J Chem Ecol (2008) 34:1392–1400
DOI 10.1007/s10886-008-9550-z
Effects of Fertilization and Fungal and Insect Attack
on Systemic Protein Defenses of Austrian Pine
Kathryn Barto & Stephanie Enright & Alieta Eyles &
Chris Wallis & Rodrigo Chorbadjian & Robert Hansen &
Daniel A. Herms & Pierluigi Bonello & Don Cipollini
Received: 27 June 2008 / Revised: 19 September 2008 / Accepted: 19 September 2008 / Published online: 7 October 2008
# Springer Science + Business Media, LLC 2008
Abstract Despite their economic and ecological importance, defense responses of conifers to pests are little
understood. In a 3-year experiment, we monitored systemic
fungal (Diplodia pinea)- and insect (Neodiprion sertifer)K. Barto : S. Enright : D. Cipollini (*)
Department of Biological Sciences, Wright State University,
3640 Colonel Glenn Highway,
Dayton, OH 45435, USA
e-mail: [email protected]
A. Eyles : C. Wallis : P. Bonello
Department of Plant Pathology, The Ohio State University,
201 Kottman Hall, 2021 Coffey Road,
Columbus, OH 43210, USA
R. Chorbadjian : D. A. Herms
Department of Entomology, The Ohio State University/Ohio
Agricultural Research and Development Center,
1680 Madison Avenue,
Wooster, OH 44691, USA
R. Hansen
Department of Food, Agriculture and Biological Engineering,
The Ohio State University,
108 Agricultural Engineering Building, 1680 Madison Avenue,
Wooster, OH 44691, USA
Present address:
A. Eyles
Cooperative Research Centre for Forestry,
TIAR/University of Tasmania,
Private Bag12,
Hobart 7001, Australia
Present address:
C. Wallis
Ecosystems Science and Management Program,
University of Northern British Columbia,
3333 University Way,
Prince George, BC, Canada V2N 4Z9
induced defense protein activities and total soluble proteins
in needles and phloem of Austrian pine (Pinus nigra)
across a soil fertility gradient. In both years, total soluble
protein content of foliage and phloem declined with
increasing fertility across induction treatments, while
defensive protein activities generally increased with increasing fertility. In 2005, total soluble protein content in
branch phloem was increased by fungal inoculation of the
stem. Peroxidase activity was suppressed in needles by
insect defoliation in 2006, while polyphenol oxidase
activity was systemically induced in branch phloem by
insect attack in 2005. Trypsin inhibitor activities in phloem
did not respond to any induction or fertility treatment.
Nutritive quality of Austrian pine tissue declined with
increasing fertility, while several protein-based defenses
simultaneously increased.
Keywords Pinus nigra . Diplodia pinea .
Neodiprion sertifer . Induction
Introduction
Conifers are ecologically and economically important
worldwide. Many fungal and insect pests attack conifers
and can severely limit their growth and value, but
interactions among these fungal and insect pests and their
host plants are poorly understood. A better understanding
of conifer defense mechanisms, including the influence of
environmental conditions on their expression, would
provide a mechanistic basis for understanding interactions
between species exploiting pines as a resource and help to
explain patterns of variation in attack across environmental
gradients in the field. It would also allow for more targeted
management to reduce the economic impacts of pest attack.
J Chem Ecol (2008) 34:1392–1400
We have been studying such interactions using a Pinus
nigra–Diplodia pinea–Neodiprion sertifer tripartite system
(Bonello and Blodgett 2003; Eyles et al. 2007). P. nigra
(Pinaceae; Austrian pine) are among the most commonly
planted trees in North America where they suffer from
Diplodia shoot blight and canker, which can kill stressed
trees (Peterson 1977). D. pinea (Ascomycetes; formerly
Sphaeropsis sapinea) inhabits the phloem of stems and
branches. This pathogen will also infect other two- and
three-needle pines, but P. nigra is the most susceptible pine
in North America. N. sertifer (Hymenoptera; Diprionidae;
European pine sawfly) feeds on 2-year-old needles of
several pine species and readily defoliates P. nigra (D.A.
Herms, personal observation). It is also an outbreak insect
known to be sensitive to host quality (Larsson et al. 2000).
In a given season, P. nigra can be attacked by both of these
pests, indicating that host-mediated interactions between
them may occur. Given their feeding locations, changes in
host plant quality in response to one attacker that are
systemic are more likely to impact the other than are local
changes in quality. In a 2-year study, we showed that D.
pinea infection of the stem of young P. nigra consistently
induced systemic resistance against the same fungus in
branches. Defoliation of branches by N. sertifer failed to
systemically affect growth of this insect in undamaged
branches in either year. In terms of cross-resistance,
however, defoliation of branches by N. sertifer induced
systemic resistance to D. pinea in an undamaged branch in
one of the 2 years of the study. Conversely, D. pinea
infection of the stem induced systemic resistance to N.
sertifer growth on foliage from an undamaged branch in
1 year, while in the other year, systemic effects of both D.
pinea infection and N sertifer feeding on N. sertifer survival
on undamaged branches were significant, but depended
upon soil fertility level (Eyles et al. 2007). These results
indicated that systemic induced resistance to D. pinea in
P. nigra by previous infection with the same fungus was
consistent and, although reciprocal cross-resistance can
occur in P. nigra in response to these attackers, the results
can be asymmetric within a single year and variable among
years.
Although regulatory mechanisms of defenses in conifers
are poorly understood, conifers produce a suite of secondary metabolites in response to attack by insects and
pathogens, notably phenolics and terpenoids (Franceschi
et al. 2005). In a companion paper that focused on fungal
resistance mechanisms in the phloem, we showed that,
when examined as a group, phenolic glycosides and
stilbenes increased systemically in the phloem of branches
of young P. nigra in response to D. pinea infection of the
stem and were positively correlated with resistance to D.
pinea in those branches. On the other hand, terpenes were
not inducible in the phloem by fungal infection as a group
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and were unrelated to fungal resistance. Only a single
terpenoid, germacrene D, was systemically inducible in the
phloem of branches by defoliation by N. sertifer, but it was
unrelated to fungal resistance (Wallis et al. 2008). Less
studied than secondary metabolites in conifers are pathogenesis-related (PR) and other defense-related proteins,
which can be associated with insect and disease resistance
in a variety of plants. In Picea sitchensis, a family of
dirigent proteins that assist in lignan formation were locally
induced by insect feeding, as were β-1,3-glucanases,
chitinase, a protease inhibitor, and peroxidases (Ralph et
al. 2006, 2007; Lippert et al. 2007). In Pinus sylvestris,
chitinases were produced constitutively and were induced
locally by endophytic microbes (Pirttilä et al. 2002), but not
pathogenic fungi (Hodge et al. 1995). In Pinus monticola,
chitinase was induced locally in needles by a pathogenic
fungus (Liu et al. 2005), as was expression of a PR10
protein (Liu et al. 2003). In P. sylvestris and Picea abies,
peroxidases and polyphenol oxidases were induced locally
after attack by pathogenic fungi (e.g., Johansson et al.
2004). P. abies also produces chitinases, chitosanases, and
glucanases locally after challenge with pathogenic fungi
(e.g., Jøhnk et al. 2005). These studies revealed substantial
variation in when and where induction of defense proteins
may be expected to occur in conifers. However, these
studies focused primarily on local induction. While certainly important in understanding local restrictions on
disease spread or insect feeding, systemic changes in plant
defenses have the potential to have broader effects on other
interacting species. Despite mounting evidence of their
importance, systemic induction of PR and other defenserelated proteins has been little studied in conifers (but see
Richard et al. 2000) and never in P. nigra in response to
fungal or insect attack.
Environmental factors, such as soil fertility, are known to
affect disease and insect resistance in plants. For example,
resistance of red pine (Pinus resinosa) to D. pinea is
decreased by high soil fertility (Blodgett et al. 2005), which
can result in increased tree mortality (Stanosz et al. 2004).
Such findings may relate to changes in host plant quality
across soil environmental gradients. Although never studied
in conifers, protein defenses in many herbaceous and
woody plants can vary with soil fertility. Constitutive
activity of trypsin inhibitors, peroxidases, chitinases, and
β-1,3-glucanases increased in response to increased fertility
in Brassica napus, Arabidopsis thaliana, and two Citrus
species (Cipollini and Bergelson 2001; Cipollini 2002;
Borowicz et al. 2003). Induced activity of trypsin inhibitors, papain inhibitors, and peroxidases were higher in
plants with higher soil fertility (Bolter et al. 1998; Cipollini
and Bergelson 2001; Cipollini 2002; Borowicz et al. 2003).
In contrast, constitutive activities of trypsin inhibitors,
chitinases, β-1,3-glucanases, and peroxidases in soybean
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and tomato decreased with increasing fertility (Inbar et al.
2001; Vollmann et al. 2003). The equivocal nature of these
results may be due to the different methods used to induce
defenses (insect feeding, wounding, or exogenous hormone
application). Furthermore, different methods of altering soil
nutrients and different methods of sampling tissues may
have yielded such varying results.
In this 3-year replicated study, we induced young
Austrian pine trees with insect defoliation of branches or
fungal inoculation of the main stem and monitored
constitutive and induced activities of defensive proteins
and levels of soluble proteins in both phloem and needles
of uninfected or undefoliated branches. The effects of these
treatments were examined across three soil fertility levels to
examine the extent to which fertility modulated protein
accumulation and the constitutive or inducible activities of
defense proteins. We expected total protein levels and
defense protein activities to increase with increasing
fertility. In addition, we expected activities of defense
proteins to be induced systemically by insect and fungal
damage.
Methods and Materials
The P. nigra–D. pinea–N. sertifer system and our experimental design are described in detail in Eyles et al. (2007).
Briefly, 4-year-old bare-root P. nigra saplings were grown
outside in 21-L containers with a commercial nursery
substrate (KB container mix, Kurtz Bros. Central Ohio,
LLC) at the Ohio Agricultural Research and Development
Center (Wooster, OH, USA). Plants were placed on a
fertigation system at either low, intermediate, or high
fertilization levels (30, 75, 150 ppm N; N/P/K 3:1:2) in
the spring of 2004. Fertilization levels were chosen to
represent recommended fertility regimes for containerized
evergreen trees in forest nursery production (Eyles et al.
2007). After a year of conditioning on the fertilization
regimes, four separate induction treatments were applied in
the spring of 2005 (D. pinea infection, mock inoculation,
defoliation by N. sertifer, and unwounded controls). The
induction of fungal inoculations occurred 5 cm above the
soil on the main stem where agar plugs containing D. pinea
were placed in a 10-mm diameter wound made with a cork
borer. Mock-inoculated trees received a 10-mm cork borer
wound and a sterile agar plug. At the same time, a separate
set of trees were induced by N. sertifer by allowing several
third and fourth instar larvae to defoliate approximately
75% of the foliage on the tree (typically 2- and 3-year-old
needles), while one branch in the top whorl of the tree
remained protected by a mesh bag. This whorl was
approximately 10 cm from the base of the trees, which
averaged about 24 cm in height when induced. We
J Chem Ecol (2008) 34:1392–1400
replicated the study by growing a set of 6-year-old
containerized trees grown under similar conditions and
exposed to the same fertility treatments beginning in 2005
that were induced in the spring of 2006. The mock
inoculation treatment was omitted in 2006 because it did
not alter response variables significantly from those of
control plants in 2005. The top whorl on trees used in the
second study was about 40 cm from the base of the trees,
which averaged about 63 cm in height. The fertility and
induction treatments in both 2005 and 2006 were assigned
to five spatial blocks with six to ten trees assigned to each
fertility and induction treatment combination in each block.
Further details on the induction procedure and our
experimental design can be found in Eyles et al. (2007).
Foliage and branch phloem were sampled 16 days after
induction treatments in 2005 and 21 days after induction
treatments in 2006 (Eyles et al. 2007). While these
timeframes are not necessarily optimal for capturing
maximal increases in protein defenses after induction, they
were timed to coincide with sampling for secondary
metabolites (Wallis et al. 2008) and for challenge bioassays
(Eyles et al. 2007). On trees induced by insect defoliation,
foliage (second year needles) and branch phloem (hereafter
phloem) were collected from a bagged branch from the top
whorl. On trees induced by D. pinea infection of the stem,
foliage and phloem were collected from a corresponding
branch on the same whorl as that collected on insectdefoliated trees. Tissues were flash frozen immediately in
liquid nitrogen and stored below −20°C until analysis.
Tissues were first weighed, then ground with a mortar and
pestle in liquid nitrogen. Needles were homogenized whole,
while phloem was dissected from branch samples prior to
homogenization. Soluble proteins were extracted from
ground samples in ice-cold 0.01 M sodium phosphate
buffer (pH 6.8) containing 5% w/v poly(vinylpolypyrrolidone). Guaiacol peroxidase (POD), polyphenol oxidase
(PPO), chitinase (CHI), trypsin inhibitor (TI) activities, and
total soluble protein content were assayed by using
spectrophotometric and radial diffusion techniques as in
Cipollini et al. (2004). β-1,3-glucanase (BGLU) activity
was measured by following the hydrolysis of laminarin
spectrophotometrically at 500 nm (modified from Abeles
and Forrence 1970). Each sample for BGLU analysis was
blanked twice, once with a blank containing extraction
buffer and laminarin, and again with one containing
extraction buffer and sample to account for color in the
protein extracts. In 2005, activities of POD, PPO, and CHI
were assessed in foliage and phloem, while TI and BGLU
were only assessed in phloem due to limited amounts of
available foliage. In 2006, activities of POD, PPO, CHI,
and BGLU were measured in both tissues. Levels of TI
were not assessed in 2006, since only very low levels were
detected in phloem in 2005. Activities of the proteins were
J Chem Ecol (2008) 34:1392–1400
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expressed both per unit of extracted biomass and per unit
extracted protein. Patterns were generally similar when
expressed either way, so only activities expressed per unit
extracted protein are reported. Total soluble protein contents were expressed per unit fresh weight of extracted
biomass.
Data were transformed as necessary and analyzed for
treatment effects using analysis of variance (SAS Version
9.1, SAS Institute). Factors in the model included fertility
level, induction treatment, and their interactions. Block
effects were examined and were negligible, so the degrees
of freedom associated with this factor were included in the
error degrees of freedom. Means were separated using
Bonferroni comparisons. Only statistical results deemed
significant at α=0.05 are presented in the descriptions
below, although all data are shown in the figures.
Results
Interactions between fertility level and induction treatment
were not significant for total protein content or for the
activity of any protein. In no case did total soluble protein
levels increase with increasing fertility or did activities of
any defensive protein decrease with increasing fertility. We
thus focus descriptions on the significant main effects of
induction treatment and fertility, although nonsignificant
patterns are also shown on the figures.
Across induction treatments, total soluble protein content
of needles decreased with increasing fertility in both years
of the study (Fig. 1a; 2005-fertility: F2,169 =6.10, P=0.003;
2006-fertility: F2,111 =7.47, P<0.001). Across induction
treatments, total soluble protein contents in phloem were
about ten times lower than in needles and tended to
decrease with increasing fertility in 2005 (Fig. 1b; fertility:
F2,135 =2.94, P=0.056). Across fertility levels in 2005, total
soluble protein levels in phloem were 40% higher in trees
induced with the fungus than in either control trees or those
receiving a mock inoculation (Fig. 1b; induction: F3,135 =
5.05, P=0.002).
Across induction treatments, POD activity in needles
increased with increasing fertility in 2005 (Fig. 2a; fertility:
F2,141 =13.70, P<0.001). Across fertility levels, POD activity
was almost 90% lower in needles of insect-induced trees
than in control trees in 2006 (Fig. 2a; induction: F2,100 =6.72,
P=0.002). In phloem, POD activities were about 400 times
higher than in needles (Fig. 2). As in needles, POD activity
in phloem in 2005 increased with increasing fertility across
induction treatments, although differences were not as
pronounced as those in needles (Fig. 2b; fertility: F2,140 =
4.65, P=0.011).
Across induction treatments, PPO activity in needles
increased with increasing fertility in 2005 (Fig. 3a; 2005-
Fig. 1 Total soluble protein content of P. nigra tissues, means
(milligrams soluble protein per gram fresh weight)±1 SE. Bars with
the same letter were not significantly different using Bonferroni
comparisons at P=0.05. If no letters are shown, results were not
significantly different. Years were analyzed separately. Numbers in
bars indicate number of replicates. C control—untreated trees, M
mock—trees challenged with a sterile agar plug for 2005 only, F
fungus—trees inoculated with D. pinea, I insect—trees defoliated by
N. sertifer. a Total soluble protein content of needles. b Total soluble
protein content of phloem
needles-fertility: F2,158 =3.24, P=0.042). Activities in phloem
were about ten times higher than in needles in 2005, but still
lowest in the lowest fertility treatment with the effect nearly
significant (Fig. 3b; 2005-phloem-fertility: F2,138 =3.03, P=
0.051). Across fertility levels, PPO levels in phloem were
40% higher in trees induced by insect feeding than in
unwounded trees in 2005 (induction: F3,133 =4.28, P=0.006).
Neither mock inoculation nor fungal attack affected PPO
levels in phloem in 2005 (Fig. 3b).
Across induction treatments, CHI activity increased with
increasing fertility in needles in 2006 and in phloem in both
years (Fig. 4; 2006-needles-fertility: F2,107 =3.08, P=0.050;
2005-phloem-fertility: F2,139 =3.09, P=0.049; 2006-phloemfertility: F2,109 =6.62, P=0.002). In 2005, CHI levels in
phloem were about five times higher than in needles, and
in 2006, levels in phloem were about ten times higher than in
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J Chem Ecol (2008) 34:1392–1400
Discussion
In 2005, we observed systemic induction of PPO in the
phloem of undamaged branches of P. nigra trees that had
been damaged by N. sertifer feeding on needles of other
branches in the same whorl. Such systemic effects of insect
feeding have never been demonstrated in conifer species,
and they implicate long-distance signaling in the phloem.
Systemic induction of PPO in upper leaves of poplar by
damage to lower leaves has been demonstrated (Haruta et
al. 2001), but systemic induction of this enzyme in the
phloem of woody plants has not been studied to our
knowledge. Despite systemic induction of PPO, defoliation
by N. sertifer did not lead to systemic resistance to N.
sertifer in the timeframe of our induction and challenge
treatments (Eyles et al. 2007). In contrast, POD activity was
Fig. 2 Peroxidase activity in P. nigra tissues, means (ΔA470 per
minute per milligram soluble protein)±1 SE. Bars with the same letter
were not significantly different using Bonferroni comparisons at P=
0.05. If no letters are shown, results were not significantly different.
Years were analyzed separately. Numbers in or immediately under
bars indicate number of replicates. C control—untreated trees, M
mock—trees challenged with a sterile agar plug for 2005 only, F
fungus—trees inoculated with D. pinea, I insect—trees defoliated by
N. sertifer. a Activity in needles. b Activity in phloem
needles (Fig. 4). Across fertility levels, no induction
treatment altered CHI levels from those seen in controls in
phloem in 2005, although levels were lower in trees induced
by fungal attack than in trees receiving a mock inoculation
(Fig. 4b; induction: F3,139 =3.07, P=0.030).
Activity of β-1,3-glucanase was very low in phloem in
2005 (Fig. 5). Across induction treatments, BGLU activity
in needles in 2006 increased with increasing fertility
(Fig. 5a; fertility: F2,105 =8.13, P<0.001). As for other
defense proteins, BGLU activities were higher in phloem
than in needles in 2006 (Fig. 5).
Trypsin inhibitor activity was only assessed in phloem in
2005 and was not significantly affected by any treatments.
Levels detected were so low that TI was not measured in
2006 (Fig. 6).
Fig. 3 Polyphenol oxidase activity in P. nigra tissues, means (ΔA470
per minute per milligram soluble protein)±1 SE. Bars with the same
letter were not significantly different using Bonferroni comparisons at
P=0.05. If no letters are shown, results were not significantly
different. Years were analyzed separately. Numbers in bars indicate
number of replicates. C control—untreated trees, M mock—trees
challenged with a sterile agar plug for 2005 only, F fungus—trees
inoculated with D. pinea, I insect—trees defoliated by N. sertifer. a
Activity in needles. b Activity in phloem
J Chem Ecol (2008) 34:1392–1400
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noticeable than systemic changes. Some of the variation in
plant responses to treatments among years may reflect age
differences of the trees used in each year.
Despite the lack of effect on insect growth, insect
induction induced systemic resistance to fungal growth in
2006 (Eyles et al. 2007). Conversely, fungal induction
induced systemic resistance to fungal growth in both 2005
and 2006, and it induced systemic resistance to insect
growth in 2005 (Eyles et al. 2007). Since fungal inoculation
did not induce systemically any of the defensive proteins
that we measured, other defenses (protein or nonprotein)
must have been responsible for the fungal-induced effects
on resistance. In the same trees as the current study, Wallis
et al. (2008) found that phenolic glycosides and stilbenes
increased systemically in the phloem of uninoculated
Fig. 4 Chitinase activity in P. nigra tissues, means (A405 per milligram
soluble protein)±1 SE. Bars with the same letter were not significantly
different using Bonferroni comparisons at P=0.05. If no letters are
shown, results were not significantly different. Years were analyzed
separately. Numbers in bars indicate number of replicates. C control—
untreated trees, M mock—trees challenged with a sterile agar plug for
2005 only, F fungus—trees inoculated with D. pinea, I insect—trees
defoliated by N. sertifer. a Activity in needles. b Activity in phloem
systemically suppressed in needles by insect feeding on
other branches in 2006. Suppression of POD could be due
to resource constraints or may reflect an ability of the insect
to directly suppress the plant’s defense response through
signal interactions (Musser et al. 2005). Inoculation with D.
pinea had surprisingly little effect on systemic defense
protein activities during the timeframe of our study in either
year, and aside from effects on PPO and POD, we saw little
systemic response to induction treatments in the other
defense proteins that we studied. Activities of rapidly
induced defense proteins often increase within a matter of
hours to days, typically followed by a return to basal levels
sometime thereafter, so our relatively delayed sampling
may have limited our ability to observe maximal changes in
protein activities. In addition, changes in these proteins
induced locally to the site of damage may have been more
Fig. 5 β-1,3-glucanase activity in P. nigra tissues, means (A500 per
milligram soluble protein)±1 SE. Bars with the same letter were not
significantly different using Bonferroni comparisons at P=0.05. If no
letters are shown, results were not significantly different. Years were
analyzed separately. Numbers in or immediately under bars indicate
number of replicates. C control—untreated trees, M mock—trees
challenged with a sterile agar plug for 2005 only, F fungus—trees
inoculated with D. pinea, I insect—trees defoliated by N. sertifer. a
Activity in needles. β-1,3-glucanase activity was not assayed in
needles in 2005. b Activity in phloem
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Fig. 6 Trypsin inhibitor levels in P. nigra phloem in 2005, means
(micrograms trypsin inhibitor per milligram soluble protein)±1 SE.
Numbers in bars indicate number of replicates. C control—untreated
trees, M mock—trees challenged with a sterile agar plug, F fungus—
trees inoculated with D. pinea, I insect—trees defoliated by N. sertifer
branches of P. nigra in response to infection of the stem and
were positively correlated with resistance to D. pinea in
those branches. In the current study, fungal inoculation
induced systemic increases in total protein levels in the
phloem, some of which with defensive roles may have
contributed to resistance. Cross-resistance to pathogens
induced by arthropod feeding has been observed in other
studies, and in these examples, the inverse was also true
(McIntyre et al. 1981; Karban et al. 1987; Inbar et al.
1998). These findings demonstrate that the outcome of
host-mediated interactions between pests can be positive or
negative and depends on environmental conditions as well
as attacker identity.
More important than systemic induction, accumulation
of total soluble proteins and the activity of most of the
defense proteins studied here were mediated strongly by
soil fertility. Declines in total soluble protein levels with
increasing fertility contrasts with reports of either increases
in total protein with increasing fertility (Borowicz et al.
2003; Vollmann et al. 2003) or constant total protein levels
across fertility levels (Cipollini and Bergelson 2001).
Although the results presented here were unexpected, they
were consistent in both years of the study in trees of
different ages and exposed to different ambient climatic
conditions. Wallis et al. (2008) also documented decreases
in total phenolics and concentrations of several individual
phenolic metabolites in the trees in the high-fertility
treatment in 2005. Assuming that our high-fertility treatment was not actually stressful to the plants, several factors
may have contributed to this unexpected result. Protein
concentrations were expressed per unit fresh weight of
tissues, so if protein production rates stayed the same or
declined as the trees grew, protein concentrations would
J Chem Ecol (2008) 34:1392–1400
decline due to dilution in greater amounts of tissue. Proteins
may also have been present but less extractable for some
reason in plants grown at high fertility. We also analyzed
only soluble proteins, and insoluble structural proteins may
respond differently to changes in fertility. In other studies,
however, increases in protein levels with fertility were seen
for total (soluble and insoluble) proteins (Vollmann et al.
2003), as well as for soluble proteins alone (Borowicz et al.
2003). This effect is unlikely to be due to the nature of the
fertilizer, since Borowicz et al. (2003) also used a complete
fertilizer and found increasing levels of total proteins with
increasing fertility. Finally, the growth conditions used here
may have been more conducive to production of certain
metabolites that may have competed for substrate with
nitrogen-rich proteins. Regardless of the mechanisms that
control protein production in conifers, soil fertility clearly
played an important role in this study.
In contrast to effects on total soluble protein levels,
defense protein activities generally increased with increasing fertility, as has been seen in other studies (e.g., Bolter et
al. 1998; Cipollini and Bergelson 2001). This was true
whether the proteins were responsive to induction and
whether their activities were expressed per unit protein or
per unit biomass. This suggests that maintenance or
increased production of some proteins is favored under
increasing fertility levels, while levels of other proteins
apparently decline. The finding that total protein levels
declined while activities of some defense proteins increased
suggests that, per unit biomass, tissues from plants in our
high-fertility treatment should be of lower quality to
herbivores and pathogens sensitive to such factors than
tissues from our low-fertility treatment. However, in our
studies, fertility had no direct effect on resistance to the
fungus or the insect (Eyles et al. 2007, Wallis et al. 2008).
This may be due to the fact that, as certain defenses
increased with fertility, such as the activity of defensive
proteins seen here, other defenses decreased with fertility,
such as concentrations of total phenolics and individual
phenolic metabolites shown by Wallis et al. (2008).
Trypsin inhibitors have been little studied in conifers, but
are a well-known constitutive and inducible defense in
many herbaceous and woody plants (e.g., Bolter et al. 1998;
Cipollini and Bergelson 2001; Major and Constabel 2008).
In 2005, we found only a low level of TI activity in phloem
and we have found no activity in needles in other studies
(D. Cipollini, unpublished data). While activities tended to
increase with fertility in 2005, as with the other defensive
proteins, TI activities were not affected significantly by any
of our treatments. The restriction of TI activity in the
phloem indicates that it could act against phloem feeding
pests, probably wood boring insects or other phloeminhabiting pathogens, but the low activity detected may
limit its importance.
J Chem Ecol (2008) 34:1392–1400
We have demonstrated systemic induction or suppression by insect feeding of PPO and POD in the phloem of
branches of P. nigra. In contrast, D. pinea infection had no
significant systemic effect on these proteins in needles or
phloem, but it did induce systemic increases in total soluble
protein accumulation in the phloem. Soil fertility strongly
decreased concentrations of total soluble protein in needles
and phloem, while it increased the activity of several
defense proteins. Variation in concentration and activities of
these proteins may be related to variation in pest attack and
performance on P. nigra across natural fertility gradients in
the field, but their impact on pests depends on correlated
responses in other plant defenses.
Acknowledgments We thank Eusondia Arnett, Bryant Chambers,
Alejandro Chiriboga, Ilka Gomez, Diane Hartzler, Anuprit Kaur,
Cherissa Rainey, Matt Solensky, Duan Wang, and Justin Whitehill for
the assistance in the field and in the laboratory and Kurtz Bros for the
nursery supplies. The comments by two anonymous reviewers
improved the manuscript. Funding was provided by the USDA
National Research Initiative Competitive Grants Program No. 200435302-14667 and by the State and Federal funds appropriated to the
Ohio Agricultural Research and Development Center, the Ohio State
University. These experiments comply with the laws of the USA.
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