Cross-talk between jasmonate and salicylate plant defense pathways: Effects on several plant parasites

Abstract

Plants are often attacked by many herbivorous insects and pathogens at the same time. Two important suites of responses to attack are mediated by plant hormones, jasmonate and salicylate, which independently provide resistance to herbivorous insects and pathogens, respectively. Several lines of evidence suggest that there is negative cross-talk between the jasmonate and salicylate response pathways. This biochemical link between general plant defense strategies means that deploying defenses against one attacker can positively or negatively affect other attackers. In this study, we tested for cross-talk in the jasmonate and salicylate signaling pathways in a wild tomato and examined the effects of cross-talk on an array of herbivores of cultivated tomato plants. In the wild cultivar, induction of defenses signaled by salicylate reduced biochemical expression of the jasmonate pathway but did not influence performance of S. exigua caterpillars. This indicates that the signal interaction is not a result of agricultural selection. In cultivated tomato, biochemical attenuation of the activity of a defense protein (polyphenol oxidase) in dual-elicited plants resulted in increased of performance of cabbage looper caterpillars, but not thrips, spider mites, hornworm caterpillars or the bacteria Pseudomonas syringae pv. tomato. In addition, we tested the effects of jasmonate-induced resistance on the ability of thrips to vector tomato spotted wilt virus. Although thrips fed less on induced plants, this did not affect the level of disease. Thus, the negative interaction between jasmonate and salicylate signaling had biological consequences for two lepidopteran larvae but not for several other herbivores tested or on the spread of a disease.

Full text

Abstract Plants are often attacked by many herbivorous
insects and pathogens at the same time. Two important
suites of responses to attack are mediated by plant hor-
mones, jasmonate and salicylate, which independently
provide resistance to herbivorous insects and pathogens,
respectively. Several lines of evidence suggest that there
is negative cross-talk between the jasmonate and salicy-
late response pathways. This biochemical link between
general plant defense strategies means that deploying de-
fenses against one attacker can positively or negatively
affect other attackers. In this study, we tested for cross-
talk in the jasmonate and salicylate signaling pathways
in a wild tomato and examined the effects of cross-talk
on an array of herbivores of cultivated tomato plants. In
the wild cultivar, induction of defenses signaled by sali-
cylate reduced biochemical expression of the jasmonate
pathway but did not influence performance of S. exigua
caterpillars. This indicates that the signal interaction is
not a result of agricultural selection. In cultivated toma-
to, biochemical attenuation of the activity of a defense
protein (polyphenol oxidase) in dual-elicited plants re-
sulted in increased of performance of cabbage looper
caterpillars, but not thrips, spider mites, hornworm cater-
pillars or the bacteria Pseudomonas syringae pv. tomato.
In addition, we tested the effects of jasmonate-induced
resistance on the ability of thrips to vector tomato spot-
ted wilt virus. Although thrips fed less on induced plants,
this did not affect the level of disease. Thus, the negative
interaction between jasmonate and salicylate signaling
had biological consequences for two lepidopteran larvae
but not for several other herbivores tested or on the
spread of a disease.
Keywords Cross-talk · Induced defense · Jasmonate ·
Salicylate · Herbivory
Introduction
Plants must protect themselves against many invading
organisms that consume, infect, and damage their tissue
in various ways (Hatcher 1995). Attack by one organism
may be associated with attack by other organisms. For
example, many herbivorous insects are vectors for plant
disease. The physical wound created by insect feeding
may itself facilitate the entry of opportunistic pathogens.
Conversely, some insects such as aphids and thrips pre-
fer the yellow color of diseased plants (Dixon 1998).
Given that insect and pathogen attack are often positive-
ly associated, plants should utilize defense systems that
are either effective against both types of attackers, or at a
minimum that do not interfere with defenses against oth-
er attackers. Some initial work supported this idea of co-
ordinated defenses against insects and pathogens (e.g.,
McIntire et al. 1980; Karban et al. 1987). However, there
is a growing body of evidence demonstrating that the
major biochemical pathways that mediate plant resis-
tance to diverse attackers interact with each other and
that these interactions can be negative.
Induced resistance involves plant-mediated changes
associated with initial attack by herbivores and patho-
gens that negatively influence subsequent attackers
(Karban and Baldwin 1997; Agrawal et al. 1999). The
jasmonate pathway (i.e., the octadecanoid pathway) and
the salicylate pathway (conditioning systemic acquired
resistance, SAR) are two of the biochemical response
mechanisms that can be triggered by various attackers,
and the components of these pathways, jasmonic and sal-
J.S. Thaler (✉)
Department of Botany, 25 Willcocks St., University of Toronto,
Toronto, M5 S 3B2 Canada
e-mail: thaler@botany.utoronto.ca
Fax: +1-416-9785878
R. Karban · D.E. Ullman · K. Boege
Department of Entomology, University of California,
Davis, CA 95616, USA
R.M. Bostock
Department of Plant Pathology, University of California,
Davis, CA 95616, USA
Present address:
K. Boege, Biology Department, University of Missouri-St. Louis,
8001 Natural Bridge Road, St. Louis, MO 63121, USA
Oecologia (2002) 131:227–235
DOI 10.1007/s00442-002-0885-9
PLANT ANIMAL INTERACTIONS
Jennifer S. Thaler · Richard Karban · Diane E. Ullman
Karina Boege · Richard M. Bostock
Cross-talk between jasmonate and salicylate plant defense pathways:
effects on several plant parasites
Received: 17 July 2001 / Accepted: 3 January 2002 / Published online: 2 March 2002
© Springer-Verlag 2002

icylic acids, function as necessary signaling molecules
that mediate such defensive responses (McConn et al.
1997; Hammerschmidt and Smith Becker 1999). Endog-
enous jasmonic acid (JA) induces putatively defensive
phytochemicals and proteins such as proteinase inhibi-
tors and oxidative enzymes. Some of these induced com-
pounds have been causally linked to increased plant re-
sistance to many insects and some pathogens (Duffey
and Stout 1996; Karban and Baldwin 1997; Vijayan et al.
1998). Endogenous salicylic acid (SA) results in the pro-
duction of a suite of phytochemicals that is correlated
with protection from many pathogen attackers (Ryals et
al. 1992; Hammerschmidt and Nicholson 1999). Jasmon-
ate and salicylate each trigger an array of biochemical
responses and products, some of which overlap, although
many are distinct (Choi et al. 1994; Niki et al. 1998;
Pieterse and van Loon 1999; Bostock 1999; Schenk et al.
2000).
The jasmonate and salicylate pathways can negatively
interact with each other at the biochemical level. Labora-
tory experiments have demonstrated an antagonistic in-
teraction between pathways in tomato, tobacco, and
Arabidopsis thaliana plants (Doherty 1988; Peña-Cortez
et al. 1993; Doares et al. 1995a, b; Sano et al. 1995; Niki
et al. 1998; Felton et al. 1999; Fidantsef et al. 1999;
Gupta et al. 2000). These pathway interactions also oc-
cur in cases where plants are attacked by biotic challeng-
ers (Preston et al. 1999; Stout et al. 1999). For example,
tobacco plants infected with tobacco mosaic virus dis-
played decreased ability to induce JA in response to me-
chanical wounding and increased susceptibility to the
herbivore Manduca sexta (Preston et al. 1999). However,
recent evidence indicates that the pathway interactions
are not always negative, depending on the dose and tim-
ing of elicitation (Doherty et al. 1988; Niki et al. 1998,
Thaler et al. 2002) and the response measured (Schenck
et al. 2000). There is also growing evidence of salicylate
and jasmonate independent plant defense responses
(Pieterse and van Loon 1999) some of which can act in a
compensatory manner when one pathway is disabled
(Boland et al. 1998). This complexity of signaling makes
the answer to the question of how cross-talk in signaling
pathways affects biotic challengers less predictable.
Thus, to understand the ecological significance of signal
interactions in plant defense we must measure the actual
level of plant resistance to herbivores rather than the
level of specific defense-related compounds.
Elicitors of the jasmonate and salicylate pathways are
being evaluated for use in agricultural pest management
and to better understand how plants coordinate defensive
responses. Two elicitors were used in the current study,
JA and the synthetic functional analog of salicylic acid,
BTH (benzothiadiazole), to induce the corresponding re-
sponse pathways. These chemical elicitors were used to
identify potential interactions between the jasmonate and
salicylate response pathways, and to avoid confounding
influences of actual leaf damage or infection by biotic
agents. Application of BTH to leaves mimics the effect
of SA, apparently by interacting with the same cellular
sites of action as SA (Lawton et al. 1996; Tally et al.
1999), and does not result in a local increase in endoge-
nous SA. Thus, all or a significant subset of the defense-
related biochemical responses induced by SA are also in-
duced by BTH.
We previously showed that simultaneous application
of both JA and BTH to field grown tomato plants result-
ed in attenuated expression of hallmark biochemical re-
sponses to these inducers compared to plants induced
with just a single elicitor (Thaler et al. 1999). Polyphenol
oxidase, a JA responsive protein, had lower activity in
plants elicited with both JA and BTH compared to plants
elicited with only JA. Accumulation of pathogenesis-
related protein 4 mRNA (P4), a SA responsive protein,
was reduced in plants elicited with both JA and BTH
compared to plants elicited with only BTH. This nega-
tive interaction in the chemical expression of the two
pathways corresponded to a reduction in resistance to a
herbivore. We found that plants simultaneously induced
with JA and BTH had compromised resistance to the
caterpillar Spodoptera exigua, compared to plants in-
duced with JA alone.
The ecological and agricultural importance of plant
signaling interactions will depend on the occurrence of
negative interactions in other plants and the range of her-
bivores that are affected by the jasmonate- and salicy-
late-mediated responses. We evaluated this by (1) testing
for signal interaction in a wild tomato variety, (2) testing
the performance of four herbivore species and a patho-
gen feeding on cultivated tomato plants that were elicit-
ed with JA and BTH singly as well as dual-elicited
plants where the two pathways showed a negative bio-
chemical interaction and (3) testing whether induced re-
sistance affected feeding behavior of thrips and the thrips
vectored spread of tomato spotted wilt virus.
Materials and methods
General methods
Tomato plants were grown in 4-inch pots containing UC soil mix
in a greenhouse. Plants were grown for approximately 1 month,
until the four-leaf stage, after which the treatments were applied.
JA and BTH were applied to plants using hand held atomizers.
The JA was prepared in aqueous suspension using acetone to help
disperse it in water (1% acetone in water). BTH was dissolved in
water. The solutions/suspensions were sprayed to runoff onto the
desired portion of the plant, shielding the rest of the leaves. Con-
trol plants were sprayed with an equal amount of water with ace-
tone. Previous experiments demonstrated that the effects of BTH
treatment are not simply due to the absence of acetone. The
growth rate of S. exigua caterpillars feeding on BTH elicited
plants was higher compared to caterpillars feeding on control
plants that had been treated with water not containing acetone
(mean±SE BTH: 0.49±0.09, Control: 0.22±0.03).
Elicitation of wild tomato
We tested for negative interactions between jasmonate- and salicy-
late- mediated induced plant resistance on a wild variety of toma-
to, Lycopersicon esculentum var. cerasiforme from Papantla, Vera-
cruz, Mexico. Var. cerasiforme is the immediate ancestor of the
228

cultivated tomato and is common in Mexico, Central and South
America (Rick 1995). This experiment was conducted to ensure
that the signal interaction was not simply an artifact of agricultural
selection.
These experiments were conducted using plants in four treat-
ments (1) control, (2) 1.5 mM JA, (3) 1.2 mM BTH and, (4) si-
multaneous 1.5 mM JA and 1.2 BTH (n=12 per treatment). The
third youngest leaf of plants at the four-leaf stage was sprayed
with the elicitors. Activity of polyphenol oxidase, performance of
S. exigua, and Pseudomonas syringae pv. tomato lesion formation
were assayed.
Polyphenol oxidase is an enzyme induced by the jasmonate
pathway in tomato plants. Polyphenol oxidase activity was chosen
as a marker of the jasmonate-induced response because of its con-
sistent systemic pattern of induction following herbivore damage
and JA spray (Thaler et al. 1996). Systemic activity of polyphenol
oxidase was determined 2 days following elicitor application in
the terminal leaflet of the fourth leaf according to the methods de-
scribed in Thaler et al. (1996). Briefly, we extracted the enzymes
from weighed leaflets that were homogenized in ice-cold buffer
and the homogenate was centrifuged to obtain a clarified extract
for enzyme analyses. The supernatant was added to a caffeic acid
solution and absorbance read at 470 nm. Polyphenol oxidase ac-
tivity in the JA and JA/BTH-treated plants was compared using
two-way ANOVA.
Herbivore bioassay
Newly hatched S. exigua caterpillars were reared on artificial diet
(Southland Products) for 3–5 days before the bioassays. Two days
following elicitor application, caterpillars were placed individually
on a single adjacent leaflet from the fourth leaf in 90-mm petri
dishes lined with moist filter paper. The caterpillars were weighed
at the beginning of the experiment, and again at the end, after
2–3 days of feeding on the specified leaflets. The assays were ter-
minated before the leaf began to deteriorate and before the cater-
pillars could consume the entire leaflet. The relative growth
rate [(final weight–initial weight)/(initial weight ×number of
days)] (RGR), and gross growth efficiency [(final weight–initial
weight)/leaf area consumed)] (ECI) were calculated (Waldbauer
1968). The effect of jasmonate and salicylate elicitation on each
performance measure was compared using two-way factorial
ANOVA.
Bacterial speck disease assay
Pseudomonas syringae pv. tomato (Pst) isolated from field-grown
tomato plants (isolate Pst23, gift of D. Cooksey, Department of
Plant Pathology, UC Riverside) was incubated at 27°C for 48–72 h
on King’s B medium and colonies were suspended in sterile water.
An aqueous suspension of 107colony-forming units per milliliter
was gently painted on the attached terminal leaflet of the fifth leaf
of intact plants with a cotton applicator, and the plants were incu-
bated in the greenhouse. There is a compatible interaction between
this Pst strain and the plant. Seven days later the number of le-
sions on the inoculated leaflet was counted as a measure of dis-
ease. There is a strong correlation between the number/amount
of lesions and bacterial populations (colony forming units/cm2)
(C. Richael, personal communication). The effect of jasmonate
and salicylate elicitation on disease was compared using two-way
factorial ANOVA.
Effect of signaling cross-talk on multiple herbivores
In many greenhouse and field experiments, noctuid caterpillars in-
cluding S. exigua and Helicoverpa zea have shown improved per-
formance on BTH-elicited plants compared to control plants and
on BTH- and JA-elicited plants compared to plants only elicited
with JA (Stout et al. 1999; Thaler et al. 1999). We examined if a
range of insects are affected by the jasmonate and salicylate signal
interaction, by testing the effects of JA and BTH application on
spider mites, thrips, hornworms, cabbage loopers, and bacterial
speck disease. The cultivated tomato (L. esculentum cv. New
Yorker) was used for these experiments. A JA and BTH elicitor
regime where the chemical signaling interactions are most consis-
tent was chosen (Thaler et al. 1999, unpublished data). These ex-
periments were conducted using plants in four treatments (1) con-
trol, (2) 1.5 mM JA, (3) 1.2 mM BTH and, (4) simultaneous
1.5 mM JA and 1.2 mM BTH. The third leaf of plants at the four-
leaf stage was sprayed with the elicitors. Nine sets of 48 plants
were used in total for these experiments. Twelve replicates of each
treatment were conducted during each experiment.
Polyphenol oxidase activity was assayed on four of the nine
sets of plants: one set that was used for a thrips, spider mite and
Pst assay, one set that was used for a hornworm assay, one set that
was used for a hornworm and a thrips assay, and one set for the
cabbage looper assay. Polyphenol oxidase activity was measured
to ensure that the negative biochemical signal interaction had oc-
curred between the jasmonate and salicylate pathways as in previ-
ous experiments. In the four sets of plants where polyphenol oxi-
dase was measured, the adjacent leaflet of the fourth leaf was ex-
cised from all 12 plants in each of the four treatments. There was
statistically significant lower polyphenol oxidase activity in the
plants that were elicited with JA and BTH compared to plants that
were elicited with JA alone. This indicates that the chemical cross-
talk had occurred (see Results).
The effects of this signal interaction on plant resistance to
western flower thrips (Frankliniella occidentalis), two-spotted spi-
der mites (Tetranychus urticae), tobacco hornworm caterpillars
(Manduca sexta), cabbage looper caterpillars (Trichoplusia ni) and
the causal agent of bacterial speck disease (Pst) were tested. These
are organisms that naturally feed on or infect tomato plants in the
field. Thrips, spider mites, cabbage loopers and Pst are general-
ists; hornworm caterpillars are specialists on Solanaceous plants.
Thrips and spider mites are cell content feeders, hornworm and
cabbage looper caterpillars are leaf chewers, and Pst is a bacteri-
um causing lesions on leaves, stems and fruits. These organisms
were obtained from laboratory colonies.
The terminal or adjacent leaflet of the fourth leaf was collected
3 days after JA and BTH applications for the herbivore bioassays.
For the thrips, hornworm, and cabbage looper caterpillar assays,
leaflets were placed in a petri dish lined with moist filter paper
and sealed with Parafilm. Either individual second instar thrips
larvae or adult thrips were placed on each leaflet and allowed to
feed for 3 days after which survivorship and the amount of leaf
area eaten (mm2) was quantified using an acetate grid. Single
newly hatched hornworm or cabbage looper caterpillars were
placed on each leaflet and allowed to feed for 3 days after which
survivorship and RGR were measured. Because the spider mite
bioassays lasted longer, the cut end of the petiole was covered
with a moist cotton ball to maintain leaf water content. Three fe-
male spider mites were placed on each leaflet and allowed to re-
produce for 10 days, after which the number of eggs, immatures,
and adults was counted using a stereo microscope. Pst was inocu-
lated onto the attached terminal leaflet of the fourth leaf and le-
sions counted 7 days later using the same methods as in the wild
tomato experiment described above. Using the nine sets of plants,
one trial of the cabbage looper and Pst assay, two trials of the spi-
der mite and hornworm assay, and six trials of the thrips assay
were conducted. Three trials were conducted using immature
thrips and three trials were conducted using adult thrips. Because
the immature and adult thrips responded in the same way to the
elicitor treatments, the results were pooled. Bioassays were con-
ducted on excised leaflets and therefore some sets of plants were
used for challenges against two or three herbivore species (hence
12 assays were conducted with 9 sets of plants).
Vector transmitted disease
We examined the effect of induction of the jasmonate pathway on
the development of disease caused by tomato spotted wilt virus
229

(TSWV), a virus frequently vectored by thrips. We tested whether
expression of the jasmonate-regulated responses or the reduced
feeding of thrips on jasmonate-induced plants would affect viral
transmission and replication. Ten-day-old greenhouse-grown to-
mato plants (cv. Celebrity) were divided into two groups of 25
plants each, half that were treated with 0.5 mM JA, the other half
left as controls. Two days later, polyphenol oxidase activity was
measured on an excised leaflet from eight control and eight
sprayed plants to ensure that the jasmonate pathway was induced.
The plants were then transferred to the laboratory and randomly
interspersed in a Plexiglas cage (2×0.9×0.9 m). Approximately
900–1,000 viruliferous thrips, obtained from a colony feeding on
Emilia sonchifolia plants infected with TSWV, were released into
the cage containing 25 induced and 25 control plants. Because all
of the plants were in a single cage, the feeding choices of the
thrips could have been influenced by neighboring plants. Four
days after the thrips were released, new leaflets were collected to
measure polyphenol oxidase activity in eight control and eight jas-
monate-induced plants. A different subset of plants was assayed
from the ones assayed for polyphenol oxidase activity prior to
thrips release. The amount (mm2) of feeding damage on all plants
was quantified using an acetate grid. The plants were then fumi-
gated to remove all thrips and allowed to grow for 3 weeks to al-
low the virus to develop. Enzyme-linked immunosorbent assay
was conducted to quantify the presence of TSWV using methods
similar to Ullman et al. (1993). TSWV infected E. sonchifolia and
tomato were used as positive controls, while healthy plants of the
same age were used as negative controls. The samples were read
at 405 nm 60 min after the start of the reaction. Plant samples with
optical density readings greater than two standard deviations be-
yond the mean of the negative controls were considered positive.
A second trial of this experiment was conducted using similar
methods except that 15 control and 15 induced plants were used
and plates were read 30 and 60 min after the start of the reaction.
Polyphenol oxidase activity and amount of thrips feeding damage
were analyzed using two-way ANOVA with induction treatment
and trial as the main effects. Virus titers were analyzed using
MANOVA with time 30 and 60 as the dependent variables.
Results and discussion
Elicitation of wild tomato
JA stimulated and BTH reduced the activity of polyphe-
nol oxidase compared to controls. Polyphenol oxidase
activity was intermediate in the dual- elicited plants
(Fig. 1 a, Table 1). This result confirmed the negative ef-
fect of SAR activation on the jasmonate pathway that we
observed previously in the cultivated tomato. Neither JA
or BTH affected the RGR or ECI of S. exigua (Fig. 1b, c,
Table 1). Surprisingly, the number of Pst lesions was
increased by BTH and not affected by JA (Fig. 1d,
Table 1). We observed much higher levels of disease on
these plants compared to what is typically observed on
cultivated tomato plants in similar greenhouse trials (see
data below).
Some features of the negative chemical signaling in-
teraction between JA and SA were seen in the wild to-
mato. Polyphenol oxidase activity was reduced on dual-
treated plants compared to JA-treated plants alone. How-
ever, this did not increase performance of S. exigua on
dual- treated plants, indicating discordance between bio-
chemical responses and biological resistance. The Pst re-
sults were surprising, with elicitation of SAR by BTH
dramatically increasing lesion number. This is contradic-
tory to our results with the cultivated tomato and may re-
present differential responses to BTH in the wild tomato.
Thus, although further studies are required, negative sig-
nal interactions between jasmonate and salicylate signal-
ing seem likely in wild systems (Preston et al. 1999).
230
Fig. 1a–d The effect of JA (1.5 mM) and BTH (1.2 mM) treat-
ment on the wild tomato, Lycopersicon esculentum cv. cerasi-
forme. aActivity of polyphenol oxidase (∆OD/g/min) (PPO), per-
formance of the herbivore Spodoptera exigua measured as b
relative growth rate [(final weight–initial weight)/(initial
weight×number of days)] (RGR), and cgross growth efficiency
[(final weight-initial weight)/mm2leaf area consumed] (ECI), and
ddisease caused by the pathogen Pseudomonas syringae pv.
tomato (mm2of lesions formed) was measured. Bars indicate
mean±SE

Effect of signaling cross-talk on multiple herbivores
A negative effect of SAR induction on jasmonate induc-
tion was detected in the plants used for the herbivore
bioassays. We measured polyphenol oxidase activity in
four of the nine sets of cultivated tomato plants that were
used for the herbivore assays. In all four sets of plants
where polyphenol oxidase activity was measured BTH
itself did not affect polyphenol oxidase activity; however
BTH elicitation reduced the polyphenol oxidase activity
in plants elicited with JA and BTH. In other words,
plants induced with jasmonate alone had higher polyphe-
nol oxidase activity than plants elicited with JA and
BTH (spider mite, thrips, Pst trial: P=0.036; hornworm
trial: P=0.045; hornworm, thrips trial: P=0.021, cabbage
looper trial: P=0.002).
Although the attenuation of resistance to S. exigua in
the dual- elicited cultivated tomato plants (Thaler et al.
1999, unpublished data) is highly repeatable, this was
not reflected in attenuation of resistance to most of the
other herbivores assayed. Resistance to the cabbage loo-
per was attenuated on the dual- elicited plants (Fig. 2 a,
Table 2). Cabbage looper RGR was increased on the
SAR-activated plants and decreased on the jasmonate-
activated plants. There was no effect of jasmonate
or SAR activation on survivorship of thrips (G=5.08,
P=0.17, n=238, Fig. 2b). The amount of damage by
thrips was decreased on jasmonate-activated plants and
was not affected by SAR activation, compared to con-
trols (Fig. 2c, Table 2). In the dual-elicited plants, the
amount of damage was similar to that of the plants elicit-
231
Table 1 Elicitation of wild tomato. ANOVA analysis of polyphe-
nol oxidase activity, the performance of herbivores and the levels
of disease on plants in four treatments: Control, JA, BTH and
BTH/JA. The elicitor (1.5 mM JA and 1.2 mM BTH) was simulta-
neously applied to plants. Polyphenol oxidase activity (PPO) was
calculated as the ∆OD/g/min. The relative growth rate (RGR) of
Spodoptera exigua caterpillars is the (final weight–initial
weight)/(initial weight) and gross growth efficiency (ECI) is (final
weight–initial weight)/ (leaf area consumed). Disease caused by
Pseudomonas syringae pv. tomato was measured as the mm2of le-
sions
Experiment Factor df F P
PPO activity JA 1 10.885 0.002
BTH 1 7.209 0.010
JA×BTH 1 2.331 0.134
Error 44 – –
RGR of S. exigua JA 1 1.497 0.228
BTH 1 0.023 0.879
JA×BTH 1 0.037 0.849
Error 42 – –
ECI of S. exigua JA 1 1.944 0.171
BTH 1 1.887 0.177
JA×BTH 1 0.025 0.852
Error 42 – –
Pst lesions JA 1 2.05 0.159
BTH 1 6.186 0.017
JA×BTH 1 2.017 0.163
Error 44 – –
Table 2 Generality of antagonism. ANOVA analysis of the per-
formance of herbivores on plants in four treatments: Control, JA,
BTH and BTH/JA. The elicitor (1.5 mM JA and 1.2 mM BTH)
was simultaneously applied to plants. The RGR of hornworm and
cabbage looper caterpillars is the (final weight–initial weight)/(ini-
tial weight). Thrip damage is measured as mm2of damage. The
number of spider mites is the sum of adults, immatures, and eggs.
Disease caused by P. syringae pv. tomato was measured as the
mm2of lesions. All interactions were included in the model; non-
significant interactions are not reported
Experiment Factor df F P
Hornworm RGR JA 1 1.555 0.216
BTH 1 1.827 0.180
Trial 1 9.095 0.003
JA×BTH 1 4.041 0.048
JA×BTH×Trial 1 4.423 0.038
Error 85 – –
Cabbage looper RGR JA 1 9.956 0.003
BTH 1 43.457 <0.001
JA×BTH 1 0.290 0.593
Error 42 – –
Thrips damage JA 1 9.378 0.002
BTH 1 0.095 0.758
Trial 5 5.042 <0.001
JA×BTH 1 0.107 0.744
Error 261 – –
Number of spider JA 1 4.680 0.033
mites BTH 1 0.907 0.334
Trial 1 0.002 0.967
JA×BH 1 0.132 0.717
Error 87 – –
P. syringae lesions JA 1 8.264 0.006
BTH 1 4.216 0.046
JA×BTH 1 1.518 0.224
Error 44 – –
ed with jasmonate alone, indicating that there was no in-
teraction between jasmonate and salicylate signaling in
terms of biological resistance to thrips. Induction of the
jasmonate response reduced the total number of spider
mites after 10 days (approximately one spider mite gen-
eration) largely due to a reduction in egg production
(Fig. 2d, Table 1). There was no detectable effect of jas-
monate treatment on the number of immature or adult
spider mites. SAR activation did not affect the number of
spider mites and did not attenuate the effect of JA in the
dual-elicited plants. Neither JA nor BTH singly affected
the RGR of hornworm caterpillars (Fig. 2e, Table 2). In
one trial, hornworm caterpillars had increased RGR on
the dual-elicited plants, but not in the other trial. The
amount of disease caused by Pst was reduced by both
jasmonate and SAR activation (Fig. 2f, Table 2).
The chemical signaling interactions previously ob-
served to affect S. exigua performance did not uniformly
extend their influence to organisms with different feed-
ing styles. Activation of the jasmonate response alone
had negative effects on four generalist herbivores, S. ex-
igua, spider mites, cabbage loopers and thrips, and the
bacteria Pst, but not on the specialist hornworm cater-
pillar. Other studies have found that hornworm larvae,
even though they are Solanaceae specialists, are nega-

tively affected by jasmonate-induced responses (Orozco-
Cardenas et al. 1993; Cipollini and Redman 1999). Acti-
vation of SAR alone did not affect spider mites, thrips,
hornworm or cabbage looper caterpillars but did reduce
the lesions caused by Pst. In the dual-elicited plants, the
performance of cabbage looper caterpillars was de-
creased but the performance of spider mites, thrips,
hornworms, and S. exigua on the wild tomato was the
same as on plants elicited with JA alone. Other studies
have found attenuation of resistance to two noctuid cat-
erpillars, S. exigua (Thaler et al. 1999) and Helicoverpa
zea (Stout et al. 1999), on BTH-treated plants. However,
a study by Inbar et al. (1998) found that treating tomato
plants with BTH actually reduced the populations of leaf
mining flies (Inbar et al. 1998).
Presumably, the explanation for this variation among
the results from different herbivores lies in the multiplici-
ty of plant products influenced by the jasmonate-salicy-
late cross-talk (Felton and Korth 2000). Schenk et al.
(2000) reported that of the 317 mRNAs positively or neg-
atively influenced by the jasmonate pathway, 55 were
synergistically and 28 were antagonistically affected by
SA elicitation. It may be that a portion of the jasmonate
pathway not negatively influenced by SAR is responsible
for resistance to spider mites and thrips. Indeed, the con-
cordance between hallmark responses of the jasmonate
pathway such as proteinase inhibitors and polyphenol ox-
idase and biological effects is not straightforward. Some
of the effects of jasmonate on herbivores may be mediat-
ed by plant nutritional quality, which may or may not be
antagonized by the activation of SAR. For instance,
Brody and Karban (1989) found that induced resistance
in cotton plants decreased spider mite fecundity, not lon-
gevity or survivorship, suggesting a decrease in plant nu-
tritional quality rather than novel secondary compounds.
Similarly, in the spider mite experiments reported here,
egg production was reduced by activation of the jasmon-
232
Fig. 2a–f The effects of JA and BTH treatment alone and in com-
bination on five parasites of tomato plants. Control plants were
treated with a water/acetone solution, JA-treated plants were elic-
ited with 1.0 mM JA, BTH-treated plants with 1.2 mM BTH, and
JA/BTH plants with 1.0 mM JA and 1.2 mM BTH at the same
time. aThe RGR [(final weight-initial weight)/(initial weight×
number of days)] of cabbage looper caterpillars (Trichoplusia ni),
bthe number of thrips (Frankliniella occidentalis) surviving on
plants in the four treatments as well as cthe amount of damage
(mm2of cleared cells) they caused the plant, dthe number of spi-
der mites (Tetranychus urticae) per leaflet in three stages, eggs,
immatures/males, and females, and the total number of spider
mites, eRGR of hornworm caterpillars (Manduca sexta), and fthe
area of lesions (mm2) caused by Pseudomonas syringae pv tomato
was determined. Bars indicate mean±SE

ate response, not survivorship, possibly consistent with a
nutritional rather than toxic mechanism of resistance.
Vector transmitted disease
Polyphenol oxidase activity was higher in the jasmonate-
treated plants compared to controls both initially and af-
ter 4 days of heavy thrips infestation (Initial: Induction:
F1, 28=24.935, P<0.001; Trial: F1, 28=4.096, P=0.053;
4 days later: Induction: F1, 28=32.796, P<0.001; Trial:
F1, 28=0.038, P=0.846; Fig. 3 a). Induced plants received
one-quarter the damage by thrips of control plants in
trial 1 and two-thirds the damage of controls in trial 2
(Induction: F1, 77=7.17, P=0.009; Trial: F1, 77=14.07,
P<0.001; Fig. 3b). However, this decrease in thrips feed-
ing did not influence the rate of infection or virus titers
of jasmonate-induced and control plants. Eighty percent
of plants in both treatments were infected with virus
(G=0.11, P=0.92). High virus titers in all plants may
have made distinguishing differences between treatments
difficult. Although the mean virus titer of the jasmonate-
induced plants was 70% compared to control plants in
the second trial, this was not statistically significant
(Wilk’s λ=0.903, F2, 28=1.49, P=0.241; Fig. 3c).
Thrips damaged all plants, control and induced, in
these experiments. The thrips feeding pressure on these
plants was very high so the likelihood that an infected
thrips did some of the feeding was very high. Given that
disease transmission only requires 5 min of feeding,
there was probably enough time on both control and in-
duced plants for the thrips to transmit the virus. It would
be worthwhile to conduct these experiments with a toma-
to variety less susceptible to thrip damage and to use
fewer thrips. We did not test the level of plant resistance
to the virus per se, but there is only a non-statistically
significant hint of reduced viral titers in the induced
plants. Other biologically similar viruses, such as to-
bacco mosaic virus, are negatively affected by SAR but
do not appear to be affected by jasmonate-induced re-
sponses in tobacco (Ajlan and Potter 1992). TSWV in-
fection of tomato plants induces markers of both the SA
and JA pathway but we do not know how each pathway
affects the virus (Sutha 1996). Resistance to thrips and
TSWV has been compared in wild and cultivated species
in the genus Lycopersicon (Kumar et al. 1995). Resis-
tance to both the insect and the virus was found in the
genus but these two were not correlated within a species,
indicating a decoupling of the resistance mechanisms to
these two plant parasites.
Conclusion
The occurrence of interactions between plant signal
transduction pathways is well documented and it is as-
sumed that this signaling overlap allows plants to deploy
defenses tailored to a given environment. The signaling
interactions reported between the jasmonate and salicy-
late pathways are known to have strong influences on the
molecular and enzymatic profile of the plant and on
some plant parasites. However, we know less about how
this signal interaction affects the plant’s interactions with
biotic challengers. We found that negative signal interac-
tions that reduced deployment of important plant second-
ary compounds did not always decrease plant resistance
to herbivores or a virus. Activation of the jasmonate
pathway reduced performance and feeding of spider
mites, thrips and cabbage loopers, but attenuation of the
jasmonate pathway due to SAR induction positively af-
fected only cabbage looper performance. Although the
generality and ecological relevance of these results re-
main to be verified, we conclude that the diversity of
233
Fig. 3a–c The effect of jasmonate induction on thrips damage and
tomato spotted wilt virus. aThe activity of polyphenol oxidase
(∆OD/g/min) 2 days after jasmonate spray (initial) and after
4 days of thrips feeding (final) in control and induced plants. Re-
sults from trial 1 and 2 are pooled. bThe amount of thrips feeding
damage (mm2) on control and induced plants. Results from trial 1
and trial 2 are shown separately. cThe viral titers in control and
induced plants 21 days following feeding by viruliferous thrips.
Optical density of the plant extracts were measured at 405 nm af-
ter reacting with substrate for 30 min in trial 1 and after reacting
for 30 and 60 min in trial 2. Bars indicate mean±SE

feeding styles among plant parasites may supercede the
dichotomous view of negative signal interactions be-
tween the jasmonate and salicylate pathways (Felton and
Korth 2000). The fact that some herbivores activate
components of the salicylate pathway and that some
pathogens activate components of the jasmonate path-
way supports this view (Fidantsef et al. 1999).
Acknowledgements We thank Hien Nguyen, Julia Quan, and
Lora Richards, for help with experiments. We thank Laura Grunert
for hornworm eggs, Novartis for BTH, D.A. Cooksey for the iso-
late of Pst, and the Tomato Germplasm Center, UC Davis for
seeds of L. esculentum var. cerasiforme. This manuscript was im-
proved by the comments of A. Agrawal, D. Viswanathan and two
anonymous reviewers. This research was supported by an operat-
ing grant from NSERC (J.S.T.), the University of Toronto Con-
naught Fund (J.S.T.), USDA-NRI Grant No. 98-02362 (R.M.B.
and R.K.), and the American Floral Endowment (D.E.U.).
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