Biochemical Systematics and Ecology 29 (2001) 10251047
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
Field experiments with manually defoliated black alders (Alnus glutinosa) showed that
defoliation aected 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 predatorpreyratios 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 aected by plant resistance thangeneralists.
Mechanisms causing this pattern, found in the eld, 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.Quantication 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), and4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). The incubation with volatiles resulted in an
*Corresponding author. Tel.: +49-551-399209; fax: +49-551-398806.
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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 signicantlyincreased the activity of proteinase inhibitors when they were associated with leaves previously
attacked by beetle larvae.In conclusion, eld 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 eects of intensied 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
Plant responses to herbivory are known from a wide range of plants and oftenaect 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 aws 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 aect 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 eld 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 eect 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 specic, widespread, univoltine and
T. Tscharntke et al. / Biochemical Systematics and Ecology 29 (2001) 102510471026
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 ushcrash uctuations 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 eld were conducted to search for further evidence
for the pattern found by Dolch and Tscharntke (2000), and to further elaborate theinterplant-communication hypothesis. Since eects of defoliation on neighbouringtrees may not only aect 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 eld. 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 1m. 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 all
arthropods 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) 10251047 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=4060 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 eld. 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 eects 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 rst desiccator (2700ml), 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, dierentdesiccators 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 (5ml)with tap water, and enclosed the leaves in a desiccator (2700ml). The chemicals,except ethylene, were dissolved in pure dichloromethane (1 mg ml1), and 5 ml of eachwas applied onto a small piece of lter paper. After brief evaporation of the solvent,we xed the lter 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 lter paper. Ethylene was applied using 1000 ml of the pure gas.Jasmonic acid was applied as a solution (1mM) 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 ve times (for the determination of activities of PPO,LOX, POD, CAT, and PI, see below) or six times (for the phenolics), using dierent
T. Tscharntke et al. / Biochemical Systematics and Ecology 29 (2001) 102510471028
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 (2700ml). 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 over
a period of 24 h on small carbon traps (1.5mg 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 (15m 0.25mm 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 for1min, ramped at 101Cmin1 to 1801C, followed by a 351Cmin1 ramp to 2801C for3min. The He carrier gas was maintained at a ow rate of 3.0mlmin1. Elutingcompounds were detected by mass spectrometry (Finnigan GCQ) with a sourcetemperature of 1801C operated in EI (70 eV) mode. GC-interface at 2651C; solventdelay: 2min; scan range 35300Da. 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 identied byretention time and mass spectra of authentic standards.Ethylene production was measured in real time with a photoacoustic laser
spectrometer 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 600ml glass cuvette which received a constant ow of1 l h1 air. One cuvette contained the leaves with continuous feeding of A. alni (veadults 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 3min for 36 h. The detection limit of this method was about 100 pptand calibration was performed with certied ethylene samples.
2.6. Quantication 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 via...