Journal of Chemical Ecology, Vol. 31, No. 11, November 2005 ( #2005)
DOI: 10.1007/s10886-005-7616-8
EFFECTS OF DROUGHT STRESS AND NUTRIENT
AVAILABILITY ON DRY MATTER ALLOCATION,
PHENOLIC GLYCOSIDES, AND RAPID INDUCED
RESISTANCE OF POPLAR TO TWO
LYMANTRIID DEFOLIATORS
BETHAN K. HALE,1,* DANIEL A. HERMS,1 ROBERT C. HANSEN,2
THOMAS P. CLAUSEN,3 and DANIELLE ARNOLD3
1
Department of Entomology, The Ohio State University/Ohio Agricultural Research
and Development Center, 1680 Madison Avenue, Wooster, OH 44691, USA
2
Department of Food, Agricultural and Biological Engineering, The Ohio State University/
Ohio Agricultural Research and Development Center, 1680 Madison Avenue,
Wooster, OH 44691, USA
3
Department of Chemistry and Biochemistry, University of Alaska Fairbanks,
Fairbanks, AK 99775, USA
(Received September 10, 2004; revised March 30, 2005; accepted July 9, 2005)
Abstract—The growthYdifferentiation balance hypothesis (GDBH) postulates
that variation in resource availability can increase or decrease allocation to
secondary metabolism, depending on how growth is affected relative to
carbon assimilation. Growth and leaf area of black poplar (Populus nigra)
increased substantially in response to increased nutrient availability, while net
assimilation rate and photosynthesis were less strongly affected. In response,
total phenolic glycoside concentrations declined, which is consistent with
GDBH. Drought stress decreased net assimilation rate and photosynthesis as
well as growth, while increasing total phenolic glycoside concentrations. This
pattern does not follow GDBH, which predicts lower secondary metabolism
when resource limitation decreases both growth and carbon assimilation.
However, there was a strong negative correlation between growth and total
phenolic glycoside concentration consistent with a trade-off between primary
and secondary metabolism, a key premise of GDBH. Drought decreased the
growth of gypsy moth (Lymantria dispar) larvae but had no effect on whitemarked tussock moth (Orgyia leucostigma). Increased nutrient availability
had a positive linear effect on growth of whitemarked tussock moth, but no
effect on gypsy moth. Treatment effects on gypsy moth corresponded closely
with effects on total phenolic glycosides, whereas effects on whitemarked
* To whom correspondence should be addressed. E-mail: [email protected]
2601
0098-0331/05/1100-2601/0 # 2005 Springer Science + Business Media, Inc.
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HALE ET AL.
tussock moth more closely tracked changes in nutritional quality. Localized
gypsy moth herbivory elicited rapid induced resistance to gypsy moth, with
the effect being independent of water and nutrient availability, but did not
affect whitemarked tussock moth, indicating that the effects of biotic and
abiotic stress on insect resistance of trees can be species-specific.
Key WordsVGrowthYdifferentiation balance hypothesis, resource availability, allocation, phenotypic plasticity, gypsy moth, whitemarked tussock moth.
INTRODUCTION
Some studies have shown drought stress to increase secondary metabolism and
decrease host quality of trees for insect herbivores (Ross and Berisford, 1990;
Craig et al., 1991; Mopper and Whitham, 1992), whereas others have found the
opposite (Wagner, 1986; Cobb et al., 1997; Roth et al., 1997). Fertilisation also
has been shown to have divergent effects on the secondary metabolites of
woody plants (Kytö et al., 1996; Koricheva et al., 1998a; Herms, 2002). The
growthYdifferentiation balance hypothesis (GDBH) (Loomis, 1932; Lorio,
1986; Herms and Mattson 1992) provides a potential explanation by predicting
a quadratic response of constitutive secondary metabolism across a resource
gradient. Thus, increased resource availability can either increase or decrease
secondary metabolite concentrations, depending on the initial status of the plant
(Herms and Mattson, 1992).
Rapidly growing plants are predicted by GDBH to have low secondary
metabolite concentrations as a result of a resource-based trade-off between
primary and secondary metabolic pathways. However, moderate water or
nutrient limitation slows growth more than carbon assimilation (Bradford and
Hsiao, 1982; Luxmoore, 1991), which can result in the accumulation of
carbohydrates in source leaves (Wardlaw, 1990; Geiger et al., 1996). This may
increase the substrate available for secondary metabolism (Waterman and Mole,
1989), resulting in a negative correlation between growth and secondary
metabolites (Herms and Mattson, 1992). However, when resource limitation is
severe enough to depress carbon assimilation, secondary metabolism is
predicted to fall because of energy and substrate constraints on biosynthesis.
In this case, increased resource availability is predicted to increase both growth
and secondary metabolite concentrations, generating a positive correlation
between them (Herms and Mattson, 1992). Few studies have simultaneously
quantified carbon assimilation rate, growth, and secondary metabolism across a
range of conditions, which is necessary to test predictions of GDBH (Stamp,
2003, 2004).
Differential effects of resource availability on constitutive and induced
resistance could influence host quality for herbivores (Lewinsohn et al., 1993;
DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
2603
Lerdau et al., 1994; Lombardero et al., 2000; Glynn et al., 2003). Rapid induced
resistance is a form of host resistance in response to herbivory that impinges on
insect generation causing the damage (Haukioja, 1990). The few studies that
have addressed the effects of nutrient availability on rapid induced resistance of
woody plants show that results vary depending on plant (Hunter and Schultz,
1995; Mutikainen et al., 2000) and insect species (Glynn et al., 2003). To our
knowledge, effects of drought on rapid induced resistance of woody plants to
folivores have not been investigated.
The objectives of this study were to test predictions of GDBH by quantifying
effects of water and nutrient availability on carbon assimilation, growth, and total
foliar phenolic glycoside concentration of black poplar, Populus nigra (L.), and
on constitutive and rapid induced resistance to gypsy moth, Lymantria dispar
(L.), and whitemarked tussock moth, Orgyia leucostigma (Smith).
MATERIALS AND METHODS
On March 29, 2003, 250 cuttings of the black poplar clone NC5271 were
taken from stock plants, wrapped in moist paper, and stored at 4-C. Relative to
other poplar clones, NC5271 is one of the fastest growing (Robison and Raffa,
1994) and most inducible to various elicitors including gypsy moth feeding
(Havill and Raffa, 1999). Ten d later, cuttings were removed from storage,
dipped into Hormex Rooting Powder No. 3\ (Brooker Chemical, Hollywood,
CA, USA) (0.3% indole-3-butyric acid), planted in Premier ProMix BX\
(Premier Horticulture Ltd., Dorval, Quebec, CA) and maintained in a greenhouse (25-C, 12:12 L/D cycle). On May 16, rooted cuttings were transplanted
to 10 l plastic pots containing the same medium and transferred to an outdoor
nursery under 50% shade cloth to acclimate.
On June 2 (d 1), 180 of the most uniform cuttings were sorted into five
blocks of 36, with assignment based on height and number of apical shoots. To
quantify initial biomass and total leaf area, six plants in each block were
selected at random for immediate harvest. Stems and foliage were harvested by
cutting plants at ground level, and roots were extracted from container media
with minimal damage using a low-pressure, high-volume air stream (Air
Spade\ , Concept Engineering Group, Inc., Verona, PA, USA). Shoots, leaves,
and roots were oven-dried at 60-C for 96 hr and weighed to the nearest
milligram. The remaining 150 plants were transferred to an outdoor gravel bed
in full sunlight, where the 30 plants in each block were subjected to one of three
fertilisation levels.
Fertility treatments were initiated by means of a computer-controlled
fertigation (irrigation and fertilisation) system (Hansen et al. 2000): 30, 75, and
150 ppm N, with N/P2O5/K2O, supplied in a ratio of 3:1:2 from calcium nitrate,
2604
HALE ET AL.
monoammonium phosphate, and potassium nitrate. Each plant was irrigated with
0.5 l of nutrient solution whenever the potting medium moisture tension (PMMT)
dried to j4 kPa, as recorded by computer-monitored tensiometers (15 cm Mini
BLT^ Remote Sensing Units (0Y25 kPa); Irrometer Company, Inc., Riverside,
CA, USA) installed in one pot per treatment in the block containing the largest
plants, because they dried out the fastest. Tensiometers, which were positioned to
a depth of 8 cm midway between the pot wall and the plant, were also installed in
one pot per treatment in two additional randomly selected blocks. This resulted in
one tensiometer per treatment combination in three of five blocks, which provided
a continuously monitored, experiment-wide estimate of mean PMMT for each
treatment. On July 22 (d 51), 10 plants from each fertility treatment were destructively harvested as described above, with two selected randomly from each block,
leaving a total of 120 plants in the experiment.
On July 24 (d 53), a drought-stress treatment was applied to half of the
remaining plants in each fertility treatment by withholding irrigation until
PMMT reached j25 kPa, whereas the rest were irrigated as described above,
resulting in two discrete treatments that were maintained over the next 75 d. The
irrigation of drought-stressed plants was controlled using scheduled events
($200 ml potj1 eventj1), with approximately four daily events required to
maintain PMMT at the required levels. Well-watered plants continued to be
irrigated according to PMMT as described above. Tensiometer readings showed
that mean PMMT of well-watered and drought-stressed plants generally
remained between j3 and j4 kPa and j10 and j25 kPa, respectively, with
precipitation having little effect (Figure 1).
On September 10 (d 101), half the plants in each treatment combination
were randomly assigned a short-term, localized herbivory treatment designed to
elicit rapid induced resistance. Three fourth-instar gypsy moths were confined
within mesh sleeves on leaves 4Y7 on the terminal shoot, with leaf 1 designated
as the youngest leaf longer than 2 cm. Larvae were allowed to feed until at least
80% of the enclosed leaves had been consumed on all plants, whereupon sleeves
and insects were removed. To control for potential effects of the enclosures on
induced responses, corresponding leaves on the remaining (constitutive) plants
were also enclosed by sleeves containing no larvae.
All remaining plants were harvested on October 7 (d 128). Thus, the first
52 d of the experiment consisted of three fertility treatments, whereas the final
75 d consisted of a 3 2 2 complete factorial design, with three fertility, two
irrigation, and two defoliation treatments, with each of 12 treatment combinations replicated 10 times within each of five blocks, for a total of 120 plants.
Plant Growth and Dry Matter Allocation. Growth analysis (Hunt, 1978;
Lambers and Poorter, 1992) was used to document treatment effects on plant
relative growth rate and dry matter allocation. Relative growth rate (mg gj1
dj1) was calculated as [(ln(final total mass j ln(initial total mass)] / time], with
DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
2605
FIG. 1. Potting medium moisture tension (PMMT) for well-watered (dark line) and
drought-stressed (gray line) plants over the course of the experiment (upper y-axis) and
daily precipitation over the experimental period (lower y-axis). The arrow indicates date
of initiation of the irrigation treatment.
initial and final mass determined from destructive harvests as described above.
Specific leaf mass (g mj2) was determined by measuring the area and dry
weight of the leaves sampled for nitrogen analysis (described below). Leaf area
was measured using a LI-3100 area meter (LI-COR, Inc., Lincoln, NE, USA).
The dry weight of foliage sampled for foliar nitrogen and phenolic glycoside
analyses (described below) was added to that subsequently obtained from the
whole plant harvest. Total leaf area per plant (m2) was estimated as total leaf
biomass / specific leaf mass. Leaf area ratio (m2 gj1) was calculated as the
quotient of total leaf area / total plant mass, and net assimilation rate (g mj2
dj1) as relative growth / leaf area ratio. Percent root mass was calculated as
(root mass / total plant mass) 100. Initial mass for each plant (used to
calculate relative growth rate and net assimilation rate) was estimated as the
treatment mean for each block at the beginning experiment, as determined from
destructive harvests before initiation of treatments.
Photosynthesis. Light-saturated net photosynthesis (mmol CO2 mj2 secj1)
was measured on d 23 and 48 (June 24 and July 19) prior to initiation of the
drought-stress treatment on d 53, and on d 72 and 100 (August 12 and
September 9) after initiation of the drought stress. Measurements were made
using a portable photosynthesis system (LI-6200, LI-COR, Inc. Lincoln, NE,
USA) on fully expanded, undamaged leaves from randomly selected plants, on
cloud-free mornings between 08:30 and 11:30 EDT. Sample sizes were 10 for
2606
HALE ET AL.
each fertility treatment (two replicates from each block) on d 23 and 48 and five
for each fertilityYirrigation treatment combination (one replicate per block) on d
72 and 100.
Phytochemistry. To quantify the effects of nutrient and water availability on
foliar nitrogen and phenolic glycoside concentrations, a subset of foliage was
sampled just prior to whole-plant harvests on d 51 from all harvested trees and on
d 128 from all trees not subjected to the defoliation treatment. For foliar nitrogen
analysis, every fourth leaf was sampled from all branches on the tree. Leaves were
dried at 60-C and then ground in a mill (Cyclotec EC 1093, Tecator AB, Hoganas,
Sweden) to pass through a 0.4-mm mesh screen. Total foliar nitrogen content
(mg gj1) was determined using a Carbo Erba CNH analyser, Model NA 1500
(Daun and DeClerq, 1994). For foliar phenolic glycoside analysis, 12Y15 fully
expanded, undamaged leaves were sampled from between leaf positions 10Y20
on random branches. Leaves were immediately frozen in liquid nitrogen and
placed on ice before being transported to the laboratory where they were freezedried at j4-C within 1 hr of sampling. Dried leaves were subsequently ground
to pass through a 0.4-mm mesh screen as described above.
Tremulacin and salicortin are dominant phenolic glycosides in Populus
species (Lindroth et al., 1987; Clausen et al., 1989a,b; Lindroth and Hwang,
1996). We quantified their combined concentration using gas chromatography
by first converting their cyclohexan-5-ene-2-one-1-ol carboxylate moiety to the
volatile compound methyl 2-methoxybenzoate by extracting 1 g of each sample
in 15 ml anhydrous methanol containing 2 mg of capric acid as an internal
standard. Capric acid is converted to methyl decanoate under the reaction
conditions, which has a similar retention time to that of methyl 2-methoxybenzoate. The solution was left to stand overnight, after which extracts were
decanted and two drops (about 100 mg) of 98% sulfuric acid were added. After
being left to stand for 12 d at room temperature, extracts were analysed using
a Hewlett-Packard 6890 gas chromatograph equipped with a flame ionization
detector and a 30 m 0.25 mm bonded EC-1 column with a 0.25-mm film
thickness (Alltech, Deerfield, IL, USA). The injector and detector temperatures were held at 275 and 300-C, respectively, the He flow rate was 2.5 ml
minj1, and the temperature program was set at 60-C for 3 min, 60Y100-C at
7-C minj1, 100Y150-C at 5-C/min, and 150-C for 2 min. In all runs, 1.0 ml
of sample was injected (splitless mode). Methyl 2-methoxybenzoate and methyl decanoate eluted at 12.62 and 12.47 min, respectively. Total phenolic glycoside concentration (mg gj1 dry weight) was calculated by summing the
molar quantities of the two individual compounds (tremulacin plus salicortin)
and expressing this as the percent mass of an equivalent molar amount of
salicortin.
Insect Bioassays. Laboratory bioassays were conducted with first instar
gypsy moth and whitemarked tussock moth to quantify treatment effects on
DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
2607
constitutive and induced resistance. Eggs of both species were obtained from
the Canadian Forest Service, Insect Production Laboratory, Sault St. Marie,
Ontario, CA. Three d after termination of the defoliation treatment, the two
leaves immediately distal to those that had been confined in mesh sleeves were
detached from plants and placed in separate Petri dishes (15 cm diam) with five
neonate larvae of one of the two species. Larvae were reared in a growth
chamber at 25-C with a 16:8 (L/D) photoperiod. To control for ontogenetic
variation, half of the replicates for each insect species within a treatment
combination received the oldest leaf (position 3), and the other half received the
youngest (position 2). A plaster base in each dish saturated with distilled water
throughout the bioassay maintained high humidity and leaf turgor. The five
larvae in each Petri dish were weighed as a group at the start of the bioassay and
again 72 hr later. Mean larval weight was calculated by dividing total weight by
number of larvae. Larval growth (mg) was calculated as the difference between
mean final and mean initial mass.
Data Analyses. Treatment effects on plant and insect variables were
analysed using ANOVA (PROC GLM, Type III sums of squares; SAS Institute,
Inc., 2000). All responses met assumptions of normality of residuals and
homogeneity of variance. Data are reported as least square means T 1 SE.
Linear and quadratic contrasts were calculated to characterise significant effects
of fertility level on treatment means (Chew, 1976; Mize and Schultz, 1985),
with coefficients for the unequally spaced treatments calculated according to
Robson (1959). Pearson correlation coefficients (PROC CORR; SAS Institute,
Inc., 2000) were used to quantify relationships between dependent variables.
RESULTS
Plant Growth and Biomass Allocation. At the intermediate harvest on d 51,
nutrient availability had significant linear and quadratic effects on plant growth
and biomass allocation (Table 1). Total plant biomass, relative growth rate, and
total leaf area increased by 50, 12, and 75%, and percent root mass and specific
leaf mass decreased by 16 and 9%, respectively, as fertilisation rate increased
from low to high (Table 2). Nutrient availability had no effect on net assimilation rate (Tables 1 and 2).
At the final harvest on d 128, nutrient availability continued to have
significant linear effects on total leaf area and percent root mass. However, the
effects of fertility on total plant biomass and relative growth rate varied within
the two irrigation treatments (significant quadratic effect and fertility irrigation interaction) (Table 3). The effect of nutrient availability of total plant
mass was stronger in the drought stress than in the well-watered treatment,
2608
HALE ET AL.
TABLE 1. F VALUES FROM ANOVA FOR GROWTH AND DRY MATTER ALLOCATION OF
POPLAR, Populus nigra, IN RESPONSE TO THREE FERTILITY LEVELS FOR 50 DAYS
Response variable
Total biomass (g)
Percentage root mass
Relative growth rate (mg gj1 dj1)
Total leaf area (m2)
Specific leaf mass (g mj2)
Net assimilation rate (g mj2 dj1)
FertilityV
main effect
FertilityV
linear contrast
FertilityV
quadratic contrast
Error df
20.1***
12.7***
21.8***
40.2***
14.6***
1.1
35.9***
21.3***
38.3***
70.6***
23.2***
1.1
5.3*
5.0*
56.6*
11.9**
7.0*
1.3
22
22
22
22
22
22
Level of significance: *P < 0.05, **P < 0.01, ***P < 0.001.
increasing 95 and 42%, respectively, as fertility increased from low to high
(Table 4). In the drought-stress treatment, relative growth rate increased 29% as
fertility increased. However, in the well-watered treatment, relative growth rate
was less than 9% higher in the intermediate than in both the high and low
fertility treatments (Table 4). When averaged across the two irrigation
treatments, total leaf area increased 28% and percent root mass decreased
12% as fertility level increased from low to high (data pooled from Table 4).
The effects of fertility on net assimilation rate were also dependent on the
irrigation treatment (significant fertility irrigation interaction). Nutrient
availability had no effect on the net assimilation rate of drought-stressed plants,
but had a quadratic effect on the well-watered plants, it being 11 and 24%
higher in the intermediate treatment than in the high and low fertility treatments,
respectively. Nutrient availability had no effect on specific leaf mass on d 128
(Tables 3 and 4).
Water availability had stronger effects on all measures of dry matter
allocation compared to nutrient availability (Table 3). Relative to droughtstressed plants, the total biomass, relative growth rate, total leaf area, and net
TABLE 2. MEAN (TSE) GROWTH AND DRY MATTER ALLOCATION OF POPLAR, P. nigra,
IN RESPONSE TO THREE FERTILITY LEVELS FOR 50 DAYS
Response variable
30 ppm N
Total biomass (g)
Percentage root mass
Relative growth rate (mg gj1 dj1)
Total leaf area (m2)
Specific leaf mass (g mj2)
Net assimilation rate (g mj2 dj1)
59.7
20.7
69.5
0.32
76.5
13.0
T
T
T
T
T
T
3.5
0.5
1.0
0.02
1.0
0.4
75 ppm N
80.6
18.2
75.6
0.49
70.6
12.4
T
T
T
T
T
T
3.3
0.5
1.0
0.02
0.9
0.3
F values and level of significance for the fertility treatment are reported in Table 1.
150 ppm N
89.8
17.4
78.0
0.56
69.3
12.5
T
T
T
T
T
T
3.3
0.5
1.0
0.02
1.1
0.3
2609
DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
TABLE 3. F VALUES FROM ANOVA FOR GROWTH AND DRY MATTER ALLOCATION OF
POPLAR, P. nigra, IN RESPONSE TO THREE FERTILITY LEVELS FOR 52 DAYS, FOLLOWED
BY THREE FERTILITY LEVELS CROSSED WITH TWO IRRIGATION LEVELS FOR 75 DAYS
Response variable
Total biomass (g)
Percentage root mass
Relative growth rate
(mg gj1 dj1)
Total leaf area (m2)
Specific leaf mass
(g mj2)
Net assimilation rate
(g mj2 dj1)
FertilityV
main effect
FertilityV
linear
contrast
FertilityV
quadratic
contrast
Irrigation
Fertility Irrigation
Error df
116.0***
19.0***
6.5**
187.0***
35.0***
7.2**
45.0***
2.0
6.3*
929.0***
27.0***
717.8***
17.0***
1.0
11.9***
110
110
106
3.0
0.8
6.0*
0.4
0.0
1.0
119.0***
7.0*
1.0
4.0*
50
50
3.7*
2.1
5.7*
184.5**
4.1*
106
Level of significance: *P < 0.05, **P < 0.01, ***P < 0.001.
assimilation rate of well-watered plants were 148, 78, 170, and 76% higher,
respectively, when averaged across all three fertility levels (data pooled from
Table 4). Percent root mass of drought-stressed plants was 9% higher than for
well-watered plants (data pooled from Table 4). In the two lowest fertility levels
(30 and 75 ppm N), specific leaf mass was 15 and 22% higher in the wellwatered relative to drought-stressed plants, respectively, but was not affected by
irrigation in the high fertility (150 ppm N) treatment (fertility irrigation
interaction) (Tables 3 and 4).
Photosynthesis. Nutrient availability had no effect on photosynthesis on
d 23, but had a positive linear effect on d 48 (Table 5) when photosynthesis rate
was 28% greater in the high relative to the low fertility treatment (Table 6). On
d 72, 19 d after the initiation of drought stress, neither treatment had any significant effect on photosynthesis (Table 5). However, on d 100, drought stress
significantly decreased photosynthesis rate by 51% relative to the well-watered
plants, whereas nutrient availability had a significant positive linear effect on
photosynthesis across both irrigation levels, being 55% greater in the high
relative to the low fertility treatment (Table 5, data pooled from Table 6). There
were no significant interacting effects of the irrigation and fertility treatments on
photosynthesis, and no significant quadratic effects of fertility (Table 5).
Phytochemistry. Foliar nitrogen content significantly increased with nutrient availability on both harvest dates and was decreased by drought stress on the
final harvest date (Table 7). On d 51, foliar nitrogen concentrations averaged 30
T 1, 36 T 1, and 39 T 1 mg gj1 for the 30, 75, and 150 ppm N fertility
treatments, respectively. On d 128, foliar nitrogen concentrations of wellwatered plants averaged 33 T 1, 38 T 1, and 36 T 0.1 mg gj1 for the 30, 75, and
Well-watered
476.0 T 17.1
32.8 T 0.7
26.5 T 0.6
0.90 T 0.07
116.8 T 8.4
16.8 T 0.8
Drought-stressed
174.5 T 17.1
37.5 T 0.7
13.3 T 0.5
0.24 T 0.07
101.4 T 4.9
10.4 T 0.7
10.4 T 0.7
0.40 T 0.07
110.2 T 3.7
247.8 T 17.1
33.5 T 0.7
15.0 T 0.5
Drought-stressed
20.8 T 0.7
0.89 T 0.07
134.9 T 11.6
741.3 T 17.1
31.4 T 0.7
28.3 T 0.5
Well-watered
75 ppm N
11.1 T 0.7
0.41 T 0.07
117.2 T 5.5
341.0 T 17.1
31.9 T 0.7
17.1 T 0.5
Drought-stressed
18.7 T 0.7
1.05 T 0.07
112.3 T 5.0
675.8 T 17.1
29.5 T 0.7
25.9 T 0.5
Well-watered
150 ppm N
Sample sizes were 10 for each treatment, except for the 30 ppm N treatment in the intermediate harvest where the sample size was 9. F values and level of
significance of treatment effects are reported in Table 3.
Total biomass (g)
Percentage root mass
Relative growth rate
(mg gj1 dj1)
Total leaf area (m2)
Specific leaf mass
(g mj2)
Net assimilation rate
(g mj2 dj1)
Response variable
30 ppm N
TABLE 4. MEAN (TSE) GROWTH AND DRY MATTER ALLOCATION OF POPLAR, P. nigra, IN RESPONSE TO THREE FERTILITY LEVELS FOR
52 DAYS, FOLLOWED BY THREE FERTILITY LEVELS CROSSED WITH TWO IRRIGATION LEVELS FOR 75 DAYS
2610
HALE ET AL.
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DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
TABLE 5. F VALUES FROM ANOVA FOR NET PHOTOSYNTHESIS OF POPLAR, P. nigra, IN
RESPONSE TO THREE FERTILITY LEVELS ON DAYS 23 AND 48 AND THREE FERTILITY
LEVELS CROSSED WITH TWO IRRIGATION LEVELS ON DAYS 72 AND 100
Source of variation
Fertility (F)
F linear contrast
F quadratic contrast
Irrigation (I )
FI
Error df
Day 23a
Day 48a
Day 72
Day 100
1.6
2.5
0.6
Y
Y
22
21.2***
41.2***
0.9
Y
Y
22
1.2
0.4
1.1
0.3
3.1
19
10.6***
20.9***
0.3
90.5***
1.8
19
Level of significance: *P < 0.05, **P < 0.01, ***P < 0.001.
a
Dashes indicate where terms were not included in the model since the irrigation treatment had not
yet been initiated.
150 ppm N fertility treatments, respectively. However, the foliar nitrogen
concentrations for the drought-stressed plants averaged only 25 T 1, 28 T 1, and
31 T 1 mg gj1, respectively, as fertility increased.
On d 51, total phenolic glycoside concentrations were not affected by
nutrient availability (Table 7). However, on d 128, fertility had a significant
negative linear effect on total phenolic glycoside concentrations, with concentrations decreasing by 32%, averaged across both irrigation treatments, as
fertilisation rate increased (data pooled from Figure 2). However, this effect was
small compared to the larger effect of the irrigation treatment (Table 7), total
phenolic glycoside concentrations being 89% higher in the drought-stressed
relative to the well-watered plants (data pooled from Figure 2). There was no
significant interaction between the drought stress and fertility treatments on total
phenolic glycoside concentration (Table 7). Total phenolic glycoside concentrations were negatively correlated with total plant biomass (Figure 3).
TABLE 6. MEAN (TSE) NET PHOTOSYNTHETIC RATE (m mol CO2 mj2 secj1) OF POPLAR, P.
nigra, IN RESPONSE TO THREE FERTILITY LEVELS ON DAYS 23 AND 48 AND THREE
FERTILITY LEVELS CROSSED WITH TWO IRRIGATION LEVELS ON DAYS 72 AND 100
Day 72
Fertility level
30 ppm N
75 ppm N
150 ppm N
Day 100
Day 23
Day 48
Droughtstressed
Wellwatered
Droughtstressed
Wellwatered
25.7 T 0.9
27.5 T 1.4
27.4 T 1.2
19.5 T 0.7
22.6 T 0.9
25.0 T 1.1
11.0 T 1.1
11.1 T 1.5
10.3 T 1.8
11.4 T 1.7
9.9 T 1.2
14.0 T 1.0
6.6 T 1.4
11.0 T 1.4
14.0 T 1.4
19.3 T 1.5
19.0 T 1.4
26.2 T 1.6
Sample sizes were 10 for each treatment on days 23 and 48 and 5 for each treatment on days 72 and
100. F values and level of significance of treatment effects are reported in Table 5.
2612
HALE ET AL.
TABLE 7. F VALUES FROM ANOVA FOR FOLIAR CONCENTRATIONS OF TOTAL
PHENOLIC GLYCOSIDES AND NITROGEN OF POPLAR, P. nigra, IN RESPONSE TO ONE OF
THREE FERTILITY LEVELS FOR 52 DAYS, FOLLOWED BY THREE FERTILITY LEVELS
CROSSED WITH TWO IRRIGATION LEVELS FOR 75 DAYS
Total phenolic glycoside concentration
Source of variation
Fertility (F)
F linear contrast
F quadratic contrast
Irrigation (I)
FI
Error df
a
Nitrogen concentration
Day 51
Day 128
Day 51a
Day 128
3.4
1.4
0.3
Y
Y
22
5.7*
11.2**
0.3
44.6***
0.1
50
36.8***
65.8***
9.7**
Y
Y
22
12.7***
18.1***
7.3**
87.0***
4.2*
50
Level of significance: *P < 0.05, **P < 0.01, ***P < 0.001.
a
Dashes indicate where terms were not included in the model since the irrigation treatment had not
yet been initiated.
Insect Bioassays. Drought stress significantly reduced the growth of first
instar gypsy moth (F1,100 = 42.01, P < 0.001), which was 36% higher on wellwatered plants (data pooled from Figure 4a). Previous defoliation by fourth instar
gypsy moth also decreased the growth of first instar gypsy moth (F1,100 = 12.46, P <
0.001), which was 27% higher on the nondefoliated than on the induced plants
(data pooled from Figure 4a). Rapid induced resistance to gypsy moth was
expressed in both well-watered and drought-stressed trees and across all levels of
FIG. 2. Mean (TSE) total foliar phenolic glycoside concentration of poplar, Populus
nigra, in response to three fertility levels for 52 d followed by three fertility levels
crossed with two irrigation levels for 75 d. P = 0.002 for linear effect of fertility and P <
0.001 for main effect of irrigation; interaction, not significant.
DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
2613
FIG. 3. Correlation between total biomass of poplar, P. nigra, and total foliar phenolic
glycoside concentration in response to three fertility levels for 52 d followed by three
fertility levels crossed with two irrigation levels for 75 d.
the fertility treatment (no significant interaction between defoliation and either
the irrigation or fertility treatments). However, neither the irrigation nor the
defoliation treatments had any effect on whitemarked tussock moth growth
(Figure 4b). Fertility had no effect on gypsy moth growth, but growth of
whitemarked tussock moth increased linearly as fertility increased (F1,98 =
6.16, P = 0.015), being 18% greater in the high relative to the low fertility
treatment (data pooled from Figure 5).
DISCUSSION
The patterns we observed conformed only in part with those predicted by
GDBH (Herms and Mattson, 1992). Plant responses to nutrient availability were
consistent, but effects of water availability diverged to some degree from responses predicted by GDBH. Consistent with GDBH, plant growth was more
sensitive to nutrient availability than was carbon assimilation, as indicated by
larger effects of the fertility treatment on plant biomass and leaf area than on net
assimilation rate and photosynthesis. In response, total phenolic glycoside concentrations declined as nutrient availability increased. This observation is also
consistent with previous studies on congeneric quaking aspen, Populus tremuloides (Michx.), in which phenolic glycoside concentrations also decreased as
nutrient availability increased (Bryant et al., 1987; Hemming and Lindroth,
1999).
Plant responses to drought did not correspond as closely with the predictions of GDBH. Concentrations of total phenolic glycosides were low in the
fast-growing, well-watered plants which is consistent with GDBH. However,
2614
HALE ET AL.
FIG. 4. Effects of short-term, localized gypsy moth herbivory and water availability on
mean growth (TSE) of first instar (a) gypsy moth and (b) whitemarked tussock moth on
poplar, P. nigra. For gypsy moth, P < 0.001 for main effect of irrigation and P < 0.001
for main effect of defoliation, interaction not significant; for whitemarked tussock moth,
no effects significant. Data pooled across fertility treatments.
the high concentrations of total phenolic glycosides observed in response to
drought stress were not consistent with GDBH, as both growth and carbon
assimilation (as indicated by net assimilation rate and photosynthesis) were
substantially reduced. In this contingency, GDBH predicts that foliar secondary
metabolite concentrations should also decrease (Herms and Mattson, 1992). The
DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
2615
FIG. 5. Effects of fertility on mean growth (TSE) of first instar gypsy moth and
whitemarked tussock moth on poplar, P. nigra. NS indicates effect is not significant.
Data pooled across watering and defoliation treatments.
high total phenolic glycoside levels in drought-stressed plants contributed to a
strong negative correlation with growth, which is consistent with the trade-off
between primary and secondary metabolism predicted by GDBH (Herms and
Mattson, 1992). Conclusions regarding effects on total phenolic glycoside
concentration were not affected by expressing the data on a leaf area basis (mg
cmj2), as water and nutrient availability had smaller effects on specific leaf
mass than on total phenolic glycoside content. Hence, treatment effects were
attributable to changes in phenolic glycoside biosynthesis per se, rather than
dilution or concentration effects resulting from variation in dry matter
accumulation (Koricheva, 1999). Phenolic glycosides have been implicated as
anti-Lepidoptera defences of Populus (Hemming and Lindroth, 1995, 2000;
Hwang and Lindroth, 1997), and the adaptive advantage of high levels of
defence was maintained even when drought-stressed plants were constrained by
a smaller carbon budget, which would substantially increase the cost of
producing and maintaining secondary metabolites. Contrary to our results, Roth
et al. (1997) observed drought stress to decrease phenolic glycoside concentrations of quaking aspen.
Some studies have shown high fertility levels to amplify the negative
effects of drought stress on plants (Walters and Reich, 1989; Miller and
Timmer, 1994; Power et al., 1998) through such mechanisms as increased soil
matric potential (Jacobs et al., 2004) or decreased root/shoot ratios (Linder and
Rook, 1984; Linder et al., 1987), therefore, also potentially increasing the
effects of drought stress on secondary metabolism and herbivore performance.
However, we observed no evidence to support this hypothesis, even when
plants were preconditioned to their respective nutrient treatment for 52 d prior
2616
HALE ET AL.
to exposure to drought stress. We did not observe a proportional reduction in
growth of high fertility relative to low fertility plants when they were
subjected to drought, nor were the nature of fertility irrigation interactions
on phytochemistry and insect performance consistent with enhanced effects of
drought stress. Although percent root mass declined in response to increased
nutrient availability, drought stress increased percent root mass, which may
have had a counteracting effect. Both responses are consistent with theories
of adaptive phenotypic plasticity (Bloom et al., 1985; Hirose, 1987; Ingestad
and Ågren, 1991, 1992), which predict that plants will shift their allocation
patterns in varying environments to increase acquisition of growth-limiting
resources.
Outbreaks of phytophagous insects have been linked to droughts (Mattson
and Haack 1987), although effects may be specific to particular feeding guilds
(Larsson, 1989; Koricheva et al., 1998b; Huberty and Denno, 2004). This study
did not provide any evidence to support the hypothesis that drought stress
enhances folivore performance (White, 1984). In contrast, drought stress
decreased the growth of gypsy moth, perhaps because drought also decreased
foliar nitrogen and increased total phenolic glycoside concentrations, both of
which are key determinants of gypsy moth performance on quaking aspen
(Lindroth and Hemming, 1990; Hemming and Lindroth, 2000; Osier et al.,
2000). Decreased foliar water content may also have contributed to lower insect
performance (Scriber, 1977).
In contrast, drought stress had no effect on the growth of whitemarked tussock
moth, perhaps because this species is less sensitive than gypsy moth to changes in
phenolic glycosides, as whitemarked tussock moth has been shown to be relatively
insensitive to other phenolic compounds (Karowe, 1989; Kopper et al., 2002).
Environmental variation does not affect all secondary metabolites within a plant
in the same way (Muzika, 1993; Reichardt et al., 1991; Kainulainen et al., 1996),
which could also generate complex patterns of herbivore response. In two willow
species, condensed tannin levels were decreased by mild drought, whereas simple
phenolic compounds were not affected (Glynn et al., 2004). Additionally, positive
effects of drought on nutritional quality, such as increases in free amino acids,
soluble proteins, and soluble carbohydrates (Brodbeck and Strong, 1987; Mattson
and Haack 1987), may counteract negative effects of increased secondary metabolite levels (Glynn et al., 2004). Nutrient availability had a positive linear effect on
whitemarked tussock moth growth and foliar nitrogen, but had no effect on gypsy
moth. This overall pattern of treatment effects on these two species is consistent
with relatively high sensitivities of whitemarked tussock moth to nutritional quality
and gypsy moth to phenolic glycosides.
Differential effects of resource availability on constitutive vs. induced
resistance may be an important source of variation in herbivore performance
(Lewinsohn et al. 1993; Lerdau et al. 1994; Lombardero et al., 2000). We are
DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
2617
not aware of other studies concerning effects of water availability on the
expression of rapid induced resistance of woody plants to folivores, and are
aware of only a few that have examined nutrient availability, all of which
revealed some effect (Hunter and Schultz, 1995; Mutikainen et al., 2000; Glynn
et al., 2003). In this study, however, rapid induced resistance to gypsy moth was
not influenced by resource availability, it being expressed in all treatments. This
study therefore emphasizes the difficulty in generalising about the effects of
abiotic and biotic stress on host quality, even for insects within the same family.
AcknowledgmentsVWe thank two anonymous reviewers for constructive comments that
improved the article. Jeremy Christman and Adam Clark helped maintain the fertigation system.
Bryant Chambers, Ann Hale, Richard Hale, Diane Hartzler, James Howell, Cathy Love, Cassie
Mahl, Ben Marthey, Kyle Theibert, and John Watson aided with field and laboratory work. Bruce
Birr (USDA Forest Service, Rhinelander, WI, USA) conducted the nitrogen analysis. Howard Lin
contributed toward the technique used to analyse phenolic glycosides. Salaries and research support
were provided by State and Federal funds appropriated to the Ohio Agricultural Research and
Development Center, The Ohio State University.
REFERENCES
BLOOM, A. J., CHAPIN, F. S. III, and MOONEY, H. A. 1985. Resource limitation in plantsVan
economic analogy. Annu. Rev. Ecol. Syst. 16:363Y392.
BRADFORD, K. J. and HSIAO, T. C. 1982. Physiological responses to moderate water stress, pp. 263Y324,
in O. L. Lange, P. S. Nobel, C. B. Osmond, and H. Ziegler (eds.) Physiological Plant Ecology. II.
Water Relations and Carbon Assimilation. Springer-Verlag, Berlin.
BRODBECK, B. and STRONG, D. 1987. Amino acid nutrition of herbivorous insects and stress to host
plants, pp. 347Y364, in P. Barbosa and J. C. Shultz (eds.) Insect Outbreaks. Academic Press,
San Diego, CA.
BRYANT, J. P., CLAUSEN, T. P., REICHARDT, P. B., MCCARTHY, M. C., and WERNER, P. A. 1987.
Effect of nitrogen fertilization upon the secondary chemistry and nutritional value of quaking
aspen (Populus tremuloides Michx.) leaves for the large aspen tortrix (Choristoneura
conflictana Walker). Oecologia 73:513Y517.
CHEW, V. 1976. Comparing treatment means: a compendium. HortScience 11:348Y357.
CLAUSEN, T. P., EVANS, T. P., REICHARDT, P. B., and BRYANT, J. P. 1989a. A simple method for the
isolation of salicortin, tremulacin and tremuloiden from quaking aspen (Populus tremuloides).
J. Nat. Prod. 52:207Y209.
CLAUSEN, T. P., REICHARDT, P. B., BRYANT, J. P., WERNER, R. A., POST, K., and FRISBY, K. 1989b.
Chemical model for short-term induction in quaking aspen (Populus tremuloides) foliage
against herbivores. J. Chem. Ecol. 15:2335Y2347.
COBB, N. S., MOPPER, S., GEHRING, C. A., CAOUETTE, M., CHRISTENSEN, K. M., and WHITHAM,
T. G. 1997. Increased moth herbivory associated with environmental stress of pinyon pine at
local and regional levels. Oecologia 109:389Y397.
CRAIG, T. P., WAGNER, M. R., MCCULLOUGH, D. G., and FRANTZ, D. P. 1991. Effects of
experimentally altered plant moisture stress on the performance of Neodiprion sawflies. For.
Ecol. Manag. 39:247Y261.
2618
HALE ET AL.
DAUN, J. K. and DECLERQ, D. R. 1994. Comparison of combustion and Kjeldahl methods for
determination of nitrogen in oilseeds. J. Chem. Ecol. 25:1427Y1440.
GEIGER, D. R., KOCH, K. E., and SHIEH, W.-J. 1996. Effect of environmental factors on whole plant
assimilate partitioning and associated gene expression. J. Exp. Bot. 47:1229Y1238.
GLYNN, C., HERMS, D. A., EGAWA, M., HANSEN, R., and MATTSON, W. J. 2003. Effects of nutrient
availability on biomass allocation as well as constitutive and rapid induced herbivore resistance
in poplar. Oikos 101:385Y397.
GLYNN, C., RÖNNBERG-WÄSTLJUNG, A.-C., JULKUNEN-TIITO, R., and WEIH, M. 2004. Willow
genotype, but not drought treatment, affects foliar phenolic concentrations and leaf-beetle
resistance. Entomol. Exp. Appl. 113:1Y14.
HANSEN, R. C., Prenger, J. J., Herms, D. A., and SHORT, T. H. 2000. Evaluation of a precision
fertigation system. Presented at the Jul 9Y12 2000 ASAE Annual International Meeting, Paper
No. 005005. ASAE, 2950 Niles Rd., St. Joseph, MI, USA.
HAUKIOJA, E. 1990. Induction of defenses in trees. Annu. Rev. Entomol. 36:25Y42.
HAVILL, N. P. and RAFFA, K. F. 1999. Effects of elicitation treatment and genotypic variation on
induced resistance in Populus: impacts on gypsy moth (Lepidoptera: Lymantriidae)
development and feeding behaviour. Oecologia 120:295Y303.
HEMMING, J. D. C. and LINDROTH, R. L. 1995. Intraspecific variation in aspen phytochemistryVeffects
on performance of gypsy moths and forest tent caterpillars. Oecologia 103:79Y88.
HEMMING, J. D. C. and LINDROTH, R. L. 1999. Effects of light and nutrient availability on aspen:
growth, phytochemistry and insect performance. J. Chem. Ecol. 25:1687Y1714.
HEMMING, J. D. C. and LINDROTH, R. L. 2000. Effects of phenolic glycosides and protein on gypsy
moth (Lepidoptera: Lymantriidae) and forest tent caterpillar (Lepidoptera: Lasiocampidae)
performance and detoxication activities. Environ. Entomol. 29:1108Y1115.
HERMS, D. A. 2002. Effects of fertilization on insect resistance of woody ornamental plants:
reassessing an entrenched paradigm. Environ. Entomol. 31:923Y933.
HERMS, D. A. and MATTSON, W. J. 1992. The dilemma of plants: to grow or defend. Q. Rev. Biol.
67:283Y335.
HIROSE, T. 1987. A vegetative plant growth model: adaptive significance of phenotypic plasticity in
matter partitioning. Funct. Ecol. 1:195Y202.
HUBERTY, A. F. and DENNO, R. F. 2004. Plant water stress and its consequences for herbivorous
insects: a new synthesis. Ecology 85:1383Y1398.
HUNT, R. 1978. Plant Growth Analysis. The Institute of Biology’s Studies in Biology No. 96.
Edward Arnold, London.
HUNTER, M. D. and SCHULTZ, J. C. 1995. Fertilization mitigates chemical induction and herbivore
responses within damaged oak trees. Ecology 76:1226Y1232.
HWANG, S. Y. and LINDROTH, R. L. 1997. Clonal variation in foliar chemistry of aspen: effects on
gypsy moths and forest tent caterpillars. Oecologia 111:99Y108.
INGESTAD, T. and ÅGREN, G. I. 1991. The influence of plant nutrition on biomass allocation. Ecol.
Appl. 1:168Y174.
INGESTAD, T. and ÅGREN, G. I. 1992. Theories and methods on plant nutrition and growth. Physiol.
Plant. 84:177Y184.
JACOBS, D. F., ROSE, R., HAASE, D. L., and ALZUGARAY, P. O. 2004. Fertilization at planting
impairs root system development and drought avoidance of Douglas-fir (Pseudotsuga
menziesii) seedlings. Ann. Sci. For. 61:643Y651.
KAINULAINEN, P., HOLOPAINEN, J., PALOMÄKI, V., and HOLOPAINEN, T. 1996. Effects of nitrogen
fertilization on secondary chemistry and ectomycorrhizal state of Scots pine seedlings and on
growth of grey pine aphid. J. Chem. Ecol. 22:617Y636.
KAROWE, D. 1989. Differential effects of tannin acid on two tree-feeding Lepidoptera: implications
for theories of plant anti-herbivore chemistry. Oecologia 80:507Y512.
DROUGHT STRESS, NUTRIENT AVAILABILITY AND INSECT RESISTANCE
2619
KOPPER, B. J., JAKOBI, V. N., OSIER, T. L., and LINDROTH, R. L. 2002. Effects of paper birch
condensed tannin on whitemarked tussock moth (Lepidoptera: Lymantriidae) performance.
Environ. Entomol. 31:10Y14.
KORICHEVA, J. 1999. Interpreting phenotypic variation in plant allelochemistry: problems with the
use of concentrations. Oecologia 119:467Y473.
KORICHEVA, J., LARSSON, S., HAUKIOJA, E., and KEINANEN, M. 1998a. Regulation of woody plant
secondary metabolism by resource availability: hypothesis testing by means of meta-analysis.
Oikos 83:212Y226.
KORICHEVA, J., LARSSON, S., and HAUKIOJA, E. 1998b. Insect performance on experimentally
stressed woody plants: a meta-analysis. Annu. Rev. Entomol. 43:195Y216.
KYTÖ, M., NIEMELA, P., and LARSSON, S. 1996. Insects on trees: population and individual response
to fertilization. Oikos 75:148Y159.
LAMBERS, H. and POORTER, H. 1992. Inherent variation in growth rate between higher plantsVa
search for physiological causes and ecological consequences. Adv. Ecol. Res. 23:187Y261.
LARSSON, S. 1989. Stressful times for the plant stressYinsect performance hypothesis. Oikos
56:277Y283.
LERDAU, M., LITVAK, M., and MONSON, R. 1994. PlantYchemical defenseVmonoterpenes and the
growthYdifferentiation balance hypothesis. Trends Ecol. Evol. 9:58Y61.
LEWINSOHN, E., GIJZEN, M., MUZIKA, R. M., BARTON, K., and CROTEAU, R. 1993. Oleoresinosis in
grand fir (Abies grandis) saplings and mature treesVmodulation of this wound response by
light and water stresses. Plant Physiol. 101:1021Y1028.
LINDER, S. and ROOK, D. A. 1984. Effects of mineral nutrition on carbon dioxide exchange and
partitioning of carbon in trees, pp. 211Y236, in G. D. Bowen and E. K. S. Nambiar (eds.)
Nutrition of Plantation Trees. Academic Press, London.
LINDER, S., BENSON, M. L., MYERS, B. J., and RAISON, R. J. 1987. Canopy dynamics and growth of
Pinus radiata. I. Effects of irrigation and fertilization during a drought. Can. J. For. Res.
17:1157Y1165.
LINDROTH, R. L. and HEMMING, J. C. D. 1990. Responses of the gypsy moth (Lepidoptera:
Lymantriidae) to tremulacin, an aspen phenolic glycoside. Environ. Entomol. 19:842Y847.
LINDROTH, R. L. and HWANG, S.-Y. 1996. Diversity, redundancy, and multiplicity in chemical
defense systems of aspen, pp. 25Y56, in J. T. Romeo, J. A. Saunders, and P. Barbosa (eds.)
Phytochemical Diversity and Redundancy in Ecological Interactions. Plenum Press, New York.
LINDROTH, R. L., HSIA, M. T. S., and SCRIBER, J. M. 1987. Characterization of phenolic glycosides
from quaking aspen. Biochem. Syst. Ecol. 15:677Y680.
LOMBARDERO, M. J., AYRES, M. P., LORIO, P. L. JR, and RUEL, J. J. 2000. Environmental effects on
constitutive and inducible resin defences of Pinus taeda. Ecol. Lett. 3:329Y339.
LOOMIS, W. E. 1932. GrowthYdifferentiation balance vs. carbohydrateYnitrogen ratio. Proc. Am.
Hortic. Sci. 29:240Y245.
LORIO, P. L. JR 1986. GrowthYdifferentiation balance: a basis for understanding southern pine
beetleYtree interactions. For. Ecol. Manag. 14:259Y273.
LUXMOORE, R. J. 1991. A source-sink framework for coupling water, carbon, and nutrient dynamics
of vegetation. Tree Physiol. 9:267Y280.
MATTSON, W. J. and HAACK, R. A. 1987. The role of drought in outbreaks of plant-eating insects.
BioScience 37:110Y118.
MILLER, B. D. and TIMMER, V. R. 1994. Steady-state nutrition of Pinus resinosa seedlingsVresponse
to nutrient loading, irrigation and hardening regimes. Tree Physiol. 14:1327Y1338.
MIZE, C. W. and SCHULTZ, R. C. 1985. Comparing treatment means correctly and appropriately.
Can. J. For. Res. 15:1142Y1148.
MOPPER, S. and WHITHAM, T. G. 1992. The plant stress paradox: effects on pinyon sawfly sex ratios
and fecundity. Ecology 73:515Y525.
2620
HALE ET AL.
MUTIKAINEN, P., WALLS, M., OVASKA, J., KEINANEN, M., JULKUNEN-TIITTO, R., and VAPAAVUORI,
E. 2000. Herbivore resistance in Betula pendula: effect of fertilization, defoliation and plant
genotype. Ecology 81:49Y65.
MUZIKA, R. M. 1993. Terpenes and phenolics in response to nitrogen fertilization: a test of the
carbon/nutrient balance hypothesis. Chemoecology 4:3Y7.
OSIER, T. L., HWANG, S.-Y., and LINDROTH, R. L. 2000. Effects of phytochemical variation in
quaking aspen Populus tremuloides clones on gypsy moth, Lymantria dispar, performance in
the field and laboratory. Ecol. Entomol. 25:197Y207.
POWER, S. A., ASHMORE, M. R., COUSINS, D. A., and SHEPPARD, L. J. 1998. Effects of nitrogen
addition on the stress sensitivity of Calluna vulgaris. New Phytol. 138:663Y673.
REICHARDT, P. B., CHAPIN, F. S., BRYANT, J. P., MATTES, B. R., and CLAUSEN, T. P. 1991. Carbon
nutrient balance as a predictor of plant defense in Alaskan balsam poplarVpotential importance
of metabolite turnover. Oecologia 88:401Y406.
ROBSON, D. S. 1959. A simple method for constructing orthogonal polynomials when the
independent variable is unequally spaced. Biometrics 15:187Y191.
ROBISON, D. J. and RAFFA, K. F. 1994. Characterization of hybrid poplar clones for resistance to the
forest tent caterpillar. For. Sci. 40:686Y714.
ROSS, D. W. and BERISFORD, C. W. 1990. Nantucket pine tip moth (Lepidoptera: Tortricidae):
response to water and nutrient status of loblolly pine. For. Sci. 36:719Y733.
ROTH, S., MCDONALD, E. P., and LINDROTH, R. L. 1997. Atmospheric CO2 and soil water availability: consequences for treeYinsect interactions. Can. J. For. Res. 27:1281Y1290.
SAS Institute 2000. SAS for Personal Computer, version 8. Carey, NC.
SCRIBER, J. M. 1977. Limiting effects of low leaf-water content on the nitrogen utilization, energy
budget, and larval growth of Hyalophora cecropia (Lepidoptera: Saturniidae). Oecologia
28:269Y287.
STAMP, N. 2003. Out of the quagmire of plant hypotheses. Q. Rev. Biol. 78:23Y55.
STAMP, N. 2004. Can the growthYdifferentiation balance hypothesis be tested rigorously? Oikos
107:439Y448.
WAGNER, M. R. 1986. Influence of moisture stress and induced resistance in ponderosa pine, Pinus
ponderosa Dougl. Ex. Laws, on the pine sawfly, Neodiprion autumnalis Smith. For. Ecol.
Manag. 15:43Y53.
WALTERS, M. B. and REICH, P. B. 1989. Response of Ulmus americana seedlings to varying
nitrogen and water status. I. Photosynthesis and growth. Tree Physiol. 5:159Y172.
WARDLAW, I. F. 1990. The control of carbon partitioning in plants. New Phytol. 116:341Y381.
WATERMAN, P. G. and MOLE, S. 1989. Extrinsic factors influencing production of secondary
metabolites in plants, pp. 107Y134, in E. A. Bernays (ed.). InsectYPlant Interactions, Vol. 1.
CRC Press, Boca Raton, FL.
WHITE, T. C. R. 1984. The abundance of invertebrate herbivores in relation to the availability of
nitrogen in stressed food plants. Oecologia 63:90Y105.