ffects of clipping and N fertilization on insect herbivory and infestationy pathogenic fungi on bilberry
oaquina Pato∗, José Ramón Obeso
esearch Unit of Biodiversity (CSIC-UO-PA), Department of Biología de Organismos y Sistemas, University of Oviedo, E-33071 Oviedo,pain
eceived 13 August 2012; accepted 13 February 2013vailable online 23 March 2013
bstract
Interactions among herbivores or between herbivores and other plant natural enemies, such as fungal pathogens, range fromompetition to facilitation. Moreover, the outcome of these interactions depends on the ecological context where they occur.n this study we examined the effects of clipping, as a surrogate of herbivory by ungulates, on the damage caused by twoypes of natural enemies (herbivorous insects and foliar fungal pathogens) on bilberry, Vaccinium myrtillus, in combination withitrogen (N) fertilization representing current N atmospheric deposition. To examine whether the responses of both these naturalnemies were mediated by changes in the plant, we estimated the effects of the treatments on bilberry growth and branching andn chlorophyll content as proxy of N content in leaves. Clipping increased the proportion of leaves damaged by herbivorousnsects regardless of whether it was combined with N fertilization or not in 2008. In 2007 and 2009 repeated damage to thehrub also facilitated insect herbivory but only under N applications. Regarding fungal infestation incidence, clipping decreasedhe proportion of infected leaves in all the years considered but only in fertilized plots. Our results suggest that vertebrateerbivores facilitate insect herbivory and reduce fungal infestation but that these effects are dependent on nutritional conditions.oreover, we found a negative residual correlation between insect herbivory and fungal infestation on bilberry leaves. Therefore,
nteractions between insect herbivores and fungal pathogens could be implicated in the final outcome of interactions betweenrowsing ungulates and both bilberry natural enemies.
usammenfassung
Die Interaktionen zwischen Herbivoren und Herbivoren und anderen natürlichen Feinden der Pflanzen wie z. B. pathogenenilzen, reichen von der Konkurrenz bis zur gegenseitigen Förderung. Darüber hinaus hängt das Ergebnis dieser Interaktionenom ökologischen Kontext ab in dem sie stattfinden. In dieser Studie untersuchten wir den Effekt des Schnitts als Ersatz für
ine Beweidung durch Ungulaten auf den Schaden, der von zwei Typen natürlicher Feinde (herbivore Insekten und pathogene lattpilze) bei Blaubeeren, Vaccinium myrtillus, verursacht wird, und kombinierten dies mit einer Stickstoff-(N)-Düngung umen derzeitigen atmosphärischen Stickstoffeintrag widerzuspiegeln. Um herauszufinden, ob die Reaktionen auf diese beidenatürlichen Feinde mit Veränderungen der Pflanzen verbunden sind, schätzten wir die Auswirkungen der Behandlungen aufas Blaubeerwachstum, die Verzweigung und auf den Chlorophyllgehalt als Näherung für den Stickstoffgehalt in den Blätternb. Der Schnitt erhöhte im Jahr 2008 den Anteil der Blätter, die durch herbivore Insekten beschädigt wurden, egal, ob er miter N-Düngung kombiniert wurde oder nicht. In den Jahren 2007 und 2009 führte die wiederholte Beschädigung der Büscheu einer Förderung der Herbivorie durch Insekten, allerdings nur, wenn die N-Düngung stattfand. In Bezug auf das Auftretenon Pilzinfektionen reduzierte der Schnitt den Anteil der infizierten Blätter in allen Jahren allerdings nur in den gedüngten
48 J. Pato, J.R. Obeso / Basic and Applied Ecology 14 (2013) 347–356
robeflächen. Unsere Ergebnisse lassen vermuten, dass die Herbivorie durch Vertebraten die Herbivorie durch Insekten fördertnd die Infektion durch Pilze vermindert, dass diese Effekte jedoch von der Nährstoffverfügbarkeit abhängen. Darüber hinausanden wir eine negative Korrelation der Residuen zwischen der Herbivorie durch Insekten und der Infektion mit Pilzen bei denlaubeerblättern. Daher können die Interaktionen zwischen den herbivoren Insekten und pathogenen Pilzen in das Ergebnis der
nteraktion zwischen grasenden Ungulaten und den beiden natürlichen Feinden der Blaubeere einbezogen werden.2013 Gesellschaft für Ökologie. Published by Elsevier GmbH. All rights reserved.
Interactions among herbivores range from competition toacilitation. When one species reduces food resources belowhe level necessary to sustain another species, both speciesompete. Alternatively, the use of resources by one speciesay increase the quantity or quality of food resources for
ther species, leading to a facilitation process (Arsenault &wen-Smith 2002). The relative importance of these two pro-
esses and their effect on plant performance remains unclear.Large mammalian herbivores not only reduce plant
iomass available for other herbivores but also activate plantnduced-defences (Bryant 1981; Bryant, Chapin, & Klein983; Karban & Baldwin 1997), which may decrease her-ivory by other plant host, e.g. insects, through a competitionrocess. On the contrary, according to Arsenault and Owen-mith (2002) feeding facilitation can arise in two differentays: first, when herbivory by one species makes plant
esources accessible to another species, or second, when her-ivory by one species enhances the nutritional quality throughlant regrowth for another species. In addition, competitivend facilitative interactions may occur between herbivoresnd other plant enemies, such as plant pathogens (Daleo et al.009). In fact, both insect herbivores and fungal pathogenstilizing the same host plant species could respond to quantitynd/or quality changes after ungulate herbivory, leading to ariple interaction system. However, the indirect interactionshat have been found between both insects and pathogens dueo synergies and/or antagonisms among signalling pathwaysn plant resistance (Lappalainen, Helander, & Palokangas995; Hatcher & Paul 2000; Mouttet, Bearez, Thomas, &esneux 2011) could disguise a direct effect of large herbi-ores on both insects and pathogens.
An increasing number of studies have shown interactionsetween mammalian and insect herbivores feeding on theame host plant. The effect of vertebrate herbivores on insectbundance and/or richness can be negative (e.g. Baines, Sage,
hitham 2006). However, in the case of studies demon-trating facilitation between vertebrate and insect herbivores,
he focus is usually on gall forming insects. With respect toungal pathogens, even though vertebrate grazing has beeneported to reduce levels of infestation by fungi in grassland
ndp
cosystems (Bowers & Sacchi 1991; Wennström & Ericson991), the possible effects of vertebrate herbivores on fungalathogens remains poorly studied.
In general, the outcome of the interactions, such as com-etition and facilitation, depends on the ecological contexthere they occur, in such a way that a potential competi-
ion process could in fact become positive under specificircumstances. For instance, Cheng and Ritchie (2006) foundhat habitat productivity and herbivore densities may medi-te shifts between facilitative and competitive interactionsetween cattle and prairie dogs. Similar results were reportedor interactions between herbivores from distantly relatedaxa. Thus, Suominen, Persson, Danell, Bergström, andastor (2008) showed that the impact of moose on inverte-rate richness depended on site productivity in such a way thatigh productivity seems to be able to buffer negative impactsf high moose density. Additionally, a spatio-temporal vari-bility in the outcome of interactions between ungulates andpigeal arthropods has been reported by González-Megías,ómez, and Sánchez-Pinero (2004).As the outcome of interactions varies according to eco-
ogical contexts, global change drivers could be tested asossible sources determining these contexts. Nitrogen (N)tmospheric deposition due to human activities is resulting inertilization of ecosystems worldwide (Vitousek et al. 1997;ala et al. 2000). Also, as a consequence of climate warm-
ng and changes in land use, it is accepted that the extent ofertebrate herbivory is increasing within ecosystems due to aider phenological window for herbivory, reduced mortalityf herbivores during mild winters (Côté, Rooney, Tremblay,ussault, & Waller 2004) and to a trophic downgrading (Estes
t al. 2011).In a scenario of global change, the ecological context
etermining plant performance could be influenced by bothxtent of vertebrate herbivory and N fertilization, which,oreover, may interact in their effects on plants (GoughGrace 1998; Strengbom, Olofsson, Witzell, & Dahlgren
003; Britton & Fisher 2007). On the other hand, besidesirect effects on plants, both factors might influence theutcome of interactions between plants and other natural ene-ies. Specifically, simulation of vertebrate herbivory under
ifferent nutritional conditions provides an appropriate sce-
ario to assess possible interactions between herbivores fromistantly related taxa, and between ungulates and foliar fungalathogens.
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The main goal of our study was to examine the importancef competition vs facilitation between ungulates and otheratural enemies (insect herbivores and fungal pathogens)haring a common host plant, bilberry Vaccinium myrtillusL.), and how these interactions can be modified by N fer-ilization. Previous studies on bilberry have reported that
fertilization increased plant quality by increasing aminocid concentration (Strengbom, Nordin, Näsholm, & Ericson002; Nordin, Strengbom, Witzell, Näsholm, & Ericson005) and decreasing certain chemical defence compoundsStrengbom et al. 2003; Witzell & Shevtsova 2004; Nordint al. 2005). Damage by foliar pathogenic fungi and from Lep-doptera larvae also increased after experimental N additionsNordin, Näsholm, & Ericson 1998; Strengbom et al. 2002;ordin et al. 2005). Furthermore, both clipping and N fertil-
zation applied together led to increases in bilberry preferencey mammal herbivores (Strengbom et al. 2003). Therefore,e examined the following questions: (1) Does clipping, assurrogate of ungulate herbivory, facilitate insect herbivory?
2) Does N fertilization increase insect herbivory and does itnteract with clipping? (3) Does clipping affect fungal infes-ation? (4) Does N fertilization increase fungal infestationnd does it interact with clipping? Our results showed thatimulated ungulate herbivory increased insect herbivory andecreased fungal infestation in bilberry leaves but that theseffects were dependent on N addition.
aterial and methods
tudy species
V. myrtillus is a deciduous clonal dwarf shrub with ever-reen stems that reaches the southern limit of its Europeaneographic range in Northern Spain, where it occurs from seaevel up to 2000 m a.s.l. In the Cantabrian Range, bilberry isncluded in the diet of some wild (roe deer Capreolus capre-lus L., red deer Cervus elaphus L.) and domestic ungulatescattle Bos taurus L.), and two endangered species, the caper-aillie (Tetrao urogallus L.) and the brown bear (Ursus arctos.). Moreover, it maintains a huge, diverse arthropod commu-ity, including herbivorous insects which feed on its leaves,owers and stems, and also acts as a host to several parasiticungus species.
tudy sites
The sample sites were located in the Cantabrian Range,sturias province, Northern Spain. The Cantabrian Range
onstitutes a scenario in which N fertilization and heavyrowsing occur simultaneously as a consequence of global
hange drivers, namely N atmospheric deposition and land-se change. N fertilization probably exceeds the critical loadn the system, owing to several carbon fuelled power sta-ions in the area and to the increase in both animal husbandry
ti2u
d Ecology 14 (2013) 347–356 349
nd wild ungulates (Rodríguez & Macías 2006). Consider-ng both domestic and wild animals, total values of biomassensity of over 4000 kg km−2 have been reported in somereas of the Cantabrian Range (Rodríguez, Naves, Fernández-il, Obeso, & Delibes 2007). Densities of wild herbivoresave undergone a 20-fold increase in the last 30 years inhe case of red deer, roe deer and chamois (Pollo, Robles,arcía-Miranda, Otero, & Obeso 2003), which is in accor-ance with global trends found by Côté et al. (2004). Hence,hereas the critical load for N in boreal forests is aroundkg N ha−1 year−1 (Nordin et al. 2005), the estimated atmo-
pheric deposition in areas near the Cantabrian Range coulde over 25 kg N ha−1 year−1 (Rodríguez & Macías 2006).
Six study sites were selected in 2007 along an altitudinalradient (from 300 to 1700 m a.s.l.). Sites between 300 and500 m consist of mature deciduous forest fragments of oakQ. petraea L., Quercus spp.), beech (Fagus sylvatica L.) orhestnut (Castanea sativa, Miller), where bilberry dominateshe understory. Above the forest line (1600 m a.s.l.), bilberryppears together with heather (Calluna vulgaris (L.) Hull).he Cantabrian Range is characterized by an Atlantic climate,ith mild winters and rainy summers. Mean precipitation and
emperature vary considerably along the altitudinal gradient.ield work was carried out in 2007, 2008 and 2009.
xperimental design
In April 2007, 12 experimental plots (2 m × 2 m) wereelected and marked with plastic tape at each site to establishfactorial clipping and N fertilization experiment. Clippingas used as a surrogate of vertebrate herbivory due to the high
patial variability in the extent of vertebrate herbivory in therea. The experimental plots were randomly assigned to onef the following four treatments: untreated (control), annualertilization, clipping and annual fertilization plus clipping.ach treatment was applied on three replicate plots per site,iving 72 plots in total.
The fertilizer, consisting of granules of ammonium nitrateNH4NO3, Fertiberia S.A.), was evenly spread by hand onsingle occasion at the onset of the growth period, i.e. the
eginning of April at the lower altitudes and after the snowelting in the other sites (May and June), in each of the
hree years from 2007 through 2009. The fertilizer level27 kg N ha−1 year−1) was chosen to be roughly equivalento the estimated current N atmospheric deposition in the area.his means that control plants received a very high N atmo-pheric deposition compared with other studies conducted inreas where N-levels in nature are low, e.g. Northern SwedenNordin et al. 2005). Clipping was done with hand shears,ith the last 5–8 cm of the current-year’s annual top shootseing removed from all the ramets within the plots assigned
o this treatment, at the same dates mentioned for fertilizationn 2007 and at the end of the growing period (September) in008. This kind of clipping emulates the type of damage thatngulates typically cause to natural bilberry in the area and
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50 J. Pato, J.R. Obeso / Basic and
hat usually occurs in autumn and winter. None of the plotsas fenced to exclude wild or domestic ungulates. This wasossible because herbivorous ungulates were particularly raren the six study sites. Areas with high densities of cattle andther domestic ungulates were far away from the study sites,nd both faecal pellets and damage on plants were hardly everbserved.
atural enemies
The experimental plots were used to assess the incidencef damage caused by herbivorous insects and parasitic fungin bilberry. Leaves of bilberry are a principal food sourceor several insect herbivore species, the main ones beingepidoptera larvae (JP, personal observation). We consid-red a leaf to be affected by insects when it showed holesr eaten areas, regardless of the area of leaf affected. Bil-erry is also infested by different pathogens. Specifically,e distinguished three different types of damage by fungin bilberry leaves under natural conditions: brown-spots,iolet-spots and reddish-brown tissue on the leaves. Theseymptoms match with three different fungal species, Valden-ia heterodoxa, Podosphaera mytillina and Pucciniastrumaccinii, respectively, and they have all been studied pre-iously in V. myrtillus (Nordin et al. 1998; Aamlid 2000).ost infected leaves (≈90%) were affected by V. hetero-
oxa, which causes premature leaf loss in summer. Due tohe dominance of V. heterodoxa we did not account for theifferent species separately; rather we merged data from thehree species considering a leaf to be infested when it showedvidence of any fungal symptom. To measure the responsef insects and fungi to simulated vertebrate herbivory and
addition, we counted the number of leaves affected byoth kinds of enemies in relation to the total number ofeaves in 10 ramets per plot. Counts on damaged leaves bynsect herbivores were taken at the end of the growing seasonSeptember) in 2007, 2008 and 2009 whereas infestation byungi was estimated in 2008 and 2009 in the middle of theeason (July) to avoid confusion with old, senescent leavesharacteristic of plants at the end of the growing season.
ilberry performance
To check whether applied N was assimilated by the plant,e measured the chlorophyll content in the plots. It has beenemonstrated that chlorophyll content in leaves can be useds a surrogate of N content and considered a useful indica-or of plant nutritional state (e.g. Ripullone, Grassi, Lauteri,
Borghetti 2003). The chlorophyll content was determinedith a chlorophyll metre SPAD 502 (Minolta® Co. Ltd.,
apan) which estimates its relative concentration in leaves.
e used three leaves per ramet from ten ramets per plot,
aking three different measurements from each leaf. Mea-urements were carried out in late spring in two consecutiveears, 2007 and 2008.
w
a(
d Ecology 14 (2013) 347–356
The effects of the treatments on bilberry performance werenalyzed by estimating the vegetative growth of V. myrtillusy means of two measurements: (1) current year’s growthCYG), estimated as the length of the apical shoot in nonlipped plots, or the length of the last shoot found before theut in clipped plots, and (2) ramification, that is, the numberf branches produced at the base of each shoot considered.oth traits were measured in 10 ramets per plot at the end of
he growing season (September) in 2008 and 2009.Given that plots were quite heterogeneous with regard to
ilberry height, and that bilberry grows on acid soils, wevaluated plant height and soil pH per plot as potential covari-tes with both bilberry performance variables and variableselated to damage on bilberry by both insects and fungi. Planteight was estimated, in five ramets per plot, as the length ofhe ramet from the ground to the highest shoot. To determineH, soil samples from the surface to 25 cm depth were col-ected using a gimlet. The samples were air-dried, groundnd sieved (2 mm diameter) prior to analysis. Soil pH waseasured in water using a ratio of 1:2.5.
tatistical analysis
To test the effects of clipping and fertilization on the extentf insect herbivory and fungal infestation on bilberry we usedeneralized Linear Mixed Models (GLMMs). Both damagey herbivorous insects and fungi consisted of proportions ofamaged leaves hence we applied binomial error distributionnd logit link function in the model. We considered “site”s a random factor since the experiment was replicated inix different sites and the extent of damage might be spa-ially correlated. To run this kind of model, we calculatedhe sum of affected leaves and the total counted leaves perombination of treatments and site. Measurements from dif-erent years were tested separately as dependent variables.inally, we included plant height and pH estimated per plot asovariates in the model, having checked previously that botheasurements were not affected by treatments. Estimated
arameters and standard errors in GLMMs were obtained byeans of Laplace approximation, and Wald Z tests were used
o determine whether the two levels of both factors, fertiliza-ion (unfertilized and fertilized) and herbivory (unclipped andlipped), differed significantly from one another.
In addition, we tested the effect of treatments on chloro-hyll content and growth variables. In this case, responseariables met the assumptions of parametric analysis andherefore Linear Mixed Models with the same model struc-ure as that described above were used instead of GLMMs.estricted maximum likelihood was used to fit the model and-ratio test to obtain the significance of the factors. Prior totatistical analyses, data from the three plots per treatment
ere summarized for each site.Finally, to establish a relationship between insect herbivory
nd fungal infestation, we used residuals from the GLMMsto avoid the effects of covariates and experimental treatments
J. Pato, J.R. Obeso / Basic and Applied Ecology 14 (2013) 347–356 351
Table 1. GLMMs results showing parameter estimation and sig-nificance for the proportion of leaves damaged by insects in threeconsecutive years. Estimates for fixed factors (fertilization and clip-ping) represent comparison of untreated vs treated and, thus, anegative estimate denotes that damage by insects was higher intreated than in control plots.
Fig. 1. Insect herbivory (proportion of affected leaves, mean ± 1Si
tfc
p
value < 0.05 indicated in bold.
reviously found) to perform correlation analyses for years008 and 2009. Analyses were performed with R 2.11.1 andPSS 19.0.
esults
Effects of clipping and N fertilization on the extent ofamage caused by insects on bilberry were dependent onhe year studied. In 2008 simulated herbivory had a signifi-ant positive effect on the proportion of leaves damaged bynsects regardless the N treatment (Table 1 and Fig. 1). How-ver, in 2007 and 2009 the significant interaction clippingN fertilization means that the effect of clipping depended
n N addition in such a way that clipping increased insecterbivory but only in fertilized plots (Table 1 and Fig. 1).his also means that N fertilization tended to reduce theamage by insects on bilberry leaves when plants were notlipped (Fig. 1). For these same years, insect herbivory wasegatively correlated with plant height whereas a negativeelationship between damage by insects and pH was found inhe three years studied (Table 1). Hence, shorter plants grow-
ng in acid soils were more vulnerable to herbivorous insectttack.
As to fungal infestation on V. myrtillus, significant interac-ions between both factors for the both years studied showed
Pcha
E, n = 6) on Vaccinium myrtillus for different clipping and fertil-zation treatments in three different years.
hat clipping only reduced infestation when the plants wereertilized (Table 2 and Fig. 2). Damage by fungi was in thisase positively correlated with soil pH in both years (Table 2).
In terms of physiological plant measurements, chloro-hyll content was higher for fertilized plots (F1,18 = 6.387,= 0.021), but only in 2007, the first year following N appli-
ation. Moreover, plots with taller plants also demonstratedigher leaf chlorophyll levels (F1,18 = 7.347, P = 0.014). Vari-bles related to growth were only significantly affected
352 J. Pato, J.R. Obeso / Basic and Applied Ecology 14 (2013) 347–356
Table 2. GLMMs results showing parameter estimation and sig-nificance for the proportion of leaves infested with fungi in 2008and 2009. Estimates for fixed factors (fertilization and clipping)represent comparison of untreated vs treated and, thus, a negativeestimate denotes that infection by fungi was higher in treated thanin control plots.
Fig. 2. Fungal infestation (proportion of infested leaves, mean ± 1SE, n = 6) by Valdensia heterodoxa, Podosphaera myrtillina andPe
onaai
tcsSefahTs
value < 0.05 indicated in bold.
y treatments in 2009, when clipping increased CYGF1,18 = 13.253, P = 0.002) but decreased the number ofranches (F1,18 = 11.906, P = 0.003, Fig. 3). In both years,nsect herbivory and fungal infestation were negatively cor-elated (Fig. 4).
iscussion
The three-year vertebrate herbivory simulation and N fer-ilization experiment showed that insect herbivory and fungalnfestation on V. myrtillus leaves were affected by the com-ination of clipping and N addition. Also, clipping alonencreased insect herbivory. These results lead us to suggesthat ungulate herbivores have the potential to facilitate insecterbivory and reduce fungal infestation.
On the whole, insect herbivory increased after clipping,ut this effect was dependent on N fertilization and variedrom year to year. Specifically, in 2008, vertebrate herbivoryimulation on bilberry plants caused an increase in the pro-ortion of affected leaves independent of N addition, whilen 2007 and 2009, the positive effect of clipping on insecterbivory was demonstrated only in fertilized plants. How-ver, these differences between years did not respond toifferences in the timeframe between clipping applicationnd leaf damage estimation as in 2007 insects respondedo clipping in the same year, whereas in 2008 and 2009hey responded to clipping carried out the previous year.ccording to the facilitation theory, facilitation of insects
y vertebrate herbivores could be mediated through changesn plant quality or quantity as a food resource, but we woulduggest that reduction in competition between insects and
iat
ucciniastrum vaccinii on their host Vaccinium myrtillus for differ-nt clipping and fertilization treatments in two different years.
ther natural enemies of plants may constitute a third expla-ation behind this theory. Hence, interactions between insectsnd fungi feeding simultaneously on bilberry could havedeterminant role on the outcome of herbivore–herbivore
nteractions.It has been proposed that N fertilization increases concen-
rations of amino acids (Strengbom et al. 2002) and reducesoncentrations of some phenolic compounds and other con-titutive defences in plants (Koricheva & Shevtsova 2002;trengbom et al. 2003; Witzell & Shevtsova 2004; Nordint al. 2005), which would result in a better quality of plantsor herbivores. Repeated damage to plants, in turn, maylso change their C/N ratios, and thus their palatability toerbivores (Bryant et al. 1983; Karban & Baldwin 1997).herefore, both reduced chemical defence and increasedhoot nutrient content in response to clipping and N fertil-
zation probably increase the palatability of plants, therebyttracting natural enemies which feed on them. However,he effects of N fertilization and repeated damage on plant
J. Pato, J.R. Obeso / Basic and Applie
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ig. 3. Effect of clipping on current-year apical growth (CYG) andumber of branches estimated in 2009 (mean ± 1 SE).
hemical composition are controversial and, in fact, there areeveral studies showing the detrimental effect of browsingn insect herbivore abundance (Baines et al. 1994; Dennist al. 1998; Allombert, Stockton, & Martin 2005). In the
ase of N, the lower insect herbivory found in N-treatedlants that were not clipped could also respond to an N-ased defence system suggested for bilberry (Selås et al.
Residuals from GLMMs for fungal infestation
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ig. 4. Relationship between insect herbivory and fungal infesta-ion in both years. Each point represents residuals obtained fromLMMs after removing covariates and fixed and random effects.
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d Ecology 14 (2013) 347–356 353
011). Nevertheless, it is worth noting that in our studyrea N-levels that plants are receiving naturally are so highhat the results may not be valid in other cases where N-evels in nature are more normal. In the current study neitherhemical compounds nor nutrients were measured in bilberryeaves. However, using chlorophyll as a proxy of N contentn leaves, we found that N fertilization increased N contentut only after the first N application and therefore N con-ent in leaves did not explain herbivory patterns obtainedor the three years. Alternatively, we can explain the indi-ect response in natural enemy activity to treatments usinghoot growth and ramification as indicators of host qualityince it has been proposed that plants spending more effortn growth will left less to defence, thereby increasing therobability for herbivore attacks (Danell, Huss-Danell, &ergström 1985; Price 1991; Roininen et al. 1997; BaileyWhitham 2006). In our study, clipping caused an increase
n CYG (current year’s growth) but a decrease in the num-er of branches, which could be related to a rapid regrowthfter damage to plants and, in consequence, to an increasednsect herbivory. However, this effect on CYG was only foundn 2009 when, furthermore, insect herbivory did not dif-er between clipped (both fertilized and non-fertilized) andontrol plants. Hence, as chlorophyll measurements, neitherhoot growth nor branching are useful to determine insect andathogen responses to treatments. Nevertheless, it is impor-ant to take into account that natural grazing by ungulates ininter could have different effects in plants and their ene-ies.Other bilberry enemies, such as fungal species, have also
een demonstrated to increase their damage on the plantfter N fertilization, and that this is related to peaks in leafmino acid concentrations (Strengbom et al. 2002; Nordint al. 2005) and decreases in the content of some pheno-ic compounds (Nordin et al. 2005). Despite these findings,ur results showed that neither N fertilization nor simulatederbivory caused a clear effect on fungal infestation whenonsidered separately. However, interaction between bothactors showed that the effect of clipping on the proportion ofnfected leaves changed from positive or null (depending onhe year) in unfertilized plots to negative in fertilized plots.herefore, we found that, under N fertilization, the effectsf clipping on fungal infestation were opposite to the effectsn insect herbivory. This leads us to suggest that, if leaf-hewing insects avoid leaves previously affected by fungalathogens (Lappalainen et al. 1995), the increase in insecterbivory under clipping and N fertilization might be due toreduced fungal attack. This hypothesis would suggest an
ndirect facilitation between ungulates and insects mediatedhrough fungal pathogens. If this hypothesis is supported,t would still remain to explain the mechanisms behind theetrimental effect of clipping on fungal attack, which could be
imilar to those exposed in the healthy herds hypothesis: pre-ation should decrease disease in host populations (Packer,olt, Hudson, Lafferty, & Dobson 2003). However, resultsay be different in the case of herbivory as, in a recent study
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54 J. Pato, J.R. Obeso / Basic and
orer, Mitchell, Power, and Seabloom (2009) demonstratedhat the presence of large vertebrate herbivores increased thenfestation risk of a pathogen group in a grassland commu-ity.
As we experimentally manipulated neither fungi nor insecterbivores, it is not possible to tell if infestation by fungi is aause or effect of ungulate-insect interactions, or whetheroth natural enemies are responding simultaneously andndependently but in opposite ways. However, we foundhat, in unbrowsed and unfertilized conditions, insect her-ivory and fungal pathogen infestation were negativelyorrelated, which means that the interaction between bothatural enemies might have a role in determining the effects ofreatments. In general, little has been reported about the effectf fungal infestation on the behaviour or performance of her-ivorous insects (see a review in Rostás, Simon, & Hilker003) whereas, in contrast, some studies have demonstratedhe inhibition of fungal activity after feeding activity of herbi-ores (Hatcher & Paul 2000; Rostás et al. 2003). However, inhe case of bilberry, it is more probable that plant pathogensnfluence insects negatively since it has been reported thatnfestation by a fungal pathogen increased the biosynthe-is of phenolic compounds, which could result in insectsvoiding infected leaves. On the contrary, simulated dam-ge by insects did not cause such effect (Jaakola, Koskimäki,iihinen, Tolvanen, & Hohtola 2008; Koskimäki et al. 2009).Besides the clipping and N fertilization effects, other
egetation factors could influence the damage both naturalnemies cause to the plant. We found that damage by herbi-ores was related to plant height in such a way that shorterlants were more affected by insect herbivory. These resultsre in contrast to other studies that found a strong correla-ion between plant height and insect abundance and diversityKruess & Tscharntke 2002). On the other hand, the leaf dam-ge caused by insects was negatively correlated with soil pH,hereas fungal infestation was positively correlated with soilH. Once again, we found different responses for both naturalnemies.
All things considered, insect and fungal responses to clip-ing (a reliable simulation of ungulate herbivory) and Nertilization showed that the outcome of the interactionsetween ungulate herbivory and both herbivorous insectsnd fungi is context dependent. Specifically, facilitationetween ungulates and insects occurred when N was addedhereas, under the same conditions of N application, clip-ing decreased fungal damage. Moreover, we suggest thatoth responses may be related and the decrease in fun-al attack in bilberry leaves after clipping may in fact acts a possible mechanism behind the facilitation of insectctivity by ungulates. This demonstrates the need for con-idering, apart from environmental stress, interactions withther elements in the system when we try to understand
he process of facilitation between two species. However,he challenge of determining the exact role that the inter-ctions between insect herbivores and fungi play on theiresponses to browsing herbivory remains. There is, therefore,
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need to conduct experiments excluding herbivore insects,athogens and/or ungulates to elucidate interactions amongngulates, insect herbivores and fungal pathogens feeding onilberry.
cknowledgements
We thank Amalia Segura and Emilie F. Ploquin forelp during the fieldwork. We also thank Ronnie Lendrumor linguistic advice and three anonymous reviewers whorovided useful and encouraging comments to improvehis manuscript. This study was financially supportedhrough a FPU fellowship (Spanish Ministry of Sci-nce and Innovation) to JP, and the projects CN-07-174Asturian Environmental Agency) and MICINN-CGL2009-1302 (Spanish Ministry of Science and Innovation) toRO.
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