Mycorrhiza changes plant volatiles to attract spider mite enemies

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1 Functional Ecology 2012, 26, doi: /j x Mycorrhiza changes plant volatiles to attract spider mite enemies Peter Schausberger*,1, Stefan Peneder 1, Simone Jürschik 2 and Daniela Hoffmann 1 1 Group of Arthropod Ecology and Behavior, Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, Peter Jordanstrasse 82, 1190 Vienna, Austria; and 2 Ionicon Analytik GmbH, Eduard Bodem Gasse 3, 6020 Innsbruck, Austria Summary 1. Indirect induced plant defence via emission of herbivore-induced plant volatiles (HIPV) to recruit natural enemies of herbivores is a ubiquitous phenomenon, but whether and how emission of above-ground HIPVs is adaptively modulated by below-ground mutualistic microorganisms is unknown. 2. We investigated the effects of the mycorrhizal fungus Glomus mosseae on chemical composition of HIPVs emitted by bean plants Phaseolus vulgaris attacked by spider mites, Tetranychus urticae, using proton-transfer mass spectrometry, and attraction of the spider mites natural enemy, the predatory mite Phytoseiulus persimilis, to these HIPVs using a Y-tube olfactometer. 3. Mycorrhiza significantly changed the HIPV composition. Most notably, it increased the emission of b-ocimene and b-caryophyllene, two compounds synthesized de novo upon spider mite attack. The constitutively emitted methyl salicylate was increased by spider mite infestation but decreased by mycorrhiza. 4. The predators responded strongly to HIPVs emitted by plants infested for 6 days and preferred HIPVs of mycorrhizal plants to those of non-mycorrhizal plants. In contrast, they were less responsive and indiscriminative to mycorrhization when exposed to volatiles emitted by non-infested plants and plants infested by spider mites for 1 or 3 days. 5. Our study provides a key example of an adaptive indirect HIPV-mediated interaction of a below-ground micro-organism with an above-ground carnivore. Key-words: arbuscular mycorrhiza, herbivore-induced plant volatiles, induced plant defence, multi-trophic interaction, predatory mites Introduction Plants may either constitutively or after induction defend themselves against herbivores, either directly through chemical substances and or morphological structures negatively affecting the herbivores or indirectly by favouring establishment and or attraction of the herbivores natural enemies (e.g. Price et al. 1980; Karban & Baldwin 1997; Sabelis et al. 1999). One form of induced indirect defence of plants is the production and release of volatiles attracting third trophiclevel natural enemies such as carnivorous predators and parasitoids. Systemic release of natural enemy attracting volatiles upon herbivore attack of above-ground plant parts is a ubiquitous phenomenon (for review, Dicke & Vet 1999; Paré & Tumlinson 1999; Hare 2011). Analogous phenomena may occur below-ground upon attack of the roots (e.g. Horiuchi et al. 2005; Rasmann et al. 2005; Wenke, Kai & * Correspondence author. peter.schausberger@boku.ac.at Piechulla 2010). However, below-ground emission of herbivore-induced plant volatiles (HIPVs) is less well documented, which is not necessarily because it is less common than above-ground emission, but possibly also because the soil environment is more difficult to study. Based on observations that below- and above-ground plant-associated processes are mutually dependent (Van der Putten et al. 2001), recent research revealed that indirect defence mechanisms such as volatile emission induced by herbivory on belowand above-ground plant parts commonly interact (Bezemer & van Dam 2005; Rasmann & Turlings 2007; Soler et al. 2007; Erb et al. 2008). Similarly, below- and above-ground micro-organisms may affect each other, mediated by induced plant defence reactions (Walters & Heil 2007). While these examples document that processes induced by above- and below-ground herbivores or by above- and below-ground micro-organisms may interfere with each other, little is known about how below-ground microorganisms affect herbivore-induced volatile production Ó 2011 The Authors. Functional Ecology Ó 2011 British Ecological Society

2 442 P. Schausberger et al. (Fontana et al. 2009; Leitner et al. 2010) and associated recruitment of natural enemies above-ground (Guerreri et al. 2004). This is an important field of research because on above-ground plant parts, the endogenous biochemical cascades (the salicylate and jasmonate ethylene-mediated pathways) and defence reactions induced by pathogenic micro-organisms and herbivores often interfere with each other due to pleiotropic effects and resource allocation trade-offs (Thaler et al. 1999, 2002; Bostock et al. 2002). The effects of mutualistic below-ground micro-organisms such as plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungi on induced indirect plant defences against above-ground herbivores are largely unknown. Van Oosten et al. (2008) did not find an effect of the plant-growthpromoting rhizobacteria Pseudomonas sp. on volatile emission of Arabidopsis thaliana attacked by caterpillars of the small white Pieris rapae or the beet armyworm Spodoptera exigua, measured in the level of attraction of the parasitoid Cotesia rubecula. Only a few studies looked into the effects of mycorrhizal symbiosis, the association between plant roots and mycorrhizal fungi, on volatiles emitted by above-ground plant parts, three of them dealt with HIPVs. Rapparini, Llusià & Penˇuelas (2008) detected constitutive changes in volatile production following mycorrhization (by Glomus spp.) of Artemisia annua but did not test for changes in volatiles induced by herbivore attacks. Fontana et al. (2009) and Leitner et al. (2010) determined mycorrhiza (Glomus intraradices)-induced quantitative and qualitative changes in the volatile blends released by above-ground plant parts of Plantago lanceolata (Fontana et al. 2009) and Medicago truncatula (Leitner et al. 2010) induced by caterpillars of Spodoptera spp. Guerreri et al. (2004) observed stronger attraction of the parasitoid Aphidius ervi to constitutive volatiles of mycorrhizal (Glomus mosseae)tomatoplantsascomparedto non-mycorrhizal plants. However, the mycorrhiza-induced behavioural changes of the parasitoids occurred independent of, and were not altered by, the presence of their hosts, the plant-attacking aphid Macrosiphum euphorbiae.this result is evolutionarily puzzling in a tri-trophic context: the parasitoids do not gain any benefits from the plants themselves and would therefore be betrayed by the mycorrhiza plant mutualism due to enhanced attraction to plants without receiving a reward, that is, potential hosts. In any case, Guerreri et al. (2004) is the only study attempting to link mycorrhizal symbiosis and prey host searching based on plant-emitted volatiles by above-ground third trophic-level natural enemies. No study to date pinpointed the chemical changes in the volatile blends and simultaneously linked them to behavioural changes of the herbivores natural enemies. Depending on the species involved and the ecological context, arbuscular mycorrhizal symbiosis is commonly but not always beneficial to both partners and hence considered mutualistic (Smith & Read 2008; Hoeksema et al. 2010). Among other (ex)changes, the mutualism mainly involves plants trading carbon for nutrients from the fungus. In some systems, mycorrhiza increases the vegetative and or reproductive growth of their host plants but can simultaneously favour plant-attacking, and thereby plant-fitness-decreasing, organisms such as herbivores. This is, for example, true for the interaction between the mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.), common bean plants Phaseolus vulgaris L. (e.g. Isobe & Tsuboki 1999) and the herbivorous two-spotted spider mite Tetranychus urticae Koch (Hoffmann et al. 2009). Spider mites are highly detrimental to both, the bean plant and the mycorrhizal fungus (Hoffmann et al. 2011), and should thus be important drivers in the evolution of the bean fungus interaction. We argue that for evolutionary optimization and stability of the fungus plant mutualism, mycorrhizal symbiosis should compensate for herbivore enhancement by simultaneously enhancing the performance of the herbivores main natural enemies, predatory mites of the family Phytoseiidae. The first chronological step in manipulating the aboveground tri-trophic plant herbivore carnivore interaction is to increase the attractiveness of herbivore-infested plants to recruit more third trophic-level natural enemies. We therefore hypothesized that mycorrhiza-induced changes in plant chemistry and associated volatile emission should lead to stronger attraction of third trophic-level natural enemies in multi-trophic systems where mycorrhiza enhances the performance of the herbivores of their host plants, as observed in the interaction between G. mosseae, P. vulgaris and T. urticae (Hoffmann et al. 2009). Accordingly, we compared (i) the composition of volatile blends released by common bean plants living in symbiosis or not with G. mosseae and attacked above-ground by T. urticae or not using protontransfer reaction time-of-flight mass spectrometry (PTR- TOF-MS), and linked possible differences in the volatile blends to (ii) the response of a specialized natural enemy of the spider mites, the predatory mite Phytoseiulus persimilis Athias-Henriot (Fig. 1), to volatiles from mycorrhizal and non-mycorrhizal plants in binary-choice situations using a Y-tube olfactometer. Fig. 1. Gravid female of the predatory mite Phytoseiulus persimilis (body length, approximately 0Æ5 mm)inaspidermitepatch(ó Peter Schausberger).

3 Adaptive indirect below- and above-ground interaction 443 Materials and methods MITES AND PLANTS The tri-trophic system of P. vulgaris, T. urticae and predatory mites such as P. persimilis is a widely used, well-studied model system in research on HIPV (e.g. Sabelis & van de Baan 1983; Dicke et al. 1990; Sabelis et al. 1999). Recent investigations documented that this system is also perfectly suited for studying multi-trophic below- and above-ground interactions between plant-associated organisms such as the interaction between mycorrhizal fungi and herbivorous and carnivorous mites living on above-ground plant parts (Hoffmann et al. 2009, 2011; Hoffmann, Vierheilig & Schausberger 2011a,b). Leaf samples used in olfactometer choice tests and mass spectrometry werederivedfromcommonbeanplants, P. vulgaris var. Taylor s Horticultural, colonized (hereafter termed +M or mycorrhizal plants) and not colonized (hereafter termed )M or non-mycorrhizal plants) by the arbuscular mycorrhizal fungus G. mosseae, and infested (+SM) or not infested ()SM) by the spider mite T. urticae. To generate mycorrhizal and non-mycorrhizal plants, we followed the protocol described by Hoffmann et al. (2009) with respect to plant growing, fungal inoculation, watering and fertilization. The G. mosseae inoculum (BEG 12) was originally obtained from the International Bank of Glomeromycota ( beg). All plants used in the olfactometer tests and for mass spectrometry were randomly chosen and checked for the degree of mycorrhizal colonization. We used detached leaves instead of leaves attached to the potted plant or whole plants because this procedure better allows for standardization of age, functional part and biomass of the plant material (Choh & Takabayashi 2006). Additionally, confounding volatiles from other parts than the leaves such as the potting substrate or the roots can be more easily excluded. HIPVs of detached leaves and leaves attached to the plants are qualitatively similar but may differ quantitatively (e.g. for bean, Dicke et al. 1990; De Boer et al. 2008; for maize, Schmelz, Alborn & Tumlinson 2003; Williams et al. 2005). Leaves were immediately used in choice tests after detachment. After leaves used in choice tests had been detached, the remaining aboveground plant parts and the roots were removed from the planting pot. For plants used for mass spectrometry, 1 week before the experiment took place, a soil sample (approximately 22 cm 3 ) was taken from each pot usinga cork borer (Ø 2Æ2 cm). In either case, the planting substrate was rinsed off the roots with cold tap water. Roots were cleared by boiling them for 10 min in 10% KOH and stained by boiling for 5 min in a 5% black ink (Schaeffer, Ft. Madison, USA) and household vinegar (equal to 5% acetic acid) solution (Vierheilig et al. 1998). The percentage of root length colonized (RLC) by G. mosseae was estimated according to the modified gridline intersect method (Giovannetti & Mosse 1980). All 36 mycorrhizal plants (+M) used for the olfactometer tests and the eight mycorrhizal plants used for mass spectrometry had >10% RLC and on average 20Æ76 ± 1Æ16 (SE) and 29Æ75 ± 3Æ70 (SE) % RLC, respectively. All non-mycorrhizal plants ()M) had 0% RLC. Plants used for the olfactometer tests and mass spectrometry were days post-inoculation with the mycorrhizal fungus. The stock population of T. urticae was maintained on whole non-mycorrhizal bean plants at 25 ± 5 C, 60 80% rh, 16:8 h L:D. Phytoseiulus persimilis subjected to olfactometer tests derived from a laboratory-reared population founded with specimens collected on clementine trees in Onda, Spain. In the laboratory, P. persimilis was maintained on T. urticae-infested bean leaves from non-mycorrhizal plants piled up on a plastic tile resting on water-saturated foam within an open plastic box half-filled with water. Prey was supplied by adding five to seven infested bean leaves three times a week (McMurtry & Scriven 1965). Predator rearing units were stored at 25 ± 1 C, 60 ± 5% rh and 16:8 h L:D. OLFACTOMETER TESTS The Y-tube olfactometer used was a modification of the olfactometer described by Sabelis & van de Baan (1983) and consisted of three glass tubes (40 mm inner diameter) of equal length (130 mm) melted together in a Y-shape (Fig. 2). The two upper arms (choice arms, Fig. 2B) joined at 75 at the intersection, leaving an angle of 142Æ5 between each choice arm and the base arm (Fig. 2C), at the bottom of which the predatory mites were released. Each choice arm was connected airtight to an acrylic jar consisting of three tubular chambers (total length, 55 mm; inner Ø, 35 mm). The middle chamber (Fig. 2A) contained the leaf (odour) sample and was connected airtight to the other chambers with female joints. Both the inner and the outer chamber were sealed at both ends with gauze. The inner chamber was connected to the choice arm by a male joint (reaching approximately 20 mm into the choice arm), and the gauze prevented the predatory mites from accessing the chamber with the leaf sample upon reaching the end of a choice arm. The outer chamber was filled with activated charcoal to purify the air sucked into the Y-tube. A Y-shaped stainless steel wire, starting 20 mm inside the bottom end of the base arm, branching at the centre of the intersection of the three arms and leading to the end of either choice arm, was placed inside the glass Y-tube in equidistance to the inner walls. The wire was fixed by a perpendicular 20-mm-long extension held in place by an inert plastic piece fitted into a small hole of the upper wall at the bottom end of the base arm. At the end of either choice arm, the wire was centrally inserted into the gauze separating the inner chamber of the acrylic jar from the middle chamber containing the leaf (odour) sample. During tests, the olfactometer was placed on a black table flush with the surface, and a cold light source was centred above the intersection of the Y-tube. Air was drawn through the Y-tube using a mini-diaphragm-vacuum pump (Laboport Ò N 86 KN.18; KNF Neuberger, Freiburg, Germany) connected to the bottom end of the base Fig. 2. Y-tube olfactometer used for the binary-choice tests, consisting of a Y-shaped cylindrical glass tube (inner diameter, 40 mm, each arm of Y 130 mm long) with a wire inside functioning as railroad for the predatory mites (Ó Peter Schausberger). Each choice arm (B) was distally connected to a chamber (A) containing a leaf sample of a mycorrhizal or non-mycorrhizal plant. The predatory mites were singly released on the wire at the bottom end of the base arm (C), which was thereafter connected to a suction pump (not shown here).

4 444 P. Schausberger et al. arm. Air was sucked in at the ends of the choice arms and was drawn through the chambers containing the charcoal and the leaf samples, down the choice arms (flow rate, 2Æ5 l min )1 per arm), and, after meeting at the intersection, down the base arm, and left the Y-tube at the bottom of the base arm (flow rate, 5Æ0 l min )1 ). Each predatory mite was given a choice between the volatiles emanating from a leaf sample from a non-mycorrhizal and a mycorrhizal plant, both of which were either infested by T. urticae or non-infested. Infested plants were created by placing 30 gravid spider mite females onto each plant (randomly distributed among the trifoliate leaves). Since HIPV emission depends on the duration of infestation (e.g. Nachappa et al. 2006), we allowed the spider mites to feed and oviposit for 1, 3 or 6 days. Different plants were used for each of the four treatments (non-infested, 1, 3 and 6 days spider mite infestation). The spider mite eggs did not hatch within 6 days keeping the density of spider mites feeding on the plants constant between mycorrhizal and non-mycorrhizal treatments over time. The three youngest fully developed trifoliate leaves with their petioles were detached from the plants and immediately used for the olfactometer tests. Before tests, gravid P. persimilis females were randomly withdrawn from the rearing units, singly caged in closed acrylic cells (Schausberger 1997) and starved for approximately 20 h. Only females laying at least one egg during the starvation period were used for the choice test. Each predatory mite was released singly at the bottom end of the wire inside the glass tube, using a moistened fine brush, and then observed for 5 min at maximum whether it moved to an end of a choice arm. Predatory mites falling from the wire were discarded from analyses. Predatory mites not reaching the end of a choice arm within 5 min after release were judged as non-responsive. For each predatory mite, we recorded the responsiveness (reached the end of a choice arm or not) and, if responding, their decision (+M or )M odour source; left or right arm). Nine plant sample pairs, each consisting of a different set of a mycorrhizal and a non-mycorrhizal plant, were used for each binarychoice combination. Five to 10 predatory mites were tested per plant sample pair. After every five to 10 predatory mites, the chambers containing the leaf samples were disconnected from the Y-tube, the +M and )M leaf samples switched between arms, to avoid any inadvertent positional effect, and the wire cleaned with ethanol to avoid any influence of traces left by the predatory mites on the wire. All olfactometer tests were carried out within a couple of consecutive days in an air-conditioned room at 25 ± 1 C. MASS SPECTROMETRY Corresponding to the olfactometer tests, we evaluated the volatiles emitted by non-mycorrhizal and mycorrhizal plants that were either infested by T. urticae or not, resulting in four treatments. Infested plants were created by placing 30 gravid spider mite females onto each plant (randomly distributed among the trifoliate leaves), which were then allowed to feed and oviposit for 6 days (corresponding to the 6 days treatment in the olfactometer tests). For analysis, three fully developed trifoliate leaves with their petioles were detached from the plant, weighed and thereafter immediately placed in a cylindrical glass container (length, 149 mm; inner Ø, 27 mm) closed at both ends with plastic screw caps. The sample container was connected airtight via tubes (length, approximately 100 mm) to a container with the charcoal filter (Supelco SupelpureÔ HC; Sigma-Aldrich, Vienna, Austria) on the outer end (air in) and the mass spectrometer on the inner end (air out). Real-time trace gas analysis of the leaves was performed using the high-sensitivity PTR-TOF-MS instrument PTR-TOF-8000 (Ionicon, Innsbruck, Austria) (for details, Jordan et al. 2009). The volatile blend of each leaf sample (four samples per treatment) was measured twice, using two modes of airflow. Airflow was either set at 130 sccm (standard cm 3 min )1 ) for 15 min (high airflow) or set at 30 sccm for 10 min (reduced airflow), immediately following the high airflow measurement. For analyses, volatiles detected during high and reduced airflow were considered autocorrelated measurements. Instrument adjustments were 80 C inlet system heat and 4 s dwell time. After the last sample of each treatment, the sample container was cleaned with acetone and allowed to air-dry for at least 1 h before being loaded with the first sample of another treatment. Additionally, we measured the air inside the empty sample container before loading with the first sample of each treatment at an airflow of 130 sccm for 13 min to determine the presence of background volatiles. The PTR-TOF-MS instrument measures the mass spectrum every 4 s. Therefore, we calculated the mean mass spectrum for each measurement (lasting 15 and 10 min for the leaf samples and 13 min for the background) using the software TOF sampler (Ionicon). We checked the mass spectra for presence of 19 compounds, known to be part of the volatile blends of common bean plants influenced by spider mite attack and to have major or minor importance for predatory mite recruitment (Dicke et al. 1990; De Boer, Posthumus & Dicke 2004; De Boer et al. 2008; Zhang et al. 2009), and determined the ion yield at the relevant atomic masses. To obtain the net ion yields, we subtracted the ionyieldsinthebackgroundvolatilesfromtheionyieldsintheleafsample volatiles. With the method used, it is not possible to distinguish between two compounds (subsequently indicated by AND OR) with the same atomic mass. STATISTICAL ANALYSES SPSS 15.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. We used generalized linear models (GLM, binomial distribution, logit link) to assess the effect of plant sample within each choice situation (non-infested, infested for 1, 3 or 6 days) on responsiveness to and preference for the mycorrhizal or non-mycorrhizal volatile blend. Similarly, the effect of choice situation (non-infested, infested for 1, 3 or 6 days) on responsiveness of predatory mite females to volatile blends (reaching the end of an arm with the mycorrhizal or non-mycorrhizal odour source or not) was assessed by a GLM (binomial distribution, logit link) and Sˇidák post hoc comparisons using the choice situation with non-infested plants as the reference category. Within each choice situation, we used two-tailed binomial tests to assess whether the preference of the predatory mites for the mycorrhizal and non-mycorrhizal odour source and the left and right arm of the Y-tube, respectively, differed from random choice. The influence of mycorrhiza and spider mite infestation on the weight of the leaf samples used in mass spectrometry was analysed by a GLM (normal distribution, identity link). To test whether the total composition of the volatiles was affected by mycorrhiza and the spider mites, we used generalized estimating equations (Hardin & Hilbe 2003) (GEE, normal distribution, identity link, exchangeable autocorrelation structure between the two modes of airflow) with compound nested within mycorrhiza, within spider mites, and within the interaction of mycorrhiza with the spider mites as between-subject variables. Subsequently, to test which compounds differed among treatments, separate GEEs (normal distribution, identity link) were used to assess the effects of mycorrhiza and spider mite infestation as between-subject factors, and mode of airflow (within-subject factor with an exchangeable autocorrelation structure) nested within

5 Adaptive indirect below- and above-ground interaction 445 mycorrhiza and spider mite infestation on emission of single volatile compounds by bean plants, P. vulgaris. Results OLFACTOMETER TESTS In neither choice situation (non-infested, infested for 1, 3 or 6 days) did plant sample have an effect on responsiveness of P. persimilis (GLM, d.f. = 8 for each situation: Wald v 2 =5Æ126, P =0Æ744 for non-infested; Wald v 2 =1Æ773, P =0Æ987 for 1 day infested; Wald v 2 =12Æ263, P =0Æ140 for 3 days infested; Wald v 2 =1Æ818, P =0Æ986 for 6 days infested). Similarly, in neither choice situation did plant sample affect the preference of the predatory mites (GLM: Wald v 2 =0Æ732, d.f. = 7, P =0Æ998 for non-infested; Wald v 2 =2Æ601, d.f. = 6, P =0Æ857 for 1 day infested; Wald v 2 =2Æ451, d.f. = 7, P =0Æ931 for 3 days infested; Wald v 2 =5Æ231, d.f. = 8, P =0Æ733 for 6 days infested). Therefore, within each choice situation, the plant samples were pooled for subsequent analyses. The responsiveness of P. persimilis differed among choice situations (GLM: Wald v 2 =52Æ321, d.f. = 3, P <0Æ001; Fig. 3). Around 90% predators made a choice when exposed to volatile blends of plants infested for 6 days, which was significantly more (Šida k: P < 0Æ001 for every pairwise comparison) than the 30% predators making a choice in situations with non-infested plants or plants infested for 1 or 3 days. Responsiveness to non-infested plants and plants infested for 1 or 3 days was similar (Šidák: P >0Æ990 for every pairwise comparison). The number of P. persimilis females preferring the volatile blend from mycorrhizal plants was significantly higher than Fig. 3. Number of predatory mite females moving to the odour emanating from a leaf sample from a mycorrhizal or non-mycorrhizal plant, both of which were infested by spider mites for either 1, 3 or 6 days or non-infested, in a Y-tube olfactometer. Numbers inside parenthesis after spider mite treatment represent the number of females tested. Ns (non-significant) and *P <0Æ001 indicate the results of two-sided binomial tests assuming an equal distribution within spider mite treatments. the number of females preferring the volatiles from non-mycorrhizal plants in the choice situation with plants infested for 6 days (two-sided binomial test: asymptotic P < 0Æ001; Fig. 3). The females did not show a preference in the other choice situations (P = 0Æ307 for non-infested, P = 1Æ000 for 1day infested, P = 0Æ122 for 3 days infested). In neither choice situation was the response of the females influenced by side (left or right, two-sided binomial test: asymptotic P = 0Æ839 for non-infested; P = 1Æ000 for 1 day infested; P =0Æ701 for 3 days infested; P =1Æ000 for 6 days infested). MASS SPECTROMETRY Neither mycorrhiza nor spider mite infestation affected the weight of the leaf samples (gram, grand mean: 1Æ48 ± 0Æ09 SE) used for mass spectrometry (GLM; Wald v 2 =0Æ423, P =0Æ515 for spider mite infestation, Wald v 2 =0Æ482, P =0Æ488 for mycorrhiza, Wald v 2 =0Æ911, P =0Æ340 for the interaction). Compound nested within mycorrhiza (Wald v 2 =323Æ434, d.f. = 19, P < 0Æ001), nested within spider mites (Wald v 2 =290Æ948, d.f. = 19, P <0Æ001) and nested within the interaction of mycorrhiza with spider mites (Wald v 2 =316Æ272, d.f. = 19, P <0Æ001) had highly significant effects on the ion yields, indicating that the composition of the nineteen compounds differed among the four treatments ()M )SM, )M +SM, +M )SM, +M +SM; Fig. 4). GEEs for each single compound revealed that mycorrhiza and spider mite infestation significantly changed 13 and 14, respectively, of the measured 19 compounds of the volatile blend emitted by bean plants, P. vulgaris. Of 19 compounds, 13 were affected by the interaction between mycorrhiza and spider mite infestation (Table 1, Fig. 4). Three of the five compounds known to play a major role in predatory mite recruitment, b-ocimene, b-caryophyllene and methyl salicylate, were affected by the interaction between mycorrhiza and the spider mites (Table 1, Fig. 4). The only compound, of all major and minor compounds, totally unaffected by mycorrhiza, spider mites and or their interaction was linalool. (E)-4,8-Dimethyl-1,3,7-nonatriene was increased by spider mite infestation and decreased by mycorrhiza, but there was no interaction between the main effects. b-ocimene and b-caryophyllene were only released by spider mite-infested plants, and their amounts were clearly higher on mycorrhizal than non-mycorrhizal plants (Table 1, Fig. 4). Both non-infested and spider mite-infested plants constitutively emitted methyl salicylate. Emission of methyl salicylate was increased by spider mite infestation but decreased by mycorrhiza (Table 1, Fig. 4). The difference between nonmycorrhizal plants with and without spider mite infestation was larger with high airflow than reduced airflow, whereas the opposite was true for mycorrhizal plants. Mode of airflow nested within spider mite infestation had an effect on linalool and methyl salicylate, that is, more ions were detected in spider mite-infested plants tested with reduced flow, and b-ocimene, which was detected in non-mycorrhizal spider mite-infested plants tested with reduced but not high flow.

6 446 P. Schausberger et al. Fig. 4. Ions of single compounds detected in the volatile blend emitted by mycorrhizal (+M) and non-mycorrhizal ()M) bean plants infested by two-spotted spider mites for 6 days (+SM) or non-infested ()SM). N = 4 for each choice situation and mode of airflow: (H) high, (R) reduced. Compound numbers represent the following: 1, 2-butanone; 2, 1-penten-3-ol AND OR 3-pentanone; 3, 2- AND OR 3-methylbutanal nitrile; 4, indole; 5, 1-octen-3-ol AND OR 3-octanone; 6, 2-methylpropanal-O-methyl oxime; 7, p-mentha-1,3,8-triene; 8, b-ocimene; 9, (Z)-3-hexen-1-ol-acetate; 10, nonanal; 11, 2- AND OR 3-methylbutanal-O-methyl oxime; 12, hexyl acetate; 13, rose furan; 14, (E)-4,8-dimethyl-1,3,7-nonatriene; 15, methyl salicylate; 16, unknown; 17, linalool; 18, decanal; 19, b-caryophyllene.

7 Adaptive indirect below- and above-ground interaction 447 Table 1. Generalized estimating equations (normal distribution, identity link function) for the effects of mycorrhizal symbiosis (yes no), spider mite infestation (yes no) and mode of airflow (high reduced) nested within the main effects on volatile compounds emitted by bean plants, P. vulgaris. Major compounds in spider mite-induced bean plant volatile blends (Dicke et al. 1990), and P values <0Æ05 are highlighted in bold Mycorrhiza Spider mites Mycorrhiza* spider mites Compound Wald v 2 P Wald v 2 P Wald v 2 P 2-Butanone 1Æ171 0Æ279 4Æ296 0Æ038 92Æ593 <0Æ001 1-Penten-3-ol AND OR 3-pentanone 4Æ683 0Æ030 0Æ039 0Æ843 2Æ280 0Æ OR 3-Methylbutanal nitrile 168Æ777 <0Æ001 41Æ833 <0Æ001 67Æ028 <0Æ001 Indole 8Æ769 0Æ003 3Æ822 0Æ051 2Æ505 0Æ113 1-Octen-3-ol AND OR 3-octanone 3Æ828 0Æ050 0Æ442 0Æ506 0Æ339 0Æ561 2-Methylpropanal-O-methyl oxime 18Æ339 <0Æ001 7Æ101 0Æ008 18Æ922 <0Æ001 P-Mentha-1,3,8-triene 21Æ929 <0Æ001 15Æ951 <0Æ001 16Æ783 <0Æ001 (E)-b-Ocimene AND OR (Z)-b-ocimene 1Æ300 0Æ254 9Æ236 0Æ002 6Æ066 0Æ014 (Z)-3-Hexen-1-ol-acetate 1Æ465 0Æ226 7Æ795 0Æ005 45Æ945 <0Æ001 Nonanal 9Æ505 0Æ002 17Æ087 <0Æ001 24Æ845 <0Æ OR 3-Methylbutanal-O-methyl oxime 9Æ857 0Æ Æ620 <0Æ001 0Æ080 0Æ777 Hexyl acetate 13Æ634 <0Æ001 0Æ001 0Æ972 13Æ679 <0Æ001 Rose furan 13Æ203 <0Æ001 18Æ738 <0Æ001 12Æ561 <0Æ001 (E)-4,8-Dimethyl-1,3,7-nonatriene 50Æ437 <0Æ001 16Æ530 <0Æ001 1Æ628 0Æ202 Methyl salicylate 61Æ087 <0Æ001 59Æ359 <0Æ001 41Æ482 <0Æ001 Unknown 3Æ109 0Æ078 8Æ922 0Æ003 24Æ351 <0Æ001 Linalool 0Æ000 0Æ998 3Æ772 0Æ052 2Æ022 0Æ155 Decanal 14Æ073 <0Æ001 8Æ130 0Æ004 16Æ232 <0Æ001 b-caryophyllene 0Æ550 0Æ458 32Æ285 <0Æ001 7Æ391 0Æ007 Airflow (mycorrhiza) had a significant effect on (E)-b-ocimene AND OR (Z)-b-ocimene, methyl salicylate, linalool and decanal (Wald v 2 >4Æ5, P <0Æ034 for all); airflow (spider mites) had a significant effect on 2-OR 3-methylbutanal nitrile, methyl salicylate and decanal (Wald v 2 >5Æ1, P <0Æ025 for all); airflow (mycorrhiza) and airflow (spider mites) did not have an effect on any other compounds (P >0Æ05). Mode of airflow nested within mycorrhiza had an effect on methyl salicylate, that is, more ions were detected with reduced flow. Every compound of minor or unknown importance for recruitment of predatory mites from the distance was affected by mycorrhiza and or the spider mites and or their interaction (Table 1, Fig. 4). For example, mycorrhiza increased the amount of 1-penten-3-ol AND OR 3-pentanone and reduced the amount of indole and 1-octen-3-ol AND OR 3-octanone. Spider mite infestation increased the amount of 2-butanone emitted by mycorrhizal plants but decreased its emission on non-mycorrhizal plants. Spider mite infestation decreased the amount of 2- AND OR 3-methylbutanal nitrile and 2-methylpropanal-O-methyl oxime on mycorrhizal plants but increased the amount on non-mycorrhizal plants. Mycorrhizal plants without spider mites did not emit p-mentha-1,3,8-triene, but plants of all other treatments did. Mode of airflow nested within mycorrhiza had an effect on the emission of 2- AND OR 3-methylbutanal nitrile and decanal: both compounds were present with more ions in the blends of mycorrhizal plants tested with reduced flow. Mode of airflow nested within spider mites had an effect on decanal, that is, more ions were detected with reduced flow (Table 1, Fig. 4). Discussion Mycorrhizal symbiosis quantitatively and qualitatively changed the emission of HIPVs. HIPVs of mycorrhizal plants were more attractive to the predatory mite P. persimilis than HIP- Vs of non-mycorrhizal plants. Whether stronger attraction of P. persimilis was due to changed overall composition of the volatile blend or to changes in single compounds needs further investigation. However, of the major compounds known to attract P. persimilis (Dicke et al. 1990), mycorrhizal symbiosis most notably increased the difference in b-ocimene emission between spider mite-infested and non-infested plants, whereas it decreased this difference in methyl salicylate emission. b-ocimene on mycorrhizal plants and 2-butanone on non-mycorrhizal plants were the most abundant among the compounds influenced by herbivory. Three compounds were synthesized de novo upon herbivore attack: 2- AND OR 3-methylbutanal-O-methyl oxime was reduced by mycorrhiza, b-caryophyllene was not changed by mycorrhiza and only b-ocimene was affected by the interaction between mycorrhiza and the spider mites. These changes were similarly detected by both modes of airflow. Our study did not allow us to distinguish between the Z and E isomers of b-ocimene. However, the finding by Dicke et al. (1990) that (E)-b-ocimene is attractive whereas (Z)-b-ocimene is repellent for P. persimilis suggests that mycorrhiza mainly increased the E isomer. No compound was synthesized de novo by mycorrhization. Methyl salicylate and (E)-4,8-dimethyl- 1,3,7-nonatriene were constitutively released by all plants but quantitatively changed by mycorrhiza, by the spider mites or by both. In comparison with our study, Leitner et al. (2010) did not detect mycorrhiza-induced changes in the emission of

8 448 P. Schausberger et al. major HIPV compounds by M. truncatula but observed that mycorrhizal plants emitted higher amounts of minor compounds such as a-gurjunene. Fontana et al. (2009) observed that mycorrhizal herbivore-infested plants emitted lower amounts of terpenoids such as b-ocimene and b-caryophyllene than non-mycorrhizal herbivore-infested plants did. Whether mycorrhiza-induced changes in HIPVs of P. lanceolata (Fontana et al. 2009) and M. truncatula (Leitner et al. 2010) would affect third trophic-level natural enemies was not assessed. All three studies, Fontana et al. (2009), Leitner et al. (2010) and our study, suggest that the mycorrhizainduced changes in volatile emission are highly sophisticated. In any case, these changes are more than a mere increase in the amount of emitted volatiles due to improved nutrient uptake. The large qualitative differences in the manipulation of single HIPV compounds by mycorrhiza observed in our study, and the studies by Leitner et al. (2010) and Fontana et al. (2009) point at strong dependency of the outcome on the involved fungus, plant and herbivore species. As in previous studies showing that mycorrhizal symbiosis amends attractiveness and prey quality of the spider mites for the predatory mites (Hoffmann et al. 2009; Hoffmann, Vierheilig & Schausberger 2011a,b) and increases plant tolerance to herbivory by spider mites (Hoffmann et al. 2011a), this study suggests yet another compensating mechanism for herbivore enhancement, that is, change in HIPVs, leading to stronger attraction of the natural enemy of the spider mites, the predatory mite P. persimilis, to mycorrhizal than non-mycorrhizal plants. The change in HIPVs is adaptive for the predator, the plant and the fungus because it guides the predators to plants with more nutritious prey, consequently increasing predator fitness (Hoffmann, Vierheilig & Schausberger 2011b), and increases fitness of the mycorrhizal fungus due to the predators negative effect on the spider mites, relaxing herbivore pressure on the plant and in turn keeping up the fungal root colonization levels (Hoffmann et al. 2011a). Our study is a key example of a sophisticated compensating mechanism via enhanced recruitment of third trophic-level natural enemies in multi-trophic systems where mycorrhizal symbiosis promotes herbivory. Assuming that the quality (positive, neutral or negative) of the effect of mycorrhizal symbiosis on the herbivores is decisive, this should also apply to systems with herbivores having different feeding modes (Gehring & Whitham 2002) and where roots are colonized by multiple AM fungi, which is common in natural settings (e.g. Smith & Read 2008). However, the ubiquity of this mechanism regarding plant, fungus and herbivore species and context (in)dependency remains to be shown. In general, the effects of mycorrhizal symbiosis on above-ground herbivores are highly variable, ranging from positive to neutral to negative (e.g. Gehring & Whitham 2002; Bennett, Alers-Garcia & Bever 2006). For inter-trophic-level signalling reliability and honesty, enhanced third trophic-level natural enemy recruitment through mycorrhiza-induced changes in volatile emission is not expected in systems where mycorrhizal symbiosis negatively affects the herbivores. 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