Herbivory, induced resistance, and interplant signal transfer in Alnus glutinosa

Biochemical Systematics and Ecology 29 (2001) 1025–1047

Herbivory, induced resistance, and interplantsignal transfer in Alnus glutinosa

Teja Tscharntkea,*, Sabine Thiessena,b, Rainer Dolcha,Wilhelm Bolandb

aAgroecology, University of G .ottingen, Waldweg 26, D-37073 G .ottingen, GermanybMax-Planck Institute for Chemical Ecology, Carl-Zeiss-Promenade 10, D-07745 Jena, Germany

Received 9 April 2001; accepted 19 April 2001

Abstract

Field experiments with manually defoliated black alders (Alnus glutinosa) showed that

defoliation affected herbivory by the major alder antagonist, the leaf beetle Agelastica alni.Herbivore damage increased with increasing distance to the defoliated tree, suggesting inducedresistance not only on the damaged tree, but also on the neighbouring trees. The beetles alsoavoided leaves from the nearest neighbours for both feeding and oviposition in a laboratory

assay, so the alders showed interplant resistance transfer. Natural enemies did not appear toshape this pattern, because the number of entomophagous arthropods and predator–preyratios even increased with increasing distance to the defoliated tree. The numbers of all

specialist, but not the generalist, herbivore species paralleled the increase in the attack of thespecialist A. alni, supporting the view that specialists are more affected by plant resistance thangeneralists.

Mechanisms causing this pattern, found in the field, were studied in more detail usingbiochemical analyses and further bioassays. Responses of alder leaves to herbivory of A. alniwere shown to include ethylene emission and the release of a blend of volatiles with mono-,

sesqui- and homoterpenes. Changes in leaf chemistry after herbivory included increases in theactivity of oxidative enzymes (polyphenoloxidase, PPO, lipoxygenase, LOX, and peroxidase,POD) and proteinase inhibitors (PIs), and an increase in the phenolic contents of the leaves.Quantification of the endogenous jasmonic acid (JA) showed the activation of the

octadecanoid pathway following herbivory.The active components in mediating a possible interplant signal transfer via airborne

volatiles may have included ethylene, b-ocimene, 4,8-dimethylnona-1,3,7-triene (DMNT), and

4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). The incubation with volatiles resulted in an

*Corresponding author. Tel.: +49-551-399209; fax: +49-551-398806.

E-mail address: [email protected] (T. Tscharntke).

0305-1978/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 0 4 8 - 5

increase in the activity of catalase (CAT) and PIs (after MeJA application) and in an increase

in the content of phenolics and PI activity (after ethylene application). Further evidence thatairborne interplant communication may be important in the response of alder trees to beetleattack came from container experiments. In airtight chambers, unattacked leaves significantlyincreased the activity of proteinase inhibitors when they were associated with leaves previously

attacked by beetle larvae.In conclusion, field experiments, bioassays in the laboratory as well as biochemical analyses

suggest the existence of interplant resistance transfer in A. glutinosa, with airborne volatiles as

a possible mechanism. However, the relative importance of airborne and possible soil-bornesignals as well as unknown effects of intensified nutrient absorption of defoliated trees,possibly reducing foliage quality of undamaged neighbours, remains to be shown. r 2001

Elsevier Science Ltd. All rights reserved.

Keywords: Talking trees; Leaf beetles; Volatiles; Proteinase inhibitors; Jasmonic acid; Phenolics; Ethylene

1. Introduction

Plant responses to herbivory are known from a wide range of plants and oftenaffect subsequent herbivory (e.g. Karban and Baldwin, 1997; Agrawal et al., 1999).Plant responses to insect herbivory may not only concern the damaged plantsthemselves but also undamaged neighbours, making them less susceptible toherbivores. Such interplant signal transfer has been hypothesized for a long time (e.g.Baldwin and Schultz, 1983; Rhoades, 1983). Communication among plantsremained a hot topic in the public, although Fowler and Lawton (1985), whosework did not support a ‘‘talking tree hypothesis’’, reviewed the published studiesdealing with interplant signal transfer and criticized most of them because ofunsuitable experimental design or statistical flaws such as pseudoreplication. Despitetheir harsh criticism, discussion has been refuelled as recent work has yielded resultsin favour of interplant communication (e.g. Bruin et al., 1995; Shonle and Bergelson,1995; Karban et al., 2000; Arimura et al., 2000). Attack of herbivores on one plantmay affect the non-attacked, neighbouring plant via connection by roots ormycorrhiza (Simard et al., 1997) or via volatiles such as methyl salicylate (MeSA)and methyl jasmonate (MeJA) (Farmer and Ryan, 1990; Shulaev et al., 1997; Bolandet al., 1998; Thaler et al., 1996; Karban et al., 2000). Two recent papers give evidencethat interplant communication may be important under natural field conditions, inboth herbs (Karban et al., 2000) and trees (Dolch and Tscharntke, 2000).

Field observations suggested the existence of resistance transfer among alders(Alnus glutinosa) (Dolch and Tscharntke, 2000). After manual defoliation, leafdamage of alder leaf beetle (Agelastica alni, Col. Chrysomelidae) increased withdistance to the defoliated tree. This effect may be attributed to interplantcommunication. Why have black alders been expected to show signal transferamong plants? Black alders were chosen, because they are known to becomeregularly and completely defoliated by their specific, widespread, univoltine and

T. Tscharntke et al. / Biochemical Systematics and Ecology 29 (2001) 1025–10471026

most important enemy, the alder leaf beetle, and such a periodic and strong selectionpressure may be associated with induced and strong defense responses. In addition tothe conspicuous flush–crash fluctuations in this system (Tscharntke and Dolch, pers.obs.), previous work had shown that herbivory caused both rapid and delayedinduced resistance in alders (Jeker, 1981; Baur et al., 1991; Seldal et al., 1994;Oleksyn et al., 1998; Tscharntke and Dolch, unpubl. data).

Additional experiments in the field were conducted to search for further evidencefor the pattern found by Dolch and Tscharntke (2000), and to further elaborate theinterplant-communication hypothesis. Since effects of defoliation on neighbouringtrees may not only affect performance of the alder leaf beetle, we also studied theresponse of the total arthropod community associated with alder leaves. In addition,the volatile compounds emitted by attacked leaves and the induced changes in leafchemistry were analysed in the laboratory. We show in this paper that damagedalder plants produce biologically active volatiles, which are possible candidatesubstances for the interplant resistance transfer observed in the field. Bioassays withunattacked leaves, neighbouring leaves attacked by beetles as well as the applicationof airborne signals such as ethylene, MeJA and MeSA revealed plant physiologicalresponses to volatile cues.

2. Material and methods

2.1. Field experiments

We selected 10 sites in the vicinity of G .ottingen (Germany) with 10 trees of blackalder growing in rows along a creek, so we studied altogether 100 alders (for detailssee Dolch and Tscharntke, 2000). At each site, one randomly selected tree waschosen for defoliation. Manual defoliation took place in early May 1994 (beforeadult beetles had colonized alders) and included stripping 20% of the trees’ leavesfrom the lower branches of the canopy. Mean distance between each of the 10 treeswas 1 m. Leaf damage was estimated as percentage of total leaf area consumed on all100 trees at six dates between May and September. In the laboratory, ovipositionbehaviour was tested using equally aged leaves from each site, and, in a furtherexperiment, beetles could choose between these leaves for consumption.

In addition to the experiments with alder leaf beetles, we took samples of allarthropods on the alder leaves from all defoliated trees, their nearest neighbours andthe farthest trees. The three lowest branches of each of the altogether 30 trees werestrongly knocked three times on four dates between May and September (before and37, 81 and 133 days after defoliation), and the arthropods dropped in an umbrellathat was held beneath these branches. Sampling the insect fauna on Alnus glutinosayielded phytophagous insects, grouped in specialists (monophagous and/or knownto prefer Alnus) and non-specialists (according to Schwenke, 1972, 1974), andentomophagous arthropods.

T. Tscharntke et al. / Biochemical Systematics and Ecology 29 (2001) 1025–1047 1027

2.2. Biochemical analyses and bioassays

For the laboratory experiments, six-months old black alder trees Alnus glutinosa(nursery ‘‘Grebenstein & Linke’’, Germany) were grown for 12 months in a plasticpot (+=10 cm) with a mixture of potting soil and sand (1 : 1, v/v) in walk-in growthchambers with 241C and a 16:8 h day : night regime. These 18-months old trees(height=40–60 cm, stem +=0.7 cm) were assigned for treatments so that eachtreatment group had plants of similar size and appearance. Agelastica alni (L.) larvaewere reared from eggs of adults sampled in the field. The larvae were fed with freshlycut A. glutinosa foliage and reared at 201C with a 16:8 h day : night regime.

2.3. Container experiments

The container experiments should show possible effects of volatiles produced byherbivore-damaged alder leaves on undamaged leaves. We placed one branch withthree leaves from intact alder plants in a clip cage to prevent the larvae fromescaping. Five larvae of A. alni were placed on each leaf and allowed to feed for 72 h.In the first desiccator (2700 ml), the A. alni-infested leaves (=‘‘A. alni’’) were kepttogether with one branch with three healthy leaves of black alder (=‘‘A-neighbour’’). We analysed the black alder physiological responses (PI activity) inthe leaves damaged by A. alni and in the leaves exposed to the volatiles. The seconddesiccator had the same container set-up, but without feeding of A. alni: one branchwith three leaves from a healthy plant (=‘‘control’’) and another healthy branch asneighbour of control leaves (=‘‘C-neighbour’’) were kept together. Assays weremaintained at room temperature (r.t.) with a 16:8 h day : night regime and 4000 lx.Experiments were repeated six times and for each repetition and treatment, differentdesiccators were used.

2.4. Application of ethylene, jasmonic acid methyl ester (=MeJA), salicylic acidmethyl ester (=MeSA), and jasmonic acid (=JA), and insect feeding

For induction experiments with volatiles (ethylene, MeJA, MeSA), we detachedthree fresh leaves from intact black alder plants, transferred them into a vial (5 ml)with tap water, and enclosed the leaves in a desiccator (2700 ml). The chemicals,except ethylene, were dissolved in pure dichloromethane (1 mg ml�1), and 5 ml of eachwas applied onto a small piece of filter paper. After brief evaporation of the solvent,we fixed the filter paper as a ‘dispenser’ below the cap of the desiccator to preventany direct contact between the chemical and the leaves. Control experiments werecarried out following the same procedure but without the application of the testcompounds onto the filter paper. Ethylene was applied using 1000 ml of the pure gas.Jasmonic acid was applied as a solution (1 mM) in tap water. For insect feeding,three leaves of black alder were kept together with 15 larvae of A. alni. The leaveswere exposed to the airborne substances, JA, and insect feeding for 48 or 72 h. Theseexperiments were repeated five times (for the determination of activities of PPO,LOX, POD, CAT, and PI, see below) or six times (for the phenolics), using different


desiccators for each treatment or replicate. The experimental set-up was maintainedat 251C and with a photophase of 16 h and 4000 lx.

2.5. Collection and analyses of alder volatiles

For the volatile induction experiments, stems of young A. glutinosa plants withthree developed primary leaves were cut and immediately transferred into glass vialscontaining a solution of the test substance in tap water. Feed-induced volatiles wereobtained by allowing 10 larvae of A. alni to feed on the alder leaves. The cut plantletswere enclosed in glass desiccators (2700 ml). The experimental set-up was maintainedat 251C and with a photophase of 16 h.

After pre-incubation of 24 h, the emitted volatiles were continuously collected overa period of 24 h on small carbon traps (1.5 mg charcoal, CLSA-Filter, Le Ruisseaude Montbrun, F-09350 Daumazan sur Arize) with air circulation according toDonath and Boland (1995). Compounds were eluted from the carbon traps withdichloromethane (2� 15 ml) and 1 ml aliquots were injected into a 2201C injector andseparated by capillary GC on a fused silica-column (15 m� 0.25 mm i.d.) coated witha 0.1 mm medium polar stationary phase (Optima-5s MS, Machery and Nagel,D .uren, Germany). Compounds were eluted under programmed conditions: 401C for1 min, ramped at 101C min�1 to 1801C, followed by a 351C min�1 ramp to 2801C for3 min. The He carrier gas was maintained at a flow rate of 3.0 ml min�1. Elutingcompounds were detected by mass spectrometry (Finnigan GCQ) with a sourcetemperature of 1801C operated in EI (70 eV) mode. GC-interface at 2651C; solventdelay: 2 min; scan range 35–300 Da. Compounds (caryophyllene, 4,8-dimethylnona-1,3,7-triene (=DMNT), 3-hexenyl acetate, indole, linalool, methyl salicylate, b-ocimene, 4,8,12-trimethyltrideca-1,3,7,11-tetraene (=TMTT)) were identified byretention time and mass spectra of authentic standards.

Ethylene production was measured in real time with a photoacoustic laserspectrometer containing a line-tunable infrared CO2 laser and a resonantphotoacoustic cell. Stems of young A. glutinosa plants with three developed primaryleaves were cut and immediately transferred into glass vials containing tap water.These plants were placed in a 600 ml glass cuvette which received a constant flow of1 l h�1 air. One cuvette contained the leaves with continuous feeding of A. alni (fiveadults on each leaf), while the second one contained only leaves (without beetles) andserved as reference cuvette. Ambient air was drawn through a platinum catalyst at4501C prior to entering the cuvette to remove hydrocarbons. After the cuvette, theair was pulled through a liquid-nitrogen trap to remove CO2 and H2O beforeentering the photoacoustic detection cell in which ethylene concentrations weremeasured every 3 min for 36 h. The detection limit of this method was about 100 pptand calibration was performed with certified ethylene samples.

2.6. Quantification of endogenous jasmonic acid (=JA) and salicylic acid (=SA)

For measuring the endogenous levels of JA and SA, we detached three fresh leavesfrom intact black alder plants, transferred the leaves into a vial (5 ml) with tap water,


and enclosed the three leaves in a desiccator (2700 ml). For feeding experiments fivelarvae of A. alni were placed on each leaf and allowed to feed continuously for thewhole time of incubation (t=0, 1, 3, 5.5, 8, 10, 12 h). Control experiments werecarried out following the same procedure but without insect feeding. Theseexperiments of a time course were repeated four times. Assays were maintained atr.t. with a 16:8 h day : night regime and 4000 lx.

The quantification of endogenous JA and SA of the leaves (1 g FW) followed theprotocol of Koch et al. (1999) adapted from the original procedure of McCloud andBaldwin (1997) and was calculated using calibration curves of the standards.

2.7. Quantification of total content of phenolic compounds

For determination of phenolic compounds, the black alder leaves were driedovernight at 501C. The dried leaves (0.5 g) were ground for 3 min with a Ultra-turraxT25 in 15 ml of methanol. Then the homogenate was heated upto 751C for 3 h. Aftercooling, the extract was centrifuged at 2500g for 3 min and the supernatant was usedfor the following measurement. A modification of the Folin–Ciocalteau method wasused for measurement of total content of phenolic compounds (Martin and Martin,1982). A 10 ml extract was delivered to an Eppendorf tube and mixed with 840 mlwater. The diluted extract was mixed with 50 ml Folin reagent (Folin–Ciocalteau’sphenol reagent, Sigma) and the mixture was allowed to incubate for 2–5 min. Then100 ml of Na2CO3 (saturated) was added. After a 30 min incubation at r.t. theabsorbance was measured at 750 nm on a Perkin-Elmer Spectrophotometer Lambda2S. Four replicates of each sample were analysed. The standard curve was preparedwith known concentrations of guaiacol.

2.8. Plant enzyme assays

To assay for foliar enzymes, the foliage, with midribs removed, was flash frozen inliquid nitrogen and homogenized in 0.1 M ice-cold NaPO4-buffer, pH 7.2, containing0.1% (w/v) sodium dodecyl sulphate (SDS) and 0.1% (w/v) polyvinylpyrrolidone(PVPP). The homogenate was centrifuged at 41C for 10 min at 12,000g, thesupernatant was frozen in liquid nitrogen, and stored at –801C until used forspectrophotometric assays of PPO, LOX, POD, and CAT activities. Protein contentof the samples was determined in duplicate using the Bradford (1976) method, andthe activities of all four enzymes were expressed as DE min�1 mg�1 protein.

To determine the polyphenol oxidase (PPO) activity, the procedure of Shermanet al. (1991) was followed. The assay solution consisted of 850 ml l-3,4dihydroxyphenylalanine (DOPA (5 mg ml�1) in 0.1 M NaPO4-buffer, pH 7.2, whichhad been aerated for 5 min prior to assay. To eliminate interfering H2O2 andperoxidase activities, catalase (280 Units ml�1, Sigma) in 100 ml H2O was added. Theassay was started by addition of 50 ml of enzyme extract. Assays were carried out foreither 2 or 10 min depending on the amount of PPO activity present in the samplesand the PPO activity was estimated spectrophotometrically at 490 nm and r.t., tofollow the conversion of l-3,4 DOPA to quinone polymers.


To determine the lipoxygenase (LOX) activity, the formation of UV-activeconjugated diene hydroperoxides was measured at 234 nm (Hildebrand andHymowitz, 1981). The reaction mixture consisted of 50 ml of enzyme extract addedto 950 ml of 1 mM linoleic acid dispersed in a 0.1 M NaPO4-buffer (pH 7.0). Changein absorbance was monitored for at least 5 min.

To determine peroxidase (POD) activity, guaiacol was used as the hydrogen donoraccording to the procedure of Ridge and Osborne (1970). Fifty microlitres of enzymesolution was mixed with a substrate solution consisting of 10 mM guaiacol in 0.1 MNaPO4-buffer (pH 7.0) with H2O2 added as a cofactor. POD activity was measuredas increase for 5 min at 470 nm.

Catalase (CAT) activity was assessed by monitoring the decrease in H2O2 bymeasuring the absorbance at 240 nm (Faccioli, 1979). The substrate was 30 mMH2O2 in 0.1 M NaPO4

- buffer (pH 7.0)

2.9. Proteinase inhibitor (=PI) extraction and quantitative determination of trypsinproteinase inhibitor (=Tryp PI) activity using a radial diffusion assay

The leaf samples for the PI analyses were weighed in a tube (1 g FW), flash frozenin liquid nitrogen, ground with a glass pestle, and thawed on ice. Before weighing,the midribs of the leaves were removed. After addition of 1 ml ice-cold proteinextraction buffer (1 ml g�1 leaf tissue; 0.1 mM Tris HCl-buffer (pH 7.6), containing5% polyvinylpolypyrrolidine, 2 mg ml�1 phenylthiourea, and 5 mg ml�1 diethyl-dithiocarbamate, 0.05 M Na2EDTA), the samples were vortexed and centrifuged at41C for 20 min at 12,000g. The supernatant was transferred to a fresh Eppendorftube and kept on ice until protein and PI analysis.

Protein content of the samples was determined in duplicate using the Bradfordmethod (Bradford, 1976). PI activities were analysed with the radial diffusion assayaccording to Jongsma et al. (1993, 1994), using bovine trypsine (type III; Fluka, 1 mgml�1) dissolved in agar. The leaf extract was allowed to incubate for 16 h at 41C inthe agar. Following incubation a 25 ml of N-acetyl-phenylalanine-b-naphtylester(APNE, a trypsin substrate, Bachem) in DMSO-/Tris buffer (0.1 mM, pH 7.6)-mixture was added to the trypsin/PI-extract mixture for 1 h at 371C.

For Tryp PI activities, a series of soybean Tryp inhibitor (STI, BoehringerMannheim, Mannheim, Germany) solutions were used to obtain a reference curve.PI activities are expressed as mg PI mg�1 protein g�1 FW.

2.10. Statistics

As the chemical assays used in this study are destructive, chemical analyses of theleaves could not be done both before and after the treatment, so differences fromcontrol plants were established. Data analysis has been done using the softwareSTATISTICA 5.0 (StatSoft). Different treatments were compared using one-wayANOVA (Figs. 1 and 5–7), the Student–Newman–Keuls multiple range test, andt-tests (Fig. 8). In the figures, we give arithmetic means + one standard error.


3. Results

3.1. Field experiments

Leaf damage of the alder trees increased with distance to the manually defoliatedtree (Dolch and Tscharntke, 2000). Distance and leaf damage were best correlated 7days after defoliation, but this correlation held up to 37–81 days. The feeding andoviposition experiments in the laboratory supported these findings from the field, asmost eggs were laid on leaves from the most distant alder tree, whereas leaves of thedefoliated tree and its nearest neighbour were avoided. Similarly, beetles (in the lab)consumed more area of the leaves from the farthest tree than from the defoliated treeor its nearest neighbour (Dolch and Tscharntke, 2000).

The response of the total arthropod community to the manual defoliation wasvery similar to that of the alder leaf beetle. Before defoliation, abundance and speciesrichness did not differ, but 81 days after defoliation, abundance of all arthropods(F ¼ 10:4; n ¼ 30; po0:001), of the phytophagous species (F ¼ 3:8; po0:05) as wellas the entomophagous species (F ¼ 9:0; po0:001) increased with distance to thedefoliated tree. This response was stronger than that after 37 or 133 days. Specialistsand non-specialists among the herbivores reacted differently (Figs. 1A and B). Whilethe non-specialists did not show a significant pattern, the specialists exhibited a cleardifference between the trees. The altogether 94 species of phytophagous insectssampled from A. glutinosa comprised 25 specialized (monophagous and/or known toprefer Alnus) and 69 non-specialized species. These species were represented by atotal of 1286 individuals, 980 (76.3%) of which belonged to the 20 most abundantspecies shown in Table 1. The altogether 75 species of entomophagous arthropods

Fig. 1. Number of individuals of phytophagous arthropods on the defoliated trees, their nearest

neighbours and their farthest neighbours (0, 1.3 and 10.6 m distance to the manually defoliated tree), 81

days after defoliation (see Table 1). Arithmetic means + one standard error are shown, different letters

indicate significant differences: (a) phytophagous non-specialists: F ¼ 0:44; n ¼ 30; p ¼ 0:65; (b)

phytophagous specialists: F ¼ 6:43; n ¼ 30; po0:005:


consisted of 361 individuals (including 187 individuals and 28 species of spiders, 110individuals and nine species of true bugs, and 33 individuals and 26 species ofparasitoids). Table 1 presents 265 individuals (73.4%) of the entomophagousarthropods belonging to the 10 most abundant species. In addition, the predator–prey ratio (abundance of predators divided by abundance of prey, 81 days after thedefoliation) increased from the defoliated tree to its nearest neighbour and the 10 mdistant tree (F ¼ 2:8; n ¼ 30; p ¼ 0:08).

Table 1

Number of arthropod individuals collected on Alnus glutinosa, 81 days after defoliation (other than

Agelastica alni)a.

Species No. of individuals

Specialist phytophagous insects

Psylla alni (Homoptera, Psyllidae) 67

Phyllonorycter alnifoliella (Lepidoptera, Gracillariidae) 29

Pterocallis alni (Homoptera, Callaphididae) 15

Alnetoidia alneti (Homoptera, Cicadellidae) 7

Ennomos alniaria (Lepidoptera, Geometridae) 7

Aphrophora alni (Homoptera, Cercopidae) 5

Oncopsis alni (Homoptera, Cicadellidae) 2

Melasoma aenea (Coleoptera, Chrysomelidae) 2

Psallus ambiguus (Heteroptera, Lygaeidae) 2

Chalcoides aurata (Coleoptera, Chrysomelidae) 1

Non-specialist phytophagous insects

Corticarina gibbosa (Coleoptera, Lathridiidae) 38

Baeopalma foersteri (Homoptera, Psyllidae) 25

Apion flavipes (Coleoptera, Apionidae) 10

Apion nigritarse (Coleoptera, Apionidae) 10

Eupteryx aurata (Homoptera, Cicadellidae) 8

Stenocranus minutus (Homoptera, Delphacidae) 5

Semiothisa alternaria (Lepidoptera, Geometridae) 4

Trioza urticae (Homoptera, Psyllidae) 3

Dicyphus epilobii (Heteroptera, Miridae) 2

Eupteryx atropunctata (Homoptera, Cicadellidae) 2

Entomophagous arthropods

Deraeocoris lutescens (Heteroptera, Miridae) 32

Singa hamata (Araneida, Araneidae) 19

Araneidae gen. sp. (Araneida) 15

Blepharidopterus angulatus (Heteroptera, Lygaeidae) 14

Tetragnatha sp. (Araneida, Tetragnathidae) 12

Araniella sp. (Araneida, Araneidae) 12

Forficula sp. (Dermaptera) 11

Metellina sp. (Araneida, Metidae) 9

Linyphiidae gen. sp. (Araneida) 6

Orius vicinus (Heteroptera, Anthocoridae) 4

a The 10 most abundant species of both Alnus specialists and non-specialists, as well as the 10 most

abundant entomophagous species are shown.


3.2. Biochemical analyses and bioassays

3.2.1. Volatile induction experimentsIn response to herbivory by A. alni, alder leaves released a blend of volatiles that

comprised the following compounds (Fig. 2): monoterpenes (e.g. b-ocimene,linalool), sesquiterpenes (e.g. b-caryophyllene, b-farnesene, a-humulene), homo-terpenes (e.g. 4,8-dimethylnona-1,3,7-triene, 4,8,12-trimethyltrideca-1,3,7,11-tetra-ene), fatty acid derivatives (e.g. 3-hexenyl acetate, decanal), and aromaticcompounds such as 2-methyl anthranilate, methyl salicylate, and indole. Herbivoryby A. alni induced a massive emission of these compounds that were not emitted by

Fig. 2. Gaschromatographic profile of the alder volatiles emitted after feeding by Agelastica alni. In

response to herbivory, black alder leaves released a blend of volatiles comprising the following

compounds: monoterpenes (e.g. b-ocimene, linalool), sesquiterpenes (e.g. b-caryophyllene, b-farnesene,

a-humulene) homoterpenes (e.g. 4,8-dimethylnona-1,3,7-triene, 4,8,12-trimethyltrideca-1,3,7,11-tetraene),

fatty acid derivatives (e.g. 3-hexenyl acetate, decanal) and aromatic compounds such as 2-methyl

anthranilate, methyl salicylate and indole. Control: gaschromatographic profile of undamaged alder

leaves.


healthy leaves. Although the collection of headspace volatiles from plants in closedsystems may lead to deprivation of CO2 and, hence may impose an additional‘‘stress’’ onto the plant, as yet, no negative impact has been observed. Moreover,headspace volatiles from control plants were collected the same way exposing thisgroup of plants to identical constraints. Volatile production was exclusively observedafter herbivore damage (or jasmonic acid treatment, Thiessen, unpublished)demonstrating that the experimental conditions do not significantly alter the plant’sresponse.

3.2.2. Ethylene emissionThe temporal pattern of ethylene emission from alder leaves over a 36 h period is

illustrated in Fig. 3. The first volatile signs of A. alni feeding were observed 8 h afterstarting the experiment. The increase in emission was relatively long-lived (slowbuild-up within about the first 10 h and waning within 24 h) in response to feeding.The ethylene production from leaf beetle-damaged tissue was not only greater than

Fig. 3. Ethylene emission (nl/l) from Alnus glutinosa plants during feeding of Agelastica alni adults,

measured by photoacoustic laser spectroscopy: (a) feeding of A. alni. Data measurement for the ethylene

emission due to the beetles’ feeding was shortly interrupted (as indicated by the double line); (b) control

(undamaged leaves).


that from the control tissue, but showed a peak of emitted ethylene (7 nl l�1) at 16 h,whereas control tissue showed a relatively steady rate of emission (1.4 nl l�1) over themeasurement time.

3.2.3. Quantification of endogenous JA and SAThe enhanced levels of endogenous JA and SA showed the activation of the

octadecanoid and the salicylate pathway (Fig. 4). Samples were taken at defined timeintervals after allowing the herbivore to feed on the leaves. The level of endogenousJA rose 2 h after the onset of the experiment, reached a maximum of ca. 1255 ng g�1

FW after 8 h, and then fell back within 12 h to the initial concentration of about13 ng g�1 FW. Control experiments without feeding larvae resulted in only a smallincrease of endogenous JA, and the maximum level was much lower (ca. 81 ng g�1

FW, due to the wounding by cutting the stem) (Fig. 4A).Temporal patterns of endogenous SA are shown in Fig. 4B. In the case of

herbivore damage, the level of endogenous SA at first decreased and then steadilyincreased to 2000 ng g�1 FW from hour 3 to 8. Later, free SA dropped to a final levelof 1295 ng g�1 FW, ca. 30% above the resting level prior to damage. Control leavesshowed a much lower increase and a lower maximum level of SA than the herbivore-damaged leaves. After a transient decrease, after the onset of the experiment, thelevel of endogenous SA reached a final concentration at about 1121 ng g�1 FW.

3.2.4. Quantification of total phenolic compoundsThe phenolic content of the alder leaves depended on the type of treatment

(Fig. 5). Control plants contained only 5.370.3 mg phenolics g�1 leaf DW. Feedingof larvae induced a significant increase to 8.570.8 mg g�1. Treatment with gaseousethylene resulted in a similar increase of phenolics (10.271.1 mg g�1), but incubationwith gaseous MeJA and MeSA did not significantly affect the level of phenolics.

Fig. 4. Jasmonic acid (JA) and salicylic acid (SA) concentrations of black alder leaves wounded at time 0

with feeding of Agelastica alni larvae, and leaves without herbivores (=control). Leaves from four alder

plants were harvested at each time: (a) JA concentration is expressed as ng JA per g leaf FW and; (b) SA

concentration is expressed as ng SA per g leaf FW. Arithmetic means 7 one standard error is shown

(n ¼ 4). (&) Control; (K) feeding of A. alni. FW= fresh weight.


Fig. 5. Phenolic content of black alder leaves treated with larvae of A. alni or with gaseous chemicals:

ethylene, jasmonic acid methyl ester (MeJA), and salicylic acid methyl ester (MeSA). Phenolic content (mg

phenolic per g leaf DW) was measured after 72 h of wounding or incubation with ethylene, MeJA and

MeSA. Arithmetic means + one standard error is given (n ¼ 6). Different letters indicate significant

differences (po0:05). DW=dry weight.

Fig. 6. Enzyme activities in black alder leaves, after 48 h feeding of A. alni larvae or incubation with

gaseous chemicals: ethylene, jasmonic acid methyl ester (MeJA), or salicylic acid methyl ester (MeSA).

Arithmetic means + one standard error are given (n ¼ 5). Different letters indicate significant differences

(po0:05): (a) polyphenol oxidase (PPO). (DE490 min�1 mg�1protein); (b) lipoxygenase (LOX)

(DE234 min�1 mg�1protein); (c) peroxygenase (POD) (DE470 min�1 mg�1protein); (d) catalase (CAT)

(DE240 min�1 mg�1protein).


3.2.5. Plant enzyme assaysAlder leaves were exposed to feeding of A. alni and incubated with gaseous

ethylene, MeJA and MeSA. After an induction period of 48 h, leaf PPO, LOX, PODand CAT activity was measured (Fig. 6). After feeding of A. alni PPO activitiesincreased 3.2-fold compared to the control. The feeding also increased the activitiesof LOX 5.7-fold and that of POD 13.6-fold. The activity of the antioxidant enzymeCAT showed a non-significant increase (1.5-fold). In contrast, treatment of alderleaves with the volatiles ethylene, MeJA and MeSA did not enhance the activities ofPPO, LOX and POD, and only application of MeJA resulted in a 2.2-fold increase inCAT activity.

3.2.6. Proteinase inhibitor activityThe effects of feeding of A. alni, elicitation by JA solution, gaseous ethylene,

MeJA, or MeSA on the activity of PI were determined by the radial diffusion assay.The data (Fig. 7) clearly indicate that alder leaves generally exhibited a low PIactivity (controls: 3.970.5 mg PI mg�1 protein g�1 FW) that is significantly enhancedby herbivory (4.7-fold), and the gaseous phytohormone ethylene (3.5-fold), withintermediate levels in the JA treatment (2.5-fold) and airborne MeJA (3-fold).

3.2.7. Container experimentsUndamaged leaves exposed to volatiles released from leaves damaged by

herbivory showed a significant, 1.9-fold increase of PI activity compared to control

Fig. 7. Proteinase inhibitor (PI) activity in black alder leaves, monitored by radial diffusion assay after

72 h of feeding of A. alni larvae or incubation with jasmonic acid (JA), ethylene, jasmonic acid methyl ester

(MeJA), or salicylic acid methyl ester (MeSA). Activity is expressed as mg PI mg�1 protein g�1 FW.

Arithmetic means + one standard error are given (n ¼ 5). Different letters indicate significant differences

(po0:05). FW=fresh weight.


leaves (p ¼ 0:0065; Fig. 8), whereas combinations of undamaged leaves withundamaged leaves did not show any significant changes in PI activity.

4. Discussion

The results of the field experiments with manually defoliated alders showed thatdefoliation affected herbivory by the major alder antagonist, the leaf beetle A. alni.Herbivore damage increased with increasing distance to the defoliated tree,suggesting induced resistance not only on the damaged tree, but also in theneighbouring trees. The beetles also avoided leaves from the nearest neighbours forboth feeding and oviposition in a laboratory assay (Dolch and Tscharntke, 2000).This pattern suggests that alders show interplant resistance transfer (inducedresistance in alders has been already shown by Jeker (1981) and Oleksyn et al.(1998)), possibly due to an airborne signal transfer with volatiles. Emission ofvolatiles to induce resistance may be advantageous for plants because of (i) a within-plant induction of resistance, as aerial dispersion of volatiles may lead to a fastertransfer to other tissues of the same tree than a systemic signal transfer within thevascular system (see Jones et al., 1993, and references therein), (ii) masking ofdefoliated trees by non-nutritious neighbours, similar to effects of ‘‘associatedresistance’’ of plants grown in polyculture rather than in monoculture (see Strong

Fig. 8. Proteinase inhibitor (PI) activity of undamaged alder leaves influenced by volatiles emitted by

leaves damaged by A. alni larvae (A-neighbour) in an airtight container. The control leaves (C-neighbour)

were associated with only undamaged leaves. PI activity (mg PI mg�1 protein g�1 FW) was monitored by

radial diffusion assay after 72 h of incubation. Arithmetic means + one standard error are given (n ¼ 6).

FW=fresh weight. Undamaged leaves (‘‘A-neighbour’’) neighbouring damaged leaves (‘‘A. alni’’) showed

a significant difference from control leaves (‘‘Control and C-neighbour’’): t ¼ 3:12; n ¼ 18; p ¼ 0:0065 (or

‘‘A-neighbour’’ vs. ‘‘C-neighbour’’: t ¼ 3:22; n ¼ 12; p ¼ 0:009). Damaged leaves and their undamaged

neighbours did also differ (‘‘A. alni’’ vs. ‘‘A-neighbour’’): t ¼ 2:85; n ¼ 12; p ¼ 0:017:


et al., 1984), or (iii) the attraction of natural enemies such as parasitoids andpredators (e.g. Dicke, 1994; Bruin et al., 1992; Turlings and Benrey, 1998). Possibleexplanations for this pattern also include a soil-borne signal transfer (e.g. Simardet al., 1997) and an intensified nutrient absorption of defoliated trees reducing foliagequality of undamaged neighbours (Tuomi et al., 1990, see Dolch and Tscharntke,2000). In the alder-leaf beetle interaction, responses of natural enemies to volatilesdid not appear to be of importance, because (i) the many rearings did not result inparasitoids and (ii) the field samples of all arthropods associated with alder leavesdid not show an aggregation of natural enemies near the defoliated tree, but just theopposite pattern, since the number of natural enemies as well as predator–prey ratiosincreased with increasing distance to the defoliated tree. Interestingly, the specialistherbivores responded similarly to the defoliation as the alder leaf beetle, which isalso a specialist. In contrast, the generalists did not show this pattern, supporting theview that specialists are more affected by plant resistance than generalists (Maddoxand Root, 1987).

Mechanisms that might have produced the patterns found in these fieldexperiments were studied in more detail using biochemical analyses and furtherbioassays. These lab experiments had, for practical reasons, to rely on very youngalder trees, although such fast-growing, young trees can be expected to invest moreenergy in growth rather than in defense (Coley et al., 1985; Karban, 1990). Dolchand Tscharntke (2000) found that damage on black alders caused by the leaf beetleswas negatively correlated with tree diameter, so the young trees in the field weremore damaged than old trees. Accordingly, the biochemical analyses and labexperiments may underestimate defensive responses which can be expected to bemore pronounced in the old (but hard to handle) trees.

Results of the laboratory studies on the mechanisms involved in the inducedresistance of black alders following attack of the alder leaf beetle suggest adifferentiation between direct, feeding-induced and indirect, volatile-induced plantresponses.

4.1. Direct, feeding-induced plant responses

Black alder proved to exhibit a multitude of inducible defense mechanisms as adirect response of alder foliage to feeding of A. alni. Results of this study showedincreases in the phenolic contents, in the activity of oxidative enzymes (PPO, LOX,and POD) and in proteinase inhibitors.

The total amount of phenolics showed a 2-fold increase, supporting the results ofOleksyn et al. (1998) and the general finding that leaves of the genus Alnus are rich inphenolic compounds (Hegnauer, 1964). The presumed role of phenolics ascomponents of plant defense is based on studies demonstrating their toxicity toherbivores when incorporated into artificial diets (Elliger et al., 1981) and on thecorrelation of the phenolic content of plants with the amount of herbivory (Dudtand Shure, 1994).

Anti-nutritive oxidative enzymes were also shown to increase in response toherbivory. PPO is a major anti-nutritive protein, known to be induced by the


jasmonic acid pathway, e.g. in tobacco and other solanaceous plants, and to causeresistance against several herbivores (Felton et al., 1992; Constabel and Ryan, 1998).LOX activity increased after herbivory and may also play a role in herbivore control.Damage to cells by feeding can cause the release of fatty acids including linoleic,linolenic, and arachidonic acids from membranes and other lipids (Gardner, 1980;Vernooy-Gerritsen et al., 1983). LOX will catalyse the oxidation of thepolyunsaturated fatty acids to form peroxy radicals, hydroperoxides, and superoxideanions (Gardner, 1980; Rustin et al., 1983; Lynch and Thompson, 1984). The freeradicals are highly reactive and can promote lignification, which may represent abarrier to some plant pests. LOX can also catalyze the production of JA fromlinolenic acid, which stimulates the expression of defense-related genes serving assecondary signals activating a subset of defense genes (see Farmer et al., 1992).Activity of the prooxidant enzyme POD was found to also increase with A. alnifeeding. Numerous studies have indicated that POD increases with insect attack orleaf tissue damage (Thaler et al., 1996). This response may be due to a direct role inplant resistance mechanisms. POD is assumed to be involved in the production oftoxic oxidative metabolites (Garner, 1984) and may promote lignification (Egleyet al., 1983). However, POD normally increases during tissue senescence in plantsand the increased concentration of lipid peroxides may simply be a response to pest-induced senescence of tissues (Matkovics et al., 1981). CAT, like POD, catalyses thereduction of toxic intermediates of the O2 metabolism (Faccioli, 1979). CAT activitydid not change after A. alni feeding. There was no response to herbivory in otherstudies (Faccioli, 1979; Matkovics et al., 1981), too, or even decreased activity insoybeans after herbivory (Bi and Felton, 1995).

Alder plants could be also shown to produce proteinase inhibitors. The PI activityincreased upon feeding 5-fold. Proteinase inhibitors are found in a wide range ofplant species and their importance as anti-herbivore compounds against insectherbivory is well known (Koiwa et al., 1997, but see Jongsma et al., 1995). PIs mayinhibit digestive enzymes and reduce body mass indices (growth, weight) (Broadway,1995; Heath et al., 1997). Alnus incana, the grey alder, which is closely related to theblack alder A. glutinosa, is known to produce inducible PIs, and the induction of PIactivity in grey alder foliage has been shown to cause retarded growth, delayedpupation, reduced egg production and low survival of the leaf beetle Galerucellalineola (Seldal et al., 1994).

4.2. Indirect, volatile-induced plant responses

Only few publications give evidence for volatile signals triggering interplantcommunication following herbivory (Bruin et al., 1992; Arimura et al., 2000; Karbanet al., 2000). Black alder responded to insect damage with the release of volatiles. Thefirst signal after herbivory was the release of the phytohormone ethylene from thewounded leaves, and ethylene production increased 7-fold in response to herbivory.Ethylene is known to play important regulatory roles in primary and secondaryplant metabolism (Abeles et al., 1992). In black alder, the volatile ethylene appearedto be an important candidate of the possible interplant signal transfer following


herbivore feeding and of the induction of plant resistance mechanisms. O’Donnellet al. (1996) suggested that the initial production of ethylene (in tomato) may act aspositive-feedback mechanism to increase jasmonate synthesis linked to wounding.

Feeding of A. alni induced not only ethylene, but also the biosynthesis of a largenumber of other volatiles. Presumably, the regurgitate of black alder leaf beetlecontains eliciting compounds inducing the production of volatiles, becausemechanical leaf wounding caused only a non-specific blend of volatiles in lowquantities (Thiessen, unpublished). The great importance of insect-specific elicitorsfor the emission of volatiles is known from many insects (Turlings et al., 1993;Mattiacci et al., 1994, 1995; Alborn et al., 1997; Par!e and Tumlinson, 1998; Arimuraet al., 2000). Preliminary chemical analyses of the regurgitate of A. alni showed thatit contains no volicitin (Thiessen, unpublished), which has been claimed to elicitvolatile biosynthesis in maize plants (Alborn et al., 1997).

JA application to leaves mediates the induction of volatiles. The inducing effect ofJA is known for various woody and herbaceous plant species (Hopke et al., 1994;Boland et al., 1995; Dicke et al., 1999; Meiners and Hilker, 2000). Many plantsrespond to herbivory, the herbivores’ oral secretions and regurgitants, to jasmonateapplication or the treatment with cellulysin with a release of a complex blend ofvolatiles including mono-, sesqui- or homoterpenes, which is often rather specific tothe attacking herbivores or treatment (Turlings et al., 1995; Takabayashi and Dicke,1996; Alborn et al., 1997; Boland et al., 1998). In black alder, application of JAinduced a volatile blend that was similar but not identical to that emitted byherbivory (Thiessen, unpublished), which supports observations on lima beans(Dicke et al., 1999). Among the many volatiles, there are signals for rapidcommunication among stressed plants: b-ocimene, DMNT, and TMTT induce theexpression of pathogen-related (PR) genes and other defense genes in undamagedlima bean leaves after exposure to the volatiles (Arimura et al., 2000). We could showthat wounded plants of Alnus glutinosa also produce the volatiles b-ocimene,DMNT, TMTT and MeSA, so these volatile signals may have also been involved ina possible interplant communication in black alders.

We exposed leaves to the gaseous elicitors ethylene, MeJA, and MeSA, generallythought to be candidates for plant–plant communication and induced resistance(Farmer and Ryan, 1990; Thaler et al., 1996; Shulaev et al., 1997; Boland et al., 1998;Karban and Baldwin, 1997; Karban et al., 2000). As we have no data for the actualvolatile concentrations in the field (typically with old trees which can be expected toshow much stronger responses than the very young trees or even isolated leaves, usedin the laboratory assays), we used these experiments to simply test whether there areany plant responses. We found an increase of CAT and PI activities after exposure ofMeJA and an increase of phenolics and PI activity after ethylene treatment.However, there was no induction of PPO by ethylene or MeJA. This was unexpectedsince an induction of PPO activity by MeJA has been reported for a number ofplants (Farmer and Ryan, 1990; Constabel and Ryan, 1998). MeSA was not foundto significantly change phenolic metabolism or protein activity in our black aldersystem. The active components in mediating the possible plant–plant interaction inthe present study may include ethylene, b-ocimene, DMNT, and TMTT, but methyl


jasmonate (MeJA) may have also been involved (although not, or not yet, detectedamong the released volatiles). The relative importance of these candidates forinterplant signal transfer in alders is still unclear.

Further evidence for plant–plant communication comes from our containerexperiments, where undamaged leaves were exposed to volatiles produced byherbivore-damaged leaves in airtight chambers. Not only the damaged leaves, butalso the undamaged neighbours of damaged leaves showed a significant increase inthe activity of proteinase inhibitors (Fig. 8). We cannot exclude that the possible CO2

enrichment in the chambers, which might be expected from the beetles’ respiration,may have affected PI activity, but as shown previously for Eucalyptus foliage, greatlyelevated CO2 levels are required to shift the metabolism to a significant extent(Lawler et al., 1997; McDonald et al., 1999). In addition, the PIs of A. glutinosa wereinducible not only after insect feeding, but also after the application of ethylene,MeJA and JA (Fig. 7). PIs have already been found to be inducible after applicationof plant hormones such as ethylene or MeJA in other systems (Pena Cortes et al.,1988; Farmer et al., 1992; Koiwa et al., 1997). Such interplant signal transfer has alsobeen shown in herbaceous plants. Lima beans become less susceptible to spider mitesafter exposure to volatiles from attacked conspecifics (Bruin et al., 1992), probablydue to the activation of defense genes by volatile terpenoids (Arimura et al., 2000).Wild tobacco plants with clipped sagebrush neighbours had increased PPO levelsand experienced reduced leaf damage by grasshoppers and cutworms (Karban et al.,2000).

Results of our experiments suggest the following scenario of herbivore-inducedplant–plant interactions among black alder trees. The alders respond to feeding of A.alni with an emission of ethylene and a blend of volatiles, followed by an activationof the JA signalling pathway. As a result, attacked alder leaves show increases (i) inthe phenolic content of the leaves, (ii) in the activity of oxidative enzymes (PPO,LOX and POD), and (iii) in the level of PIs. When exposed to ethylene, the phenoliccontent and the level of PIs were even induced in undamaged alder trees. However,we need more experiments to solve some inconsistencies, such as the increase in PIactivity, but not in phenolics (Thiessen, unpublished) when leaves are exposed to thevolatiles emitted by damaged neighbours.

In conclusion, field experiments, bioassays in the laboratory as well as biochemicalanalyses suggest the existence of interplant resistance transfer in Alnus glutinosa, withairborne volatiles as a possible mechanism. However, we did not show the relativeimportance of airborne and possible soil-borne signals as well as unknown effects ofreduced foliage quality of neighbouring trees, due to a possible soil nutrientdepletion as a result of compensatory growth after defoliation (Dolch andTscharntke, 2000).

Acknowledgements

Comments of Ian T. Baldwin, Jan Bruin, and Marcel Dicke improved the papergreatly. We would like to thank Frank K .uhnemann (Institute for Applied Physics,


University of Bonn) for his help with the photoacoustic laser spectrometer. Financialsupport came from the German Science Foundation (Deutsche Forschungsge-meinschaft, DFG).

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Herbivory, induced resistance, and interplant signal transfer in Alnus glutinosa