when the infection
units are located in their original positions,
while induced resistance is assessed by use of
a challenger, located in random positions dif-
ferent from those where the resistance actually
was induced.

Correlation between induced resistance and
host fluorescence at the site of the inducer

In a later work (Thordal-Christensen and
Smedegaard-Petersen in press, b) inducer
fluorescence (host fluorescence at the site of
the inducer) was investigated in the barley
powdery mildew interactions. A fluorescent
halo appeared in the epidermal cell at the site
of primary germ tubes of the inducer conidia.
The diameter of the halo was 2 and 4 /jm five
hours after inoculation with barley and wheat
powdery mildew, respectively. 10 hours after



inoculation the fluorescent haloes had doubled
their diameter, and fluorescent papillae had
developed in the center of the haloes. At this
time the diameters of the papillae at the site
of barley powdery mildew were about 3 /cm
and of the site of wheat powdery mildew it
was 5 jum. The diameter of these fluorescent
papillae at the site of the primary germ tube
only increased slightly until 24 hours after
inoculation. Ten hours after inoculation, in-
ducer fluorescence (fluorescent haloes and
papillae) at the appressorial lobes had only
developed to a small extent, but in the case
of barley powdery mildew it developed con-
siderably in the next 14 hours. In the case of
wheat powdery mildew, inducer fluorescence
at the site of the appressorial lobes only had
a weak development in this period.

When comparing the two assessments of
host response to powdery mildew infection,
i.e. induced resistance (Figs. 1 and 2) and in-
ducer fluorescence, it appears that wheat
powdery mildew causes stronger responses
than barley powdery mildew during the first
approximately 10 hours after inoculation.
This suggests that the primary germ tube is
essential in the process of resistance induction.
As the inducer fluorescence present 10 hours
after inoculation is mainly situated at the
primary germ tube, and as resistance induced
by virulent barley powdery mildew and wheat
powdery mildew only increases to a small ex-
tent after 10hours of induction, it seems that
this resistance mainly correlates with the in-
ducer fluorescence at the primary germ tube.

Localization of induced resistance

In an attempt to assess the extension of in-
duced resistance, inducer fluorescence was
used to determine the previous location of the
inducer (Thordal-Christensen and Smede-
gaard-Petersen in press, b). Three results are
of importance: 1) No correlation between the
infection success of the challenger and the
average distance to the ten nearest inducer
fluorescences could be detected. 2) Countings
of the number of inducer fluorescences in the

epidermal cells attacked by the challenger
showed that successfully attacked epidermal
cells contained only half the number of in-
ducer fluorescences found in cells unsuccess-
fully attacked. 3) About one-third of the
unsuccessful challenger infection units were
attacking cells that contain no inducer fluores-
cence. In relation to (3) it is indicated above
that a virulent race may have an infection
efficiency of almost 100 % when no resistance
is activated by other infection units in the local
leaf area (low inoculum density).

These results indicate that the induced re-
sistance is principally localized to the cells
attacked by the inducer, but it is to some ex-
tent also translocated to the epidermal cells
not attacked by the inducer. This is consistent
with the results of Woolacott and Archer
(1984), who found that infection units of
virulent barley powdery mildew had a lower
infection success when the primary germ tube
previously had been in contact with the at-
tacked barley leaf cell.

Correlation between induced resistance and
host fluorescence at the site of the challenger

Thordal-Christensen and Smedegaard-Pe-

tersen (in press, b) also studied the fluores-
cence at the challenger infection sites in relation
to induced resistance. It was found that the
diameters of the fluorescent papillae beneath
unsuccessful challenger infection units were
increased when resistance was induced in the
leaf. The result agrees with the suggestion
made by Sahashi and Shishiyama (1986) that
papilla formation at the challenger is a major
factor of induced resistance. Further, the
result supports the assumed importance of the
papillae in the resistance process (e.g. Kita
et ai. 1980, Skou et ai. 1984, Heitefuss and
Ebrahim-Nesbat 1986, Smart et ai. 1986).

It thus appears that resistance is induced,
although no fluorescence or other host re-
sponses are visible in the microscope before
challenge inoculation. This indicates that in-
duced resistance is a state of sensitivity, where
the resistance reaction is partially completed.

239



The resistance reaction is fully expressed when
the challenger is attacking. One of the major
factors of this induced resistance could be the
formation of larger papillae.

Inducer removal versus no inducer removal

Cho and Smedegaard-Petersen (1986)
found that resistance induced without remov-
ing the avirulent inducer race resulted in a fairly
large number of visible necrotic spots, contrary
to when the inducer race was removed. This
indicates that resistance induction with and
without inducer removal caused different
types of resistance reactions. With inducer
removal the resistance was expressed as a
reduction in number of mildew colonies, while
the resistance reaction without inducer removal
furthermore includes a change in infection
type towards an infection type with necrotic
lesions.

Significance of the primary germ tube

Kunoh et ai. (1978, 1982) investigated the
incidence of host response at the primary
germ tube. They found the first response to
be host fluorescence at 2 to 3 hours after
inoculation. The appearance of fluorescence
was almost instantly followed by cytoplasmic
aggregation, and later by papillae forma-
tion. These responses were found to have
negative effect on the infection efficiency
of the appressorium some hours later, where
the primary germ tube and the appressorium
were in contact with the same epidermal cell
(Woolacott and Archer 1984). Cho and
Smedegaard-Petersen (1986) and Thordal-
Christensen and Smedegaard-Petersen (in
press, a and in press, b) found that induced
resistance could be detected as early as 1 hour
after inoculation, and that the level of induced
resistance seems to be proportional to the
diameter of the host fluorescence beneath
the primary germ tube of barley and wheat
powdery mildew. These results suggest that
the host response at the primary germ tube
is essential to infection by barley powdery

mildew. As the resistance induced by the non-
host pathogen, wheat powdery mildew, seems
to be induced mainly by the primary germ
tube, the interaction between this germ tube
and the barley epidermal cell may be deleteri-
ous to the later infection attempt from wheat
powdery mildew appressorium. The finding
that the infection efficiency of virulent barley
powdery mildew in a single inoculation pro-
cedure is reduced as inoculum density is in-
creased, may also be a consequence of re-
sistance induced by the primary germ tube.

Conclusions of the work on induced
resistance at our Department

Both virulent and avirulent races of barley
powdery mildew can induce resistance in
double inoculation procedures, and a signifi-
cant part of the resistance seems to be induced
by the primary germ tube. In single inocula-
tion procedures with virulent races this induc-
tion of resistance probably leads to infection
failure. Thus, consistent with Fig. 2, the dif-
ference between virulent and avirulent races
may be that avirulent races induce a small
amount of resistance in addition to the level
induced by virulent races, which then causes
high rates of infection failures of the avirulent
races.

Wheat powdery mildew induces most resist-
ance shortly after inoculation, suggesting that
a significant part of resistance in barley to
wheat powdery mildew may be induced by the
primary germ tube.

Induced resistance seems to be localized
principally to the cells attacked by the in-
ducer, but a significant part of the resistance
is translocated to other epidermal cells.

One of the mechanisms of induced resist-
ance is possibly an increased formation of
fluorescent papillae at the challenger infection
sites.

Energetic consequences of active resistance
on plant growth and yield

Although disease resistance has proved in-

240



valuable in plant breeding and plant produc-
tion, research at our Department has shown
that active defence processes in plants occur
at the expense of the energy resources of the
host and hence at the expense of yield.

Obviously resistance reactions are asso-
ciated with enhanced biological activities in-
cluding the accumulation of host-synthesized
antimicrobial substances, synthesis and dep-
osition of lignin-like materials, enhanced
synthesis of postinfectional polypeptides, in-
creases in certain hydrolytic enzymes such as
chitinase, accumulation of hydroxyproline-
rich glycoproteins and increased quantities of
new mRNA species (Sequeira 1983, Manners
et al. 1985, Davidson et al. 1987).

That such resistance related increases in
biosynthetic activity require considerable
amounts of energy was demonstrated by com-
paring the respiration of incompatible and
compatible interactions between barley and
the barley powdery mildew fungus (Smede-
gaard-Petersen and Stolen 1981, Smede-
gaard-Petersen 1982). After a single inocula-
tion resistant leaves reacted with a rapid tem-
porary respiratory increase, already detectable
eight hours after inoculation, returning to the
level of the non-inoculated control after three
days. When plants were subjected to three suc-
cessive inoculations spaced with intervals of
two days, the oxygen uptake initially followed
the same pattern but instead of returning to
the normal level the rate stabilized at a level
significantly higher than that of the non-
inoculated controls. Thus, repeated inocula-
tion of barley with an avirulent race of the
powdery mildew fungus causes a permanent
increase in the rate of respiration. The pro-
nounced increase in oxygen uptake coincided
in time with the appearance of papillae beneath
the appressoria (Kunoh et al. 1978), and the
synthesis and accumulation of resistance re-
lated mRNA species and proteins (Manners
et al. 1985, Davidson et al. 1987).

The high correlation between increases in
oxygen uptake and synthetic activity indicated
that the enhanced respiratory rate in incom-
patible barley powdery mildew interactions is

part of energy-requiring biosynthetic processes,
probably defence reactions, against the path-
ogen.

To investigate whether the increased energy
demand in inoculated, resistant plants is suf-
ficient to reduce plant yield, experiments were
carried out in growth chambers (Smedegaard-
Petersen and Stolen 1981). Although re-
sistant plants did not show any visible disease
symptoms after continuous inoculation with
an avirulent race, the grain yield was sig-
nificantly reduced by 7 % and the kernel
weight by 4 %. The yield of grain protein was
reduced by 11 % and straw length by 5 %.

The fact that highly resistant barley plants
did not show any visible symptoms after in-
oculation with the pathogen does not, however,
mean that the plants are not affected. The
results suggest that mildew resistant barley
plants respond to inoculation by energy-
demanding defence reactions that drain the
stored host-energy otherwise available for
build up of yield components.

Recent extensive studies (Tolstrup 1984,
Smedegaard-Petersen and Tolstrup 1985),
have demonstrated that commonly occurring
leaf saprophytes have the capacity for reducing
crop yield by inducing energy-requiring defence
reactions in much the same way as do in-
compatible races of powdery mildew. Leaf
saprophytes are present in large amounts on
the aerial parts of field crops where they
colonize the lower dead leaves and deposit
large quantities of spores on the upper green
leaves. In general, these saprophytic leaf fungi
are unable to infect vigorously growing green
leaves, but the studies referred to above clearly
suggest that saprophytic filamentous leaf
fungi, especially species of Cladosporium,
elicit active, energy-consuming defence reac-
tions similar to those elicited by spores of
avirulent barley powdery mildew. These reac-
tions cause a drain of stored host energy,
advanced senescence, and significantly less
grain yield than would appear if these reac-
tions did not happen. The yield-reducing ef-
fect of saprophytes is most marked in dense

241



crops with a humid microclimate that promotes
fungal growth and propagation.

Induced resistance as a model for molecular
studies of resistance

The previous sections have indicated that in-
duced resistance includes the same reactions as
elicitation of the natural defence mechanisms
of the plant.

With the use of biotically or abiotically in-
duced resistance an excellent tool for the
characterization of the plant response to treat-
ments is available. Thereby the molecular
genetics of the system can be investigated. In
this context we are focusing on the resistance
to barley powdery mildew induced either by
the barley powdery mildew fungus itself or by
non-host pathogenic fungi (Cho and Smede-
gaard-Petersen 1986, Thordal-Christensen
and Smedegaard-Petersen in press, a).

These experiments are based on a double
inoculation procedure as mentioned earlier.
By following the changes in the gene expres-
sion of the plant in response to the inducing
fungus, and observing the level of resistance
induced it is possible to correlate the com-
position of the mRNA population to the
level of resistance. Differences in the mRNA
population between induced and non-induced
lines of barley can be isolated and their oc-
currence investigated and quantified. These
differences should also be compared to the
changes observed during incompatible and
compatible interactions conditioned by the
presence or absence of resistance genes respec-
tively. This is necessary in order to separate
infection related responses occurring in both
resistant and susceptible plant lines from
resistance related responses occurring only in
resistant lines. This is essential when con-
sidering the similarities between induced and
resistance gene conditioned powdery mildew
resistance.

Resistance, in the case of powdery mildew
in barley is governed by resistance genes which
are normally dominant and display a 3 : 1

ratio when crossed to a recessive allele partner.
It is observed in the case of powdery mildew

on barley thatalso susceptible plants do display
a fairly large degree of resistance when ob-
serving single penetration events. Andersen
and Tore (1986) found that on the susceptible
line »Pallas» only around 30 % of the conidia
applied had formed ESH (Elongating Sec-
ondary Hyphae) 48 h after inoculation in-
dicating a successful penetration event with a
functional haustorium, compared to around
2 % on the line with the Ml-a gene. When ob-
serving the development of the fungus on
»Pallas», and the Ml-a line with the same gene
background, it was seen that on both lines the
most limiting stage was at penetration. Studies
of thereactions in the plants have also shown
that the penetration stage is the limiting stage
and that the most significant reaction is the
formation of papillae (Bushnell and Berg-
quist 1975, Kunoh et al. 1978, Johnson et al.
1979, Kita et al. 1980, Koga et al. 1980). The
difference between the lines appears to a dif-
ference in the level of resistance rather than
strictly resistance and susceptibility. The re-
sistance genes can then be called regulatory
loci amplifying the response over the level seen
in susceptible plants (Bennetzen 1984).

Considering the induction of resistance in
susceptible plants, we are able to simulate
the presence of a resistance gene and trigger
a resistant response indiscernible from the
response when a resistance gene is present.
This indicates that the susceptible plant also
contains all the genetic information needed
to express resistance at a level comparable to
the resistant line. Only the initial triggering ap-
pears to be different. The resistance gene can
then be postulated to be responsible for the
triggering in the genetically resistant line.

It seems that the interaction with the fungus
is controlled by interaction with both primary
gene products exerting an effect on the ex-
pression of resistance or being effective on
their own, and the secondary metabolism of
the plant (formation of physical and chemical
compounds having an effect on the pathogen).

242



7

Primary geneproducts involved in resistance

Constitutively expressed. Due to the lack of
precise information on how the elicitation of
resistance actually takes place, certain assump-
tions must be made.

1) It is reasonable to assume that plants con-
tain sensing mechanisms that are able to detect
attacking organisms or elicitating compounds
in the cell environment, and start counter-
measures against ingress of the pathogen.

2) It is also plausible that the sensing mech-
anisms at least to some extent are able to
distinguish between fungi. In the case of the
most specialized systems to which the powdery
mildew in barley belongs, a level of fungal
race/host gene specific interaction exists that
is essential for the final expression of resistance
(Ellingboe 1972).

At least part of this sensor/trigger system
needs to be present at all times in the plant,
and therefore the gene(s) involved very likely
are constitutively expressed. Whether there is
induced expression of additional genes or the
expression of the system as a whole is increased
when resistance is induced or a resistance gene
controlled incompatible interaction develops,
is not known.

Investigations on the molecular genetics of
the sensing process and triggering of resistance
should be performed using non-inoculated
plants, due to at least two reasons. Firstly, one
should expect that the constitutive level of
expression of the resistance gene is low con-
sidering that there are probably many systems
for the pathogens the plants are in contact
with. The energy demand on the plant would
be large if a high level of expression was to
be maintained. Secondly, using inoculated
plants the bulk of changes in gene expression
is very likely to mask subtle differences between
lines mediated by the presence of resistance
genes.

The response to infection after induction of
resistance in susceptible plants appears indis-
cernible from the response when a resistance
gene is present. How far these similarities ex-
tend when we study the induced resistance at

the level of gene expression is not known, but
most of the observable responses are derived
from secondary metabolism in the plant (see
next section).

Induced expression of PR-proteins. Many
dicotyledonous plants have been reported to
produce a special class of proteins as a response
to viral infection. These proteins occurred
at the time of hypersensitive necrosis forma-
tion and are characterized by low molecular
weight (15 —20 kD), solubility at low pH,
protease resistance and their presence in the
intercellular space in cells undergoing hyper-
sensitive collapse (Van Loon 1985). The pro-
teins have been called pathogenesis-related
(PR) proteins, as they appeared to be more
involved in the general response to pathogenic
conditions than in the expression of resistance
as such. A role has been proposed in hyper-
sensitive collapse reactions, but no proof has
been presented (Van Loon 1985).

Until recently these proteins were con-
sidered to be unique to dicotyledonous species
responding to viral infection, but recent studies
at Rothamsted have shown the presence of PR-
proteins in barley undergoing hypersensitive
collapse reactions (White et al. 1987). That
is a reaction in a monocotyledonous plant to
a fungus.

Until recently no biochemical function could
be suggested for these proteins, but Richard-
son et al. (1987) have observed homology
between a maize protein that inhibits bovine
trypsin and a-amylase in vitro and a tobacco
PR-protein. PR-protein-like compounds have
also been found in wounded tomato plants
after squeezing the leaf blade. These proteins
had a molecular weight of only around half
the average molecular weight of the tobacco
PR-proteins, but they also possessed a strong
inhibitory effect on proteinases (Graham et
al. 1985). These proteins occurred in leaves
that were undergoing collapse reactions in
order to limit the wounded area from the
healthy leaf area. This somewhat extends the
definition of PR-proteins as only being in-
volved in pathogenesis to rather being in-
volved in collapse reactions more generally.

243



If the PR-proteins were to be assigned a func-
tion in protein/protein interaction it would
make sense in many respects. The synthesis
and transport to the intercellular space results
in inhibition of an intercellular, cell wall or
outer membrane protein essential for the func-
tion of the cellular membrane resulting in loss
of electrolytes from the cell ending with death
of the cell (Van Loon 1985).

Secondary metabolism involved in resistance

Resistance to powdery mildew is observed
in the plant cell as a sequence of responses.

The first visible reaction is the formation
of a cytoplasmatic aggregate with a papilla
at the site of the primary germ tube as ob-
served by Kunoh et al. (1982). This papilla
may also contain substances that fluoresce in
UV light (Kunoh et al. 1982). The second
visible reaction is the formation of similar
structures at appressorial germ tubes at the site
of penetration attempts (Bushnell and Berg-

quist 1975, Aist and Israel 1977). The pres-
ence of substances fluorescing under UV-light
in these papillae seems to enhance the level of
resistance to penetration (Kita et al. 1980,
Koga et al. 1980). The fluorescence is initially
limited to the papilla and the surrounding
halo, but in case of penetration of the papilla
the fluorescence of the papilla can spread all
over the plant cell, followed by disintegra-
tion (hypersensitive collapse) of the host cell,
leading to death of the host cell (Johnson et
al. 1979, Koga et al. 1980, Kita et al. 1981).

Studies of different powdery mildew resist-
ance genes have shown that the fluorescence of
whole cells and host cell collapse is a common
phenomenon in particular in postpenetration
events (Koga et al. 1980). These authors
showed that the later the collapse reactions
started, the more cells were affected. This was
also found by Andersen and Jiang (1984),
who furthermore reported intense reactions in
the mesophyll leading to both visible chlorosis
and necrosis in the case of the resistance genes
Ml-p and Ml-(1402).

Unfortunately very little is known about the
pathways of secondary metabolism in barley.
The papillae have been shown to contain
lignin in wheat (Ride and Pearce 1979), but
not in barley (Smart et al. 1986). Smart et al.
(1986) failed to detect any suberin deposi-
tion in papillae. Lignin and suberin obtain
the main part of their monomers from the
shikimic acid pathway, but also the informa-
tion on the enzymes frequently reported to be
stimulated in dicotyledonous plants are very
limited in barley. Suberin furthermore con-
tains a large fraction of fatty acids, presumably
mobilized from an endogenous pool.

Callose has been demonstrated in barley
papillae by Smart et al. (1986) and Skou
(1982). The key enzyme in callose deposition
is UDP glucose: glucan synthetase whose
product is /31-3 glucans. Very scarce informa-
tion is available on this enzyme, and nothing
is to our knowledge published on barley. The
articles reporting on the presence of callose in
papillae do not go into deeper studies of the
biochemistry of papilla formation.

In the barley/powdery mildew system no
fungitoxic (phytoalexins) compounds have
been identified, although initial reports by
Oku and Ouchi (1976) and Oku et al. (1975)
have been addressing the subject. They ob-
tained results from exudates of inoculated
leaves stating that substances having an in-
hibitory effect on the germination of powdery
mildew conidia were present in the plant,
with a peak of activity in incompatible in-
teractions around 12 h after inoculation.
These substances have not to our knowledge
been dealt with further, but the occurrence of
phytoalexins from other cereals have been
reported after theseresults were published. In
rice the momilactones (A and B) were identified
by Cartwright et al. (1977) and in oats the
avenalumins was identified by Mayama et al.
(1981).

These substances are not derivatives of the
shikimic acid pathway indicating that this
pathway has a lower significance in cereals
than in the dicotyledonous species mostly
studied.

244



Comparing this with what was reported on
the chemically induced resistance in rice (Oryza
saliva) to the rice blast fungus (Pyricularia
oryzae) by applying compounds of the di-
chlorocyclopropane group might give some in-
formation on the reactions taking place in
barley. These investigations showed some
characteristic features in the treated plants be-
fore inoculation. Treated plants had peroxidase
activities 12 times higher than non-treated
plants, lAA-oxidase activity 1.7 times higher
and catalase 0.5 times the activity of non-
treated plants (Langcake and Wickins 1975).
These authors reported that they found no
changes in PAL, TAL and (3-galactosidase in
response to treatment neitherbefore nor after
inoculation.

These changes were apparently sufficient to
condition the susceptible rice cultivar to be
resistant and resulted in a much faster forma-
tion of brown pigment, said to be melanin
(Langcake and Wickins, 1975), although the
main monomer of melanin (catechol) could
not be shown before inoculation.

It is significant to note that these authors did
not observe any visible symptoms in the plants
after treatment (Langcake and Wickins,
1975), which corresponds to our failure to ob-
serve any changes in the cells, such as diffuse
cellular fluorescence, after induction (Thor-
dal-Christensen and Smedegaard-Petersen,
in press, b).

Hislop and Stahmann (1971) reported that
the activity of peroxidase increased in a range
of host lines tested after inoculation with the
barley powdery mildew fungus. The increase
was observed in both compatible and incom-
patible interactions, being slightly greater at
24 hours after inoculation in incompatible in-
teractions. The pattern of peroxidase increase
during the first 24 hours followed the same

line in both compatible and incompatible in-
teractions, and comparing to induced resistance
it follows the trend of the data presented in
Fig. 2, with the avirulent and virulent isolate
being equally efficient as resistance inducers
before 10—12 hours after inoculation and the
avirulent isolate being best at induction periods
longer than 12 hours.

This is no evidence for the involvement of
peroxidase in induced resistance, but it is
suggested that peroxidase could be used as
a marker enzyme for the biochemical and
molecular studies of resistance. Much knowl-
edge is lacking on other enzyme systems being
involved in the response to fungal infection.
The studies of the gene expression associated
with induced resistance and resistance gene
conferred resistance are just beginning, and
hopefully we will be able to employ and com-
bine the new technology with the current
knowledge to get a more complete picture of
the genetic machinery involved in resistance.

Transgenic plants with improved resistance

For the purpose of making transgenic plants
with improved resistance a number of factors
need to be resolved before success is to be
expected. First of all, our transformation
systems need to be improved, but in the light
of recent developments on cereal transforma-
tion such possibilities may have improved
(De La Pena et al. 1987). Second, we need to
localize and characterize the sensor/trigger
system and to study the molecular mechanisms
in its function. Third, we must try to modulate
the effect to give an improved effect against
more races of the pathogen and a better am-
plification of the resistant response, and
fourth, we need to know more about the
complex mechanisms regulating the resistant
response in the later stages.

245



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Ms received August 19, 1987

248



SELOSTUS

Kasvin puolustusmekanismien
aktivoituminen

Hans Thordal-Christensen,
Per L. Gregersen, Jan B. Andersen
ja Viggo Smedegaard-Petersen
Department of Plant Pathology, The Royal Veterinary
and Agricultural University, Thorvaldsensvej 40,
1871 Frederiksberg C, Copenhagen, Denmark

Katsauksessa tarkastellaan kasvien indusoituvaa resis-
tenssiä. Se perustuu fysikaalisiin ja kemiallisiin mekanis-
meihin, jotkaalkavat toimia patogeenin tai saprofyytin
aiheuttaman infektion tai näiden aineenvaihduntatuottei-
den vaikutuksesta. Indusoituvan resistenssin ja kasvin nor-
maalin taudinkestävyyden perusmekanismit lienevät sa-
mankaltaiset. Indusoituvan resistenssin aktivoivat elisi-
torit, kemialliset aineet, joita on eristetty useista sieni-
patogeeneistä jakasvin soluseinästä. Useimmat elisitorit
ovat epäspesifisiä ja vain muutamat spesifisiä eli aktivoivat
vain tiettyjen lajikkeiden resistenssin. Toistaiseksi ei tie-
detä, miten elisitorien laukaisema viesti välittyy kasviso-
lun reseptoriin ja saa resistenssimekanismit toimimaan.

Tanskan maatalouskorkeakoulun kasvipatologian lai-
toksella tutkitaan ohran indusoituvaa härmäresistenssiä.

Sekä virulentit että ei-virulentit isolaatit indusoivat resis-
tenssin mutta ei-virulenttien aikaansaama resistenssi on
alkuvaiheessa tehokkaampaa. Vehnän härmäisolaatti in-
dusoi ohrassa voimakkaamman resistenssin kuin vastaa-
va ohran isolaatti. Indusoituva resistenssi paikallistuu pää-
asiassa infektiokohdan epidermisoluihin mutta se voi il-
metä myös infektiokohdan ympäristössä.

Härmäresistenssi lisää kasvin energian tarvetta, min-
kä on havaittu pienentävän ohran jyväsatoanoin 7 %.

Resistenssi perustuu yksittäisiin major-geeneihin ja sen
ilmenemiseen liittyy lähetti RNA:n japroteiinien syntee-
siä. Nykyisen molekyylibiologian, erityisesti yhdistelmä-
DNA -tekniikan, avulla voidaan tutkia indusoituvan re-
sistenssin molekulaarista perustaa, aktivoitumista ja il-
menemistä, mikä ei aiemmin ole ollut mahdollista.

249