REVIEWARTICLE

Volatile Organic Compound Mediated Interactionsat the Plant-Microbe Interface

Robert R. Junker & Dorothea Tholl

Received: 14 May 2013 /Revised: 3 July 2013 /Accepted: 10 July 2013 /Published online: 24 July 2013# Springer Science+Business Media New York 2013

Abstract Microorganisms colonize the surfaces of plant roots,leaves, and flowers known as the rhizosphere, phyllosphere,and anthosphere. These spheres differ largely in a number offactors that may determine the ability of microbes to establishthemselves and to grow in these habitats. In this article, wefocus on volatile organic compounds (VOCs) emitted by plants,and we discuss their effects on microbial colonizers, with anemphasis on bacteria. We present examples of how growth-inhibiting properties and mechanisms of VOCs such as terpe-noids, benzenoid compounds, aliphatics, and sulfur containingcompounds prevent bacterial colonization at different spheres,in antagonism with their role as carbon-sources that support thegrowth of different bacterial taxa. The notion that VOCs repre-sent important factors that define bacterial niches is furthersupported by results for representatives of two bacterial generathat occupy strongly diverging niches based on scent emissionsof different plant species and organs. Bacteria are known toeither positively or negatively affect plant fitness and to inter-fere with plant-animal interactions. Thus, bacteria and othermicrobes may select for VOCs, enabling plants to controlmicrobial colonizers on their surfaces, thereby promoting thegrowth of mutualists and preventing the establishment of detri-mental microbes.

Keywords Anthosphere . Epiphytic bacteria . Microbiota .

Niches . Plant volatiles . Phyllosphere . Rhizosphere .

Terpenes

Introduction

Humans and other animals sustain intimate relationshipswith diverse microbiota, as demonstrated by the recent char-acterization of the human gut microbiome (Huttenhoweret al. 2012; Spor et al. 2011). Similarly, plants do not interactwith a random set of microbial species, but rather, have theirspecific interacting partners among microbes (Bulgarelliet al. 2013). For example, bacterial communities colonizingleaf surfaces in a Brazilian tropical forest show remarkabledifferences across tree species and also, to a lesser extent,within species (Lambais et al. 2006). According to BaasBecking’s famous notion “everything is everywhere, but theenvironment selects” (1934), the composition of microbialcommunities is defined mainly by habitat filtering, and tosome extent by mechanisms of their dissemination (Belisleet al. 2012; Lachance et al. 2001; Lindström and Langenheder2012) rather than by the dispersal abilities of microbes (Beisneret al. 2006; Östman et al. 2010). Plant species and genotypes(Berendsen et al. 2012; Berg and Smalla 2009; Whipps et al.2008) differ strongly in a number of traits, and thus representselecting environments for the colonization by microorgan-isms. In individual plants, microbes inhabit tissue surfaces(and intercellular spaces) of organs above and below ground(flowers – anthosphere, leaves – phyllosphere, roots – rhizo-sphere), which offer niches for establishing different commu-nities (Fuernkranz et al. 2012; Ibekwe and Grieve 2004; Junkeret al. 2011). Moreover, with the age of plant organs, bacterialcommunities pass through successions (Shade et al. 2013) ofspecies composition, density, and diversity (Ibekwe and Grieve2004).

Robert R. Junker and Dorothea Tholl contributed equally to themanuscript.

R. R. JunkerDepartment of Organismic Biology, University Salzburg,Hellbrunnerstrasse 34, 5020 Salzburg, Austria

D. Tholl (*)Department of Biological Sciences, Virginia Tech,Blacksburg, VA 24061, USAe-mail: tholl@vt.edu

J Chem Ecol (2013) 39:810–825DOI 10.1007/s10886-013-0325-9

One of the most important traits influencing plant-microbe associations is the availability of nutrients (pri-mary metabolites such as sugars and amino acids) andtheir distribution over plant surfaces (Lindow and Brandl2003). For example, when exuded by roots, primary me-tabolites help establish communities of beneficial bacteriathat in turn positively affect plant health and inhibit thegrowth of other microbes (Berendsen et al. 2012; Berg andSmalla 2009). The effect of secondary or specialized me-tabolites on plant-associated microbial communities is lesswell understood. Many secondary metabolites includingterpenes, phenylpropanoids, benzenoids, nitrogen and sul-fur containing compounds are emitted as volatile organiccompounds (VOCs) from above and belowground planttissues where they have long and short distance effects ondifferent interacting organisms (Dudareva et al. 2006).Other plant VOCs of ecological value include C1 com-pounds, such as methanol and other short chain oxygen-ated compounds (Seco et al. 2007) and low molecularweight fatty acid derivatives (Dudareva et al. 2006). Therole of VOCs in flower-visitor interactions has been stud-ied intensively and their functions in interactions betweenplants and herbivores has been well established (Dickeand Baldwin 2010; Junker et al. 2010; Raguso 2008).Additionally, growth inhibiting effects of VOCs on mi-crobes have been described in numerous cases (Kalembaand Kunicka 2003; Tripathi et al. 2011). There now isgrowing evidence that plant VOCs constitute importanttraits in structuring plant-microbe interactions (DelGiudice et al. 2008; Huang et al. 2012; Junker et al.2011; Karamanoli et al. 2005). However, how the activi-ties of VOCs affect compositions of microbial communi-ties in vivo together with growth promoting effects ofVOCs as possible carbon sources requires furtherattention.

This review focuses on the impact of VOCs in establishingand structuring plant-microbe associations. Particularly, wepay attention to the role of plant-derived volatiles in interac-tions with bacteria, but also include VOC-mediated relation-ships with beneficial or pathogenic fungi and fungal likemicrobes where appropriate. For each of the three microbialhabitats (anthosphere, phyllosphere, rhizosphere), we describehow VOCs affect the establishment of microorganisms bygrowth inhibiting and promoting effects (Fig. 1), and wedefine how these factors may contribute to the niche develop-ment of microbes on plant tissues. Later, we discuss theunderlying molecular mechanisms of how plant VOCs affectthe growth of microbes (and vice versa), and present studiesshowing how microbes alter scent emissions by plants andhow these activities and direct emissions of volatiles by bac-teria affect interactions with animals. We conclude with futureperspectives to stimulate research on VOC-mediated interac-tions at the plant-microbe interface.

VOC-Mediated Interactions in the Rhizosphere

Roots release a diverse mixture of low and high molecularweight organic compounds, which make root tissues anutrient-rich environment for a diverse community of mi-crobes (Badri and Vivanco 2009). Soil-borne microorgan-isms associate with roots at different spatial scales they:colonize the rhizosphere, which is the narrow layer of soilimmediately surrounding the root; invade the intercellularspace as commensalistic or mutualistic endophytes (Hardoimet al. 2008; Reinhold-Hurek and Hurek 2011); or reside in roottissues as intracellular endosymbionts (Bonfante and Genre2010; Desbrosses and Stougaard 2011). Recent metagenomicsstudies of the Arabidopsis root microbiome have demonstrat-ed that a small number of bacterial taxa such asActinobacteriaand several Proteobacteria families are recruited as endo-phytes as opposed to other common taxa residing in the soiland rhizophere (Acidobacteria,Verrucomicrobia, andGemmatimonadetes) (Lundberg et al. 2012). The same studieshave provided evidence that the composition of root microbialcommunities depends on the soil environment but also isaffected by host-specific factors (Bulgarelli et al. 2012;Lundberg et al. 2012). Such host-specific traits include com-plex mixtures of primary metabolites (carbohydrates, aminoacids and other carboxylic acids) and specialized or secondarymetabolites. Volatile organic compounds (VOCs) producedby roots may exert short and long distance effects on microbesin the rhizosphere and endophytic compartment.Phytochemical studies have presented numerous examplesof the production of VOCs in roots or rhizomes includingterpenes and phenolics as well as low molecular weight alco-hols, aldehydes, and acids (e.g., Cecchini et al. 2010; DelGiudice et al. 2008; Kpoviessi et al. 2009; Steeghs et al.2004; Yeo et al. 2013). Bioactivities of root-specific volatileterpenes and phenolic compounds have been associated pri-marily with growth inhibiting effects based on in vitro assays(reviewed by Wenke et al. 2010). However, in contrast tostudies demonstrating roles of non-volatile secondary metab-olites as signals in mutualistic interactions or as defensesagainst microbial pathogens (Akiyama et al. 2005; Bressanet al. 2009; Oldroyd 2013; Osbourn et al. 2003; Papadopoulouet al. 1999; Weston and Mathesius 2013), not much directevidence has been provided on the functions of VOCs in situ.Terpenes and other root-derived VOCs most likely servemultiple roles as C-sources, defense metabolites and chemo-attractants. Degradation of monoterpenes such as geraniol bysoil microbial activity has been demonstrated (Owen et al.2007), and rhizobacteria such as Pseudomonas fluorescens andAlcaligenes xylosoxidans have been shown to metabolize α-pinene as their sole carbon source (Kleinheinz et al. 1999).Moreover, Del Giudice et al. (2008) found that bacteria asso-ciated with the roots of Vetiver grass (Vetiveria zizanioides) usesesquiterpenes of the essential root oil as a carbon source.

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Microscopic examinations and molecular identification of thebacteria associated with the cells that synthesize the sesquiter-penes revealed communities of strains belonging to familiescommonly found on root, leaf and flower surfaces, e.g.,Pseudomonadaceae and Enterobacteriaceae (Del Giudiceet al. 2008). Cultivation experiments showed that most of thesebacteria were able to grow with the sesquiterpenes as sole C-source. Consequently, catabolism of these compounds by bac-teria was found to strongly alter the plant-produced terpenebouquets and thus affect the final volatile blend (Del Giudiceet al. 2008). This example demonstrates clearly that bacteriaassociated with plants have the ability to use volatile secondarymetabolites as resources and alter their profile, which may have

consequences for interactions of plants with other organisms.Comparative studies with other plant species that produce rootessential oils, such as lemon grass (Cymbopogon spp.) orginger (Zingiber officinale), could provide further insight inthe role of terpenes as carbon sources in root-microbeassociations.

Defensive functions of volatile terpenes in the rhizospherehave been described primarily in direct and indirect interac-tions with root herbivores (e.g., see Erb et al. 2012; Johnsonand Nielsen 2012). For instance, the insect-induced sesqui-terpene, (E)-β-caryophyllene, is known to promote indirectdefense responses inmaize roots by recruiting entomopathogenicnematodes (Turlings et al. 2012). In Arabidopsis roots, diterpene

Fig. 1 Volatile organic compound mediated interactions at the plant-microbe interface. Right panel: Flowers, leaves, and roots emit a varietyof volatile organic compounds that inhibit specific bacterial strains intheir growth and thus prevent the colonization of these bacteria in theanthosphere, phyllosphere, or rhizospere, respectively. a Effect of floral(E)-β-caryophyllene emission on seed production of Arabidopsisthaliana wild-type Col-0 and (E)-β-caryophyllene synthase mutants(tps21-1, tps21-2) after inoculation of inflorescences with Pseudomo-nas syringae pv. tomato DC3000 (Pst DC3000), a bacterial pathogen ofbrassicaceous plants. The average weight of 100 seeds of each line isshown. Results represent mean values±SE (N=5). Different lettersindicate significant differences (analysis of variance, Tukey’s HSD test(P<0.05)). Figure modified from Huang et al. (2012). b Results of agar-diffusion assays using bacteria isolated from leaves and petals ofSaponaria officinalis (image) and volatiles either emitted from flowersor leaves. Diameter of inhibition zones was dependent on the substance

tested, the origin of bacteria (leaf or petal) and the interaction of bothfactors substance×origin (ANOVA: substance: F4, 740=142.0***, ori-gin: F1,740=9.5**, substance×origin: F4, 740=3.0*). Different lettersdenote differences in the growth-inhibiting effect of volatiles accordingto Tukey HSD. Asterisks denote differences in the growth-inhibitingeffect of individual volatiles on bacteria from different origins(** P<0.01). Mean±SE of inhibition zones is shown. Figure modifiedfrom Junker et al. (2011). c Volatile monoterpenes, sesquiterpenes, andditerpenes with known or putative antibacterial activity produced byArabidopsis roots. A schematic cross section of a root is shown withconcentric layers of different cell types. Volatiles are produced in theepidermis (orange – 1,8-cineole), cortex and endodermis (yellow andlight red – (Z)-γ-bisabolene), or the stele (pericycle in red – rhizathaleneand DMNT). Left panel: Plant and bacterial volatiles with growthpromoting effects. Footnote: 1(Sy et al. 2005); 2(Del Giudice et al.2008); 3(Leroy et al. 2011)

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hydrocarbons named rhizathalenes exhibiting an unusual tricy-clic spiro-hydrindane structure recently have been identified(Vaughan et al. 2013). Similar to other diterpene hydrocarbons,rhizathalenes exhibit lower volatility (lower vapor pressure) thanmonoterpenes and sesquiterpenes, but their emission is readilydetected at room temperature in the headspace of Arabidopsisroot tissue (Vaughan et al. 2013). Using rhizathalene synthasemutants, it was shown that these compounds contribute to con-stitutive chemical defenses against root feeding insects such asthe opportunistic root herbivore Bradysia (Vaughan et al. 2013).While direct evidence is yet missing, it is possible thatrhizathalenes and structurally related diterpenes found in otherArabidopsis ecotypes (Wang and Tholl, unpublished results)function as genotype-specific chemical factors that fine-tuneecotype-specific root microbiomes. Arabidopsis roots also pro-duce small amounts of the monoterpene 1,8-cineole and thesesquiterpene (Z)-γ-bisabolene (Chen et al. 2004; Ro et al.2006; Steeghs et al. 2004; Tholl and Lee 2011), both of whichare known for their antimicrobial effects as single compounds orconstituents of essential oil mixtures (Aligiannis et al. 2004;Kalemba and Kunicka 2003; Simic et al. 2005; Vilela et al.2009). It should be noted that the terpene synthases that catalyzethe formation ofmonoterpenes, sesquiterpenes, and diterpenes inArabidopsis roots, are largely expressed constitutively, whichresembles expression profiles in Arabidopsis floral tissues butdiffers from those of related genes with induced expression toherbivory and pathogen infection in leaves (Attaran et al. 2008;Chen et al. 2003; Huang et al. 2010; Tholl et al. 2005; Tholl andLee 2011). Moreover, the composition of volatile terpene mix-tures in Arabidopsis roots shows no overlap with that of above-ground tissues. These differences may reflect the importance ofmaintaining distinct chemical profiles in reproductive and be-lowground tissues throughout the life cycle of the plant in orderto mediate attractant, repellent, or defensive activities in interac-tion with different microbiota in floral and root tissues.Bodenhausen et al. (2013) observed that leaves and roots ofnaturally grown Arabidopsis share many bacterial genera butin different proportions. Specifically, leaf and root endophyticcompartments were found to be similar in bacterial communityrichness and diversity, but different in community composition.

Gene transcript maps of Arabidopsis roots indicate thatvolatile terpenes are produced in specific cell types (Tholland Lee 2011) with rhizathalenes being primarily biosynthesizedin the stele while 1,8-cineole and (Z)-γ-bisabolene are made inthe epidermis and the cortex/endodermis, respectively (Fig. 1).Cell type specificity is a recurring feature in the formation ofterpenes in roots (Field and Osbourn 2008), and may optimizemetabolic investments among different cells. Since volatilecompounds diffuse between cells, concentric overlapping gra-dients can be established that may provide distinct cues forrecruiting or confining opportunistic and competent bacterialtaxa (Hardoim et al. 2008) to specific niches in the endosphereor the vascular tissue. Mutant-based studies in different plant

model systems that manipulate root volatile blends in vivo arerequired to determine the effects of volatiles on the rootmicrobiome and their interaction with beneficial and patho-genic bacteria and fungi. Such a reverse genetic attemptrecently has been made to decipher the role of the irregularacyclic C11-homo(nor)-terpene 4,8-dimethylnona-1,3,7-triene(DMNT) in Arabidopsis roots. DMNT and its larger analog4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) are com-mon plant volatiles emitted constitutively from floral tissuesand in induced responses of foliage to herbivore and microbialpathogen attack (Tholl et al. 2011). Arabidopsis roots emitDMNT constitutively in trace amounts but show increasedtransient emission of this terpene in the early phase of infec-tion with the oomycete root rot pathogen Pythium irregulare(Sohrabi and Tholl, unpublished). In vitro assays that applieddifferent concentrations of DMNT showed inhibitory effectson Pythium oospore germination and mycelial growth,suggesting that formation of infection hyphae on the rootsurface is slowed by the presence of the volatile compound.In addition, mutants impaired in DMNT formation were moresusceptible to the pathogen in soil-based assays (Sohrabi andTholl, unpublished). These studies further support the notionthat volatile terpene hydrocarbons can be functionally activein the rhizosphere. While the specific mode of action ofDMNTon Pythium spores and mycelium currently is not wellunderstood, interference of this terpene with cellular mem-branes and proteins (see section on molecular targets of VOCsbelow) is likely to occur.

Plant VOC-Mediated Interactions in the Phyllosphere

Plant foliage represents not only a large biotic habitat forbacteria – known as the phyllosphere (Vorholt 2012) – butalso strongly contributes to global VOC emission (Guentheret al. 1995). Plant carbon emissions are estimated to be above1,000 Tg year-1 worldwide, most of it bound in isoprene,monoterpenes, methanol, and other volatile organic com-pounds (Galbally and Kirstine 2002; Guenther et al. 1995).Leaves emit a large variety of molecules ranging from smallC1-compounds, such as methane, to more complex compounds,such as sesquiterpenes and fatty acid derivates (lipoxygenasepathway), the latter constituting typical “green leaf volatiles”(Matsui 2006).

One important source for epiphytic microbial communi-ties are airborne bacteria that reach densities up to 10,000viable CFU m−3 air (Fahlgren et al. 2010). However, Vokouet al. (2012) demonstrated that only a small proportion ofbacterial strains carried by air have the ability to establishthemselves in the phyllosphere, suggesting that bacteria re-quire adaptations to this habitat (Bulgarelli et al. 2013;Vorholt 2012). The ability to cope with secondary metabolitesincluding volatile compounds that have growth-inhibiting

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effects on some strains may be an important adaptation ofepiphytic bacteria. Accordingly, volatile compounds emittedby some coniferous species inhibit the growth of airbornebacteria (Gao et al. 2005) and thus prevent their establishmenton needles. Besides direct effects on the epiphytic communi-ties, the density of bacteria in the air surrounding the coniferstands was strongly reduced (Gao et al. 2005). Particularlyefficient in inhibiting bacterial growth were the monoterpeneslimonene and β-pinene and three compounds with aldehydefunctional groups (Gao et al. 2005). Likewise, aldehydes suchas acetaldehyde, benzaldehyde, and cinnamaldehyde havebeen found to inhibit the growth of epiphytic bacteria atcomparably low doses (Utama et al. 2002). In comparison toalkanes, aldehydes (and alcohols) are considered to be moreeffective in disturbing microbial membranes and their associ-ated proteins (see section on molecular targets of VOCs be-low). In this context, it is interesting to note that trans-2-hexanal, an aldehyde derived from the lipoxygenase pathway,possesses strong growth-inhibiting activity against phytopath-ogenic bacteria, and it is released in high amounts after path-ogen attack (Croft et al. 1993). As green leaf volatiles arestrongly emitted after the damage of plant tissues that after-wards are no longer mechanically protected by cuticles, theemission of these compounds may be a response not only toprotect plants against herbivores (Unsicker et al. 2009) butalso to avoid infections by pathogens (Matsui 2006).

Terpenoids, phenylpropanoid and benzenoid compoundsare dominant volatile constituents of essential oils with dem-onstrated growth-inhibiting properties (e.g., Ashour 2008;Rossi et al. 2007), and they may strongly influence bacterialcolonizers of the phyllosphere. This notion is supported byresults that demonstrate that ethanol extracts of Salviaofficinalis leaves have stronger growth-inhibiting effects thanextracts from stems and flowers of the same species(Velickovic et al. 2003). Aromatic plants such as S. officinalisare common in the Mediterranean region, and the epiphyticbacterial communities of their leaves have been investigatedunder consideration of the content of secondary metabolites.The density of epiphytic bacteria isolated from 19 plant speciesshowed a pronounced inter-specific variability despite the sameenvironmental growth conditions (Karamanoli et al. 2005). Theimportance of volatile essential oil compounds for the leafbacterial colonization of these plants was demonstrated by thefinding that the density of bacteria decreases with an increase inoil content (Karamanoli et al. 2000, 2005; Yadav et al. 2005).This also is true for ice nucleation active bacteria (Karamanoliet al. 2005) that cause severe frost damages (Lindow et al.1978). Essential oil constituents may either act additively onthe survival and growth of bacteria or synergistically(Karamanoli et al. 2000; Kubo et al. 1992), which may reducethe chance of bacteria to adapt to individual compounds andthus circumvent the chemical defense of plants. Since manyaromatic plants produce and store essential oil compounds in

glandular trichomes, these metabolites are not homogenouslydistributed over leaf surfaces, which is reflected in the distribu-tion of bacteria that do not grow in the direct proximity oftrichomes (Karamanoli et al. 2000). In contrast to leaves ofOriganum vulgare that possess glandular trichomes, bacteriahave been found to be relatively homogenously distributed overthe surface of bean leaves that lack these structures (Karamanoliet al. 2005). Nonetheless, a more detailed examination ofbacteria associated with glandular trichomes of O. vulgare hasrevealed that some bacteria (e.g., members of theEnterobacteriaceae and Pseudomonadaceae) seem to be spe-cialized on these habitats because they tolerate even high con-centrations of essential oils and indeed may benefit from thesecompounds (Karamanoli et al. 2012). Despite strong evidencethat volatile components of essential oils decrease the abun-dance of epiphytic bacteria, other studies in Mediterraneanplant communities have shown that the density and diversityof bacteria on leaves of aromatic plants exceeds those found onnon-aromatic plants (Yadav et al. 2005, 2008), whichchallenges the generality of the antimicrobial effect of thesemetabolites.

The results summarized above suggest that the ability ofbacteria to tolerate secondary metabolites emitted by plantsurfaces opens niches, which are otherwise not accessible tobacteria due to the inhibitory properties of these compounds.Likewise, the ability to metabolize plant resources that arenot available to other bacterial strains may be advantageousfor a successful colonization of these surfaces that have arather limited nutrient supply (Wilson and Lindow 1994).VOCs may constitute a reliable carbon source, which is alimiting factor in the phyllosphere (Lindow and Brandl2003). For instance, the emission of methanol is highlypredictable, as it is a byproduct of the stabilization of primarycell walls, and thus is released by plants in amounts as highas 100 Tg y−1 (Galbally and Kirstine 2002). Accordingly,methylotrophy, the ability to metabolize methanol, repre-sents a selective advantage for the establishment of bacteriain the phyllosphere (Sy et al. 2005). Bacteria from the genusMethylobacterium – also known as pink-pigmented faculta-tive methylotrophic bacteria (PPFM) – are common colo-nizers of plant surfaces that often reach high densities(Madhaiyan et al. 2005; Wellner et al. 2011). These bacteriaare not only able to utilize methanol as C-source but alsoother C1 compounds such as formaldehyde and multicarboncompounds (C2-4) (Madhaiyan et al. 2005, 2009), suggestingadaptation to this nutritional niche. Methylobacterium bac-teria are thus commonly found in the phyllosphere wherethey facultatively assimilate methanol produced and emittedby plants or use it as an energy supply (Sy et al. 2005). Forsome bacteria, methanol even represents the sole C-source(Fall and Benson 1996). The importance of the methylotrophyfor Methylobacterium extorquens has been demonstrated byusing mutants with a non-functional methanol dehydrogenase

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that oxidizes methanol to formaldehyde or with the inability tooxidize formaldehyde to CO2 due to a lack of the cofactortetrahydromethanopterin (Sy et al. 2005). Bacterial commu-nities with an initially balanced composition of wildtype andmutant strains showed a dramatically uneven distribution infavor of wildtype bacteria a few days after colonization indi-cating that bacteria metabolizing methanol as C-sourcesucceeded much better in colonizing plant surfaces than thoseimpaired in methanol catabolism (Sy et al. 2005). The effi-ciency of using methanol by Methylobacterium extorquensbacteria has been demonstrated by Abanda-Nkpwatt et al.(2006): Seedlings of Nicotiana tabacum emitted methanol at0.005 to 0.01 ppbv (parts per billion by volume) in thepresence ofM. extorquens, while plants not colonized by thesebacteria showed much higher emissions (0.4 – 0.7 ppbv).Methanol emission is not homogenously distributed on sur-faces; stomata show the highest emissions of methanol, aswell as the highest density of PPFMs (Abanda-Nkpwatt et al.2006; Fall and Benson 1996), indicating that stomata providea niche for methylotrophic bacteria. In many cases, PPFMs areno commensalistic colonizers of plants but support the germi-nation and growth of many plant species (Madhaiyan et al.2005), thus suggesting a mutualistic relationship betweenbacteria and plants supported by volatile nutrients.

VOC-Mediated Interaction in the Anthosphere

Flowers offer distinct micro-habitats, namely petals, stylesand stigmas, stamens, and pollen and nectar that are colonizedby microorganisms (Huang et al. 2012; Junker et al. 2011;Fridman et al. 2012; Fuernkranz et al. 2012). Microbes thatinhabit these generative plant parts experience fundamentallydifferent conditions from those that colonize other above-ground plant tissues. Accordingly, bacterial communities iso-lated from flowers and those established on leaves showmarked differences in the diversity of bacteria and the identityof community members (Junker et al. 2011). One strikingdifference between flowers and leaves is the permanence ofthe habitat. Flowers are ephemeral structures, usually with amuch shorter lifespan than that of leaves, which may not besufficient for some – slowly growing – bacteria to establish.Nonetheless, the microbiomes associated with apple flowersshowed a pronounced variability within the few days offlowering (Shade et al. 2013). The succession was mostlycharacterized by a set of taxa that persisted during the lifespanof the flowers but altered in their relative proportions to eachother (Shade et al. 2013). The short lifespan of flowers may becompensated by more frequent visits of diverse animals(Wardhaugh et al. 2012) and thus by a more directed trans-mission from one habitat to another with the same properties.Flower fidelity of various pollinators is pronounced due toinnate preferences or associative learning (Wright and Schiestl

2009). It has been shown that flower visitors transport yeastbetween flowers (Belisle et al. 2012), and that the compositionof yeast communities in nectar is partly explainable by thevisitation of different vectors (Lachance et al. 2001).

A major difference between the phyllosphere and theanthosphere is the distinct profiles of secondary metabolites.Flowers emit a variety of substances from diverse biochem-ical pathways including aromatics, terpenoids, and aliphatics(Knudsen et al. 2006), but they also accumulate non-volatilemetabolites such as alkaloids, phenolics, and proteins in theirnectar (Adler 2000). Many of these compounds have beenshown to be antimicrobial in other contexts than floral ecol-ogy (Cowan 1999; Karapinar and Aktug 1987). However,direct evidence that volatile and non-volatile floral second-ary metabolites filter the bacterial taxa that are able to colo-nize floral surfaces and nectar and thus potentially serve asdirect defense remains scant. Nectarins, proteins associatedwith nectar, that generate high levels of hydrogen peroxidehave been proposed to serve as protective means againstmicrobial colonization (Carter and Thornburg 2004).Additionally, the presence of alkaloids, phenolics, terpe-noids, and saponins in nectar has been interpreted as adap-tation against microbial infestation (Adler 2000; Kessler andBaldwin 2007). Pollen, another pollinator reward, but mostimportantly the vector for male gametes (and also bacteria,Fuernkranz et al. (2012)), may also require antimicrobialproperties to remain viable. Accordingly, it has been sug-gested that pollen odors evolved as defense against pathogens(Dobson and Bergström 2000), a hypothesis finding supportin a study showing that the methanol extract of pollen fromone or some unknown plant species inhibits the growth of 13bacterial pathogens even in high dilutions (Basim et al. 2006).

The most conclusive evidence for the growth inhibitingeffect of floral volatiles and its adaptive value for plantreproductive success comes from a study of the function ofthe common floral volatile sesquiterpene, (E)-β-caryophyllene(Knudsen et al. 2006), which is produced in the stigmas ofArabidopsis thaliana flowers. Huang et al. (2012) usedArabidopsis mutant lines that lacked (E)-β-caryophyllenesynthase activity, and compared the growth of Pseudomonassyringae – a pathogen of A. thaliana – on stigmas and thesubsequent seed set of the investigated plants (Fig. 1a). Theauthors demonstrated clearly that high levels of (E)-β-caryophyllene reduced the growth of P. syringae on the stig-mas, which translated into more viable seeds. The reductionin bacterial density was due to a direct growth inhibitingeffect of (E)-β-caryophyllene rather than an indirect defenseresponse induced by the volatile compound (Huang et al.2012). Therefore, that study strongly suggests that the emis-sion of floral volatiles affects the colonization of phytopatho-genic bacteria that reduce plant fitness.

Apart from effects on individual strains of pathogenicbacteria, another study implies that floral scents play a role

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in the structuring of bacterial communities colonizing petals.Junker et al. (2011) found that bacterial communities arerather plant organ specific than plant species specific: Thecompositions of cultivatable bacteria isolated from Saponariaofficinalis and Lotus corniculatus flowers were more similarto each other than those of the communities found on flowersand leaves of the same species. Moreover, the bacterial com-munities on petals of S. officinalis and L. corniculatus werenot only taxonomically different from those found on leavesbut were also less diverse (Junker et al. 2011) withEnterobacteriacea dominating the floral communities and witha limited presence of Pseudomonas strains. Nonetheless, den-sities of bacteria (cfu cm-2) were unaffected by plant organ(Junker et al. 2011). Agar diffusion assays revealed that VOCsemitted by leaves of S. officinalis did not severly inhibit thegrowth of bacteria. However, the volatiles emitted by flowers– phenylacetonitrile and 2-phenylethyl alcohol – had stronggrowth inhibiting effects on bacterial strains exclusively foundon leaves (Fig. 1b), while bacteria isolated from flowers wereless affected by these scents (Junker et al. 2011). These datasuggest that strains tolerating and potentially metabolizingfloral volatile compounds are able to colonize flowers, whilethose strains that are inhibited in their growth by the samecompounds cannot use this niche. Adaptation to niches shapedby volatile organic compounds is an important aspect inunderstanding the composition of plant microbial communi-ties and is further explored in the following section.

VOCs as Niche Dimensions

An ecological niche may be defined as n-dimensionalhypervolume where each resource and each other bioticand abiotic factor represent one niche dimension (Hutchinson1957). Each species occupies a specific n-dimensionalhypervolume, which is its niche (Hutchinson 1957). The factorscharacterizing roots, leaves, and flowers may be viewed as suchdimensions that determine whether bacterial strains are able tocolonize these surfaces, i.e. whether they find a niche that allowsthem to establish themselves and to grow (Bulgarelli et al. 2013).For example, bacteria that differ in their ability to utilize differentC-sources occupy different niches, accounting for differences incommunity structure when plant surfaces provide variablesources of carbon (Yadav et al. 2008). As reviewed in theprevious sections, volatile compounds emitted by plants repre-sent significant factors in determining the presence or absence ofindividual strains and in shaping bacterial communities, and are,thus, important niche dimensions that affect the microbiomesassociated with these habitats. Theoretically, bacterial strains thatare able to tolerate growth-inhibiting volatiles and colonizesurfaces emitting these volatiles have a wider niche dimensioncharacterized by these compounds than bacteria that are inhibitedin growth by even small concentrations of the same compound.

Junker et al. (2013) proposed a statistical approach toquantify species-specific trait-widths ⟨Si⟩ that are proportion-al to the expansion of a single niche-dimension. Small valuesindicate a specialization on a trait, i.e., the species uses only asmall fraction of the potentially available spectrum of thecharacteristics of that trait (e.g., low to high emission rates ofa volatile compound). Large values indicate a generalizedoccupation of this dimension suggesting that the trait underconsideration does not strongly affect the distribution of thespecies on different habitats, i.e., the bacteria can be foundon a surface regardless of the emission of the volatile underconsideration. The product of all trait-widths ⟨Si⟩ was de-fined as trait-volume Vi representing a quantitative measureof Hutchinson’s (1957) n-dimensional hypervolume (Junkeret al. 2013). We used the dataset of Junker et al. (2011) on thedistribution of bacterial genera on petals and leaves of Lotuscorniculatus and Saponaria officinalis, as well as the scentemission of these bacterial habitats to quantify the trait-widths ⟨Si⟩ of two bacterial genera based on the emissionrates [ng/h/g dryweight] of individual benzenoid compounds,fatty acid derivates, monoterpenoids, sesquiterpenoids, andnitrogen containing compounds. As an example, we chosethe bacterial genera Serratia (Enterobacteriaceae) andPseudomonas (Pseudomonadaceae) because representativesof these genera were nearly exclusively distributed on petalsor leaves of both plant species tested, respectively, andaccounted for most of the plant organ-specific differences inbacterial colonization (Junker et al. 2011). For each of thevolatile compound classes, we established a distance matrixwith Euclidean distances djk between habitat j and k (i.e. petalsand leaves of the two plant species) based on quantitativedifferences in the emission of individual compounds belong-ing to that chemical class. Using the weight wjk ¼ aij

Ai⋅aikAi

with

aij and aik as the observed number of colony forming units ofgenus i on habitat type j and k, respectively, and Ai as the total

number of colony forming units of genus i on all plant parts,

which corrects for different absolute numbers of colony

forming units of the genera on the four habitats, we calculated

the weighted mean distance ⟨Di⟩ ¼ ∑ wjk ⋅djk� �

∑wjk, followed by

the calculation of the standardized weighted mean distance

⟨Si⟩ ¼ ⟨Di⟩−min djkð Þð Þmax djkð Þ−min djkð Þð Þ (trait-width). Thus, ⟨Si⟩ is propor-

tional to the similarity of the colonized habitats based on thequantitative emission of benzenoid compounds, fatty acidderivates, monoterpenoids, sesquiterpenoids, or nitrogen con-taining compounds, respectively, corrected for the quantitativedistribution of the representatives of the genera Serratia andPseudomonas. ⟨Si⟩ was compared to standardized mean ran-

dom distances Ri that represent the trait-width expected ifbacteria colonized the four habitats randomly, i.e., withoutany effects of habitat filtering e.g., due to growth-inhibiting

816 J Chem Ecol (2013) 39:810–825

volatiles. We randomly drew djk-values from the pool of djk-values, with n=number of djk used for the calculation of ⟨Si⟩ ofgenus i, and calculated their mean and standardized it as above(see ⟨Di⟩ and ⟨Si⟩), resulting in standardized random distancesRi. This procedure was replicated 1,000 times for each genus iand each compound class; the mean of the 1,000 Ri was used

as the standardised mean random distance Ri . If compound

class-specific ⟨Si⟩ did overlap with<5 % of the Ri -values(n=1,000) for the same compound class, the difference wasregarded as significant. For a detailed description of the sta-tistical approach, please refer to Junker et al. (2013).

The trait-volume Vi of Serratia (niche width) based onvolatile emission of the plant parts was larger than the onecalculated for representatives of the genus Pseudomonas(2·107 fold, Fig. 2), indicating that Pseudomonas strainswere much more restricted in their distribution by VOCsthan Serratia strains. The difference was due mainly to thesmall trait-widths ⟨Si⟩ based on benzenoids and nitrogencontaining compounds of Pseudomonas that significantlydiffered from a random expectation (Fig. 2). These smalltrait-widths ⟨Si⟩ indicate that Pseudomonas species colonizeonly those surfaces that are highly similar in the emission ofthese chemical classes, i.e., in this case no emission or onlytrace-amounts (compare to Junker et al. 2011). In contrast,representatives of the genus Serratia did not select habitatswith defined emission rates of any of the chemical classes,

reflected in the fact that ⟨Si⟩ did not deviate from Ri (Fig. 2).Generally, it becomes evident from the spider charts vi-

sualizing the trait-volumes Vi of the two bacterial genera(Fig. 2) that the representatives of both genera have differentVOC-related requirements on their habitats, and thus occupydifferent niches. Serratia spp. are able to establish them-selves in habitats that are not suited for other strains due togrowth-inhibiting plant volatiles. This notion is supported byagar-diffusion assays demonstrating that bacteria from aSerratia strain were significantly less inhibited in theirgrowth by any of the tested volatiles (including benzenoidsand nitrogen-containing compounds) than other bacterialstrains with a less generalized distribution among plant sur-faces (Junker et al. 2011). Unfortunately, such data areunavailable for representatives of the genus Pseudomonas,but the absence of these bacteria on petals of Saponariaofficinalis, the plant organs with the strongest emission ofbenzenoids and nitrogen-containing compounds, both quan-titatively and qualitatively (Junker et al. 2011), supports thehypothesis that these compounds strongly influence theniche of Pseudomonas strains.

Niche partitioning among bacteria due to secondary me-tabolites of plants also may explain the finding thatPseudomonas putida bacteria were found in high densitiesclose to glands producing monoterpenoid-dominated essentialoils in Origanum vulgare plants (Karamanoli et al. 2012).

Further experiments demonstrated that this bacterial specieswas not affected in its growth by the essential oils in agardiffusion assays and was able to establish much larger popu-lations on O. vulgare leaves than other bacteria that are com-monly associated with plant surfaces (Karamanoli et al. 2012).These results correspond to large niche-dimensions ofPseudomonas strains based on monoterpenoids (Fig. 2,Junker et al. 2011). This is not to say that volatiles (individualcompounds or products of different pathways) are the sole ormost important niche-dimension determining factors, but theresults summarized in this review and the data presented hereindicate that these compounds are important determinantsshaping epiphytic bacterial communities.

Molecular Targets of Plant VOCs in Microbes

Given the complexity and structural diversity of plant vola-tile mixtures, elucidating their mode of action with respect tospecific targets in microbes has remained a challenge. Mostknowledge on how VOCs affect microbial cells has beengained from components of essential oils with antimicrobialactivity. Because many VOCs have lipophilic properties,their primary mechanism of interfering with microbial cellsis to interact with the cytoplasmic membrane (Radulovicet al. 2013). In many cases, VOCs affect the structure andstability of the phospholipid bilayer resulting in a distur-bance of the membrane integrity and an increase in ionpermeability. For instance, the monoterpene carvacrol, anessential oil constituent with potent microbial growthinhibiting activity, increases membrane fluidity and perme-ability by inserting between the acyl chains of the phospho-lipids. This effect leads to an efflux of ions and ATP, and themembrane potential and pH gradient are disturbed (Ulteeet al. 1999, 2000). Similar effects have been demonstratedfor the carvacrol isomer, thymol, (Cristani et al. 2007; Walshet al. 2003), and many other hydroxylated monoterpenes andphenylpropanoids as summarized in a comprehensive reviewby Radulovic et al. (2013). While essential oil componentswith polar moieties such as aldehyde or alcoholic groups areusually more potent in disturbing membranes or interactingwith membrane bound proteins, terpene hydrocarbons such asγ-terpinene and limonene, which are more representative ofvolatiles emitted from non-storage pools in flowers andleaves, have also been reported to interfere with membraneproperties (Cristani et al. 2007; Di Pasqua et al. 2007).Transcriptome and proteome analyses of yeast and plant orhuman bacterial pathogens further indicate that VOCs altermembrane protein compositions and change core metabolic orregulatory processes inside the cell (Di Pasqua et al. 2010;Horvath et al. 2009; Parveen et al. 2004) . For instance,exposure of yeast to 0.02 % α-terpinene induced genes in-volved in the biosynthesis of ergosterol, a major sterol of

J Chem Ecol (2013) 39:810–825 817

fungal membranes, indicating inhibitory effects of the mono-terpene on the formation of ergosterol similar to effects ob-served by exposure to thymol and carvacrol (Parveen et al.

2004; Ahmad et al. 2011).α-Terpinene also induced transcrip-tional changes of genes related to lipid metabolism, cell wallstructure and function, detoxification, and cellular transport.

Fig. 2 Niches of bacteria belonging to the genus Serratia (upper chart)and Pseudomonas (lower chart) based on volatile organic compoundsvisualized as trait-volumes Vi. For each bacterial genus and volatilederiving from a common biochemical pathway, the observed trait-widths ⟨Si⟩ and the trait-widths Ri are shown that were expected ifbacteria randomly colonized plant surfaces with characteristic scentprofiles. Colored areas denote the observed trait-volumes Vi determinedby trait-widths ⟨Si⟩. Small ⟨Si⟩ -values indicate a specialization on thattrait, i.e., the genus uses only a small fraction of the potentially availablespectrum of emission rates of the respective compounds; large valuesindicate a generalized occupation of this dimension suggesting that the

trait under consideration does not strongly affect the distribution of thegenus among different habitats. Expected trait-volumes Vi based on Ri

are framed by a black line. Significant negative deviations of theobserved ⟨Si⟩ from the expected trait-widths Ri are indicated by aster-isks (*** P<0.001). Significantly smaller observed than expected trait-widths (⟨Si⟩ < Ri ) indicate that bacteria display some degree of special-ization regarding the characteristics of scents. Data on bacterial coloni-zation of petals and leaves of Lotus corniculatus and Saponariaofficinalis as well as the scent emission of these bacterial habitats areextracted from Junker et al. (2011). The statistical approach described inJunker et al. (2013) was used to calculate niche-widths

818 J Chem Ecol (2013) 39:810–825

Terpene-elicited modifications of fatty acid compositions ofbacterial cell membranes were shown to have negative effectson bacterial cell aggregation (de Carvalho and de Fonseca2007). Effects on intracellular processes also are known forsulfur-containing volatiles. Allicin, the main volatile com-pound of garlic oil, inhibits enzyme activities by reacting withfree amino acid thiol residues (Miron et al. 2000). Allylisothiocyanate, the volatile breakdown product of the aliphaticglucosinolate sinigrin, affects protein function by oxidativecleavage of disulfide bonds (Luciano and Holley 2009).

Effects of volatile terpenes might also depend on their rapidreaction with particular oxygen species in the atmosphere(Atkinson and Arey 2003). The resulting oxidized productscan have enhanced activity against microbes as was shown inthe case of photo-oxidized (E)-β-caryophyllene (Kim et al.2008). This mode of action may occur when the oxidizedcompound is deposited back on the tissue surface (Himanenet al. 2010). In general, the activities of VOCs onmicrobes canbe considered to be dose-dependent, although inhibitory ef-fects might be confined to a specific concentration range. Forexample, Inoue et al. (2005) reported that the inhibitory activ-ity of diterpenes such as geranylgeraniol was dependent on adefined concentration range, and even had growth acceleratingeffects above a certain threshold. Concentration-dependentand blend-specific effects also have been demonstrated forattractive and deterrent activities of VOCs on insects (Bruceand Pickett 2011; Davis et al. 2013) and are of high signifi-cance in determining and understanding the mode of action ofplant VOCs.

Taken together, plant VOCs target microbial cells at dif-ferent molecular levels. While the presence of specific VOCreceptors in microbes seems unlikely, differential effects ofVOCs on microbial populations probably depend on severaloverlapping factors. These include the physicochemical be-havior of volatile compounds under the environmental con-ditions at the plant cell surface, their synergistic and dose-dependent activities together with other volatile components(Kubo et al. 1992), and their effects on protein targets at themembrane surface or inside the cell. Moreover, differentlipid compositions of bacterial and fungal membranes andtheir changes in adaptation to sub-lethal concentrations ofvolatiles (Di Pasqua et al. 2006) are important factors of howmicrobes cope with structurally different compounds.Finally, degradation of VOCs by enzymatic activities or theirremoval by efflux pumps (Wiggins 2004) undoubtedly in-fluence the ability of different microbial taxa to reside inparticular tissue compartments and niches.

Effects of Microbes on Plant VOC Emissions

Having discussed the impact of plant volatiles on microor-ganisms, the question remains how microbes can manipulate

volatile emissions by plants. As indicated above, one way formicrobes to change volatile compositions of plants is thedegradation and consumption of volatile compounds as car-bon sources. In addition, the effect of microbes on plantVOC production may depend on whether a microorganismelicits an immune response by the plant or if its colonizationis tolerated by the immune system. Several examples havebeen described for the induction of volatile emissions bybacterial and fungal pathogens (e.g., Toome et al. 2010;Wenda-Piesik et al. 2010). These responses typically aremediated by defense signaling pathways involvingjasmonate, ethylene, and salicylate, and they depend on thecompatibility of host and pathogen. For instance, infection oftomato plants with the avirulent strains P. syringae pv.maculicola ES4326 (Psm ES4326) or pv. tomato DC3000resulted in the release of a volatile blend that differs from thatelicited by the virulent pathogen P. syringae pv. tabaci (Pstb)(Huang et al. 2003). Interestingly, VOC emissions inducedby P. syringae in tobacco do not depend on jasmonate incontrast to Pseudomonas-induced emissions of terpenes inArabidopsis (Herde et al. 2008).

Symbiotic (mycorrhiza, rhizobia) and non-symbiotic ben-eficial microbes associated with roots are believed to sup-press immune responses that are triggered by their microbe-associated molecular patterns (MAMPs) (Zamioudis andPieterse 2012). This would imply that rhizobacteria and rootendophytes minimize the release of defense-related volatilesin root tissues, although no clear evidence has been provided.Instead, beneficial microbes are recognized for priming de-fenses in above ground tissues, a response known as inducedsystemic resistance (ISR).Pseudomonas fluorescens associatedwith Arabidopsis roots was found to modify aphid-inducedvolatile profiles in leaves in a jasmonate dependent manner,which resulted in the reduced attraction of parasitoids of theleaf herbivore (Pineda et al. 2013). Modifications of herbivore-induced VOCs aboveground also have been observed for my-corrhizal associations of Medicago truncatula (Leitner et al.2010). Moreover, it has been reported that mycorrhizal fungiand rhizobacteria can change essential oil production, and anincrease in essential oil formation can be elicited byrhizobacteria-derived volatiles (Banchio et al. 2009;Rapparini et al. 2008). These observations indicate that micro-bial communities in the rhizopshere directly or indirectly mod-ify VOC concentrations and their effect on microbes in above-ground tissues (see also Jung et al. 2012; Soler et al. 2012;Song et al. 2013).

Effects of Microbial VOCs on Plants and Functionsof Microbial and Plant VOCs at Other Trophic Levels

There is growing evidence that plant-microbe associationsinvolve complex chemical communication networks with

J Chem Ecol (2013) 39:810–825 819

signal exchanges from both sides. Among the metabolic sig-nals that are produced by microbes in interaction with plants,volatile compounds have caught increased attention. Severalin vitro studies have investigated VOC emissions from soilborne bacteria and fungi revealing a diverse array of com-pounds including alkanes, alkenes, alcohols, aldehydes, ke-tones, esters, pyrazines, lactones, and sulfides (Effmert et al.2012). Beneficial rhizobacteria were shown to produce mix-tures of VOCs, of which particular compounds such as 2,3-butanediol and acetoin, according to experiments with bio-synthetic mutants, appear to promote plant growth and in-crease pathogen and abiotic stress resistance (Cho et al. 2008;Farag et al. 2006; Ryu et al. 2003, 2004). Similar to elucidat-ing specific targets of plant VOCs in the microbial cell, acentral question about bacterial or fungal volatiles is howthese compounds are perceived by plants (or other microbes)and which signaling pathways they may elicit that result indistinct responses at gene and protein levels. One of the firstmolecular studies addressing effects of VOCs emitted by plantgrowth inhibiting bacteria (Serratia plymuthica andStenotrophomonas maltophilia) revealed distinct changes ingene transcript profiles that overlap with those affected byabiotic stress, and supported the role of specific transcriptionfactors mediating these responses (Wenke et al. 2012). Furtherdiscussion of the mechanistic effects of microbial VOCs goesbeyond the scope of this article and the reader is referred torecent reviews (Wenke et al. 2010) including a contribution byRyu and colleagues in this special issue (Farag et al. 2013).

Volatile-mediated interactions between plants and microbesalso may affect responses of third parties above- and below-ground. For example, volatiles can be considered importantsignals in the communication of plant associated bacteria andfungi in the rhizo- and endosphere. The role of VOCs in positiveand antagonistic interactions between rhizobacteria and mycor-rhizal fungi and their ecological significance has been describedrecently in an excellent review by Effmert et al. (2012). Anintriguing study by Hountondji et al. (2005) revealed the role ofplant VOCs in the life cycle of entomopathogenic fungi.Volatiles released by cassava plants upon feeding of greenmites,which are hosts of the entomopathogenic fungus N. tanajoae,induce conidiation and spore release by the fungus; however,spore germination is suppressed in the absence of herbivore-elicited volatiles.

Instead of responding to plant VOCs directly, fungi andbacteria can indirectly change plant VOC emissions and theolfactory behavior of pests and parasitoids as has been re-ported by Pineda et al. (2013) (see above) and in otherstudies (Fontana et al. 2009; Jallow et al. 2008; Piesik et al.2011). Insects may also use microbial volatiles as directchemical cues to locate resources (see Davis et al. 2013).For example, volatiles emitted by Staphylococcus sciuribacteria inhabiting the honeydew of aphids attract naturalenemies of the aphids (Leroy et al. 2011). Neither sterile

honeydew nor honeydew inoculated only with Acinetobactercalcoaceticus (another common colonizer of the honeydew)attract the predator Episyrphus balteatus, a fact that identifiesa single bacterium (S. sciuri) as the source of the kairomone(Leroy et al. 2011). Finally, volatiles mediating antagonisticplant-microbe interactions also may be exploited by higheranimals. Clark and Mason (1985) found that EuropeanStarlings select specific plants for the construction of theirnests, while avoiding other plants that are also available in thesame range. A chemical analysis of the selected and avoidedplants revealed that plants collected by the birds emittedsignificantly more mono- and sesquiterpenoids than plantsthat were not used for nest-construction. Additionally, agardiffusion assays demonstrated that the preferred plantsinhibited the growth of bacterial pathogens associated withthe birds, indicating that the birds utilize growth-inhibitingproperties of plants for hygienic reasons (Clark and Mason1985). Likewise, bees collect resins that are offered as solepollinator reward by flowers of Clusia plants (Lokvam andBraddock 1999). Agar diffusion assays have demonstrated thegrowth-inhibiting activity of these resins, which suggests thatthe bees use this material, thus protecting their nests againstbacterial contamination (Lokvam and Braddock 1999). Theexamples described here indicate that volatiles emitted byplants or microbes may affect the behavior or physiologicalprocesses of third-party organisms with positive or negativeeffects for all involved organisms.

Future Perspectives

Plant volatiles contribute to the shaping of microbial,especically bacterial communities on their surfaces viagrowth inhibiting and promoting effects. As we outlinedhere, the effects of volatiles are variable depending on thebacterial strain that enters the plant surface. Future studiesmay use next generation sequencing to search themetagenome of bacterial colonizers for loci encoding formetabolic abilities that enable them to establish and growin these habitats. The metabolic prerequisites of bacteria todwell in the anthosphere, phyllosphere, and rhizospheremay include enzymes necessary to tolerate growth-inhibiting volatiles or to utilize volatiles as C-source, butalso to exploit other nutrients and to cope with the oftenharsh conditions in these environments. This informationmay provide detailed insights into the mechanisms of theestablishment of epiphytic bacterial communities that in-teract with plants and plant parts that may filter theirmicrobial communities. To date, we are not aware of astudy assessing the relative role of VOCs on the compo-sition of bacterial communities or the establishment ofindividual strains in relation to other factors. Future stud-ies should address the issue that plant traits, dissemination,

820 J Chem Ecol (2013) 39:810–825

and environmental factors cannot be viewed in isolation,and that their combined effects may act additively orsynergistically.

Apart from the paper by Huang et al. (2012) that clearlydemonstrated that the emission of a single volatile can havepositive effects on plant fitness, little is known about whethervolatile emissions that affect microbial colonization of plant-surfaces have an adaptive value or whether these effectsrepresent a byproduct of an adaptation to other (eukaryotic)interacting partners. To reveal whether bacteria select forVOCs, studies should compare how bacteria that colonizeplants and those that do not because their growth is inhibitedby VOCs directly and indirectly or positively and negativelyinterfere with plant growth and reproduction. Alternatively,as done by Huang et al. (2012), mutants of plants with alteredscent profiles should be used to study the interplay betweenplants and bacteria mediated by volatile compounds.

Additionally, more insight should be gained as to whatextent plant volatiles indirectly affect microbes by primingor stimulating different signaling pathways. Priming effects ofinduced volatiles have been demonstrated convincingly inintra- and inter-plant interactions upon herbivore attack (Heiland Karban 2010; Heil and Silva Bueno 2007). However, it isunclear whether VOCs emitted constitutively in theanthosphere and phyllosphere exhibit similar activities.Likewise, VOCs such as rhizathalenes that diffuse from thecentral stele of the root may change gene expression profilesin surrounding concentric cell layers. It has become increas-ingly obvious that VOCs – whether of plant or microbialorigin – elicit transcriptional changes in plants, and it will beworthwhile deciphering transcriptional signatures that are as-sociated with the exposure to particular VOC mixtures, andwhich may be important for establishing or minimizing inter-actions of plants with different microbes.

Acknowledgments We thank Maren Höfers for help with Fig. 1 andAfroditi Kantsa for valuable comments on the manuscript. Research byR.R.J. on bacterial communities on petals and leaves was funded by theDeutsche Forschungsgemeinschaft (BL960/1-1). Work by D.T. wassupported by a National Science Foundation Advance Virginia Techresearch and development grant, National Science Foundation GrantMCB-0950865, Thomas and Kate Jeffress Memorial Trust Grant J-850,and a US Department of Agriculture Cooperative State Research, Ed-ucation, and Extension Service National Research Initiative Grant2007-35318-18384 (to D.T.).

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