APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2011, p. 1000–1008 Vol. 77, No. 30099-2240/11/$12.00 doi:10.1128/AEM.01968-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Volatile-Mediated Killing of Arabidopsis thaliana by Bacteria Is MainlyDue to Hydrogen Cyanide�†

Dirk Blom, Carlotta Fabbri, Leo Eberl, and Laure Weisskopf*Department of Microbiology, Institute of Plant Biology, University of Zurich, Zurich, Switzerland

Received 19 August 2010/Accepted 19 November 2010

The volatile-mediated impact of bacteria on plant growth is well documented, and contrasting effectshave been reported ranging from 6-fold plant promotion to plant killing. However, very little is knownabout the identity of the compounds responsible for these effects or the mechanisms involved in plantgrowth alteration. We hypothesized that hydrogen cyanide (HCN) is a major factor accounting for theobserved volatile-mediated toxicity of some strains. Using a collection of environmental and clinicalstrains differing in cyanogenesis, as well as a defined HCN-negative mutant, we demonstrate that bacterialHCN accounts to a significant extent for the deleterious effects observed when growing Arabidopsis thalianain the presence of certain bacterial volatiles. The environmental strain Pseudomonas aeruginosa PUPa3 wasless cyanogenic and less plant growth inhibiting than the clinical strain P. aeruginosa PAO1. Quorum-sensing deficient mutants of C. violaceum CV0, P. aeruginosa PAO1, and P. aeruginosa PUPa3 showed notonly diminished HCN production but also strongly reduced volatile-mediated phytotoxicity. The doubletreatment of providing plants with reactive oxygen species scavenging compounds and overexpressing thealternative oxidase AOX1a led to a significant reduction of volatile-mediated toxicity. This indicates thatoxidative stress is a key process in the physiological changes leading to plant death upon exposure to toxicbacterial volatiles.

Bacteria interact with plants in many different ways. Overthe past few years, the significance of volatile compounds asmediators of bacterium-plant interactions has become increas-ingly evident (14, 18, 33, 46, 50, 54). Using compartmental petridish assays with a bacterial culture on one side and Arabidopsisplants on the other, bacterial volatiles have been shown toeither promote the growth of plants (14, 36) or to reduce it (16,18, 43, 46). Despite the strong effects observed, very little isknown at present about the identity of the compounds involvedin this volatile-mediated impact of bacteria on plants. In addi-tion to carbon dioxide (17), 2,3-butanediol and its precursoracetoin are, to our knowledge, the only organic volatiles whichhave been put forward as candidates to explain the beneficialeffects of bacterial volatiles on plants (14, 36). Likewise, thecompounds responsible for the “killing” effect of some bacte-rial strains on Arabidopsis thaliana are yet to be discovered.

In the course of a study to assess the volatile-mediatedimpact of a collection of rhizosphere bacterial isolates on themodel plant A. thaliana, we observed contrasting effects, rang-ing from 6-fold growth promotion to plant killing. We noticedthat the most virulent strains were known producers of hydro-gen cyanide (e.g., Pseudomonas or Chromobacterium species).We therefore hypothesized that hydrogen cyanide (HCN), apotent inhibitor of cytochrome c oxidase and of other metal-

containing enzymes, might be responsible for the observedplant-killing effects.

Few bacterial species are known to produce cyanide, andthese are restricted to members of the genera Pseudomonas,Chromobacterium, and Rhizobium (5). Cyanide is usually pro-duced at the end of exponential phase, when the oxygen con-centration is reduced and cells have reached a density at whichquorum sensing is activated. The involvement of quorum sens-ing in the control of cyanogenesis is not a general phenomenonbut appears to be strain specific: in the extensively studiedclinical strain Pseudomonas aeruginosa PAO1, both acyl-homo-serine lactone (AHL)-based quorum-sensing systems (RhlI/Rand LasI/R) are necessary for HCN production (31). Similarly,the disruption of quorum sensing in Chromobacterium violaceumCV0 was reported to abolish cyanogenesis (44). Conversely, quo-rum sensing does not seem to regulate cyanogenesis in Pseudo-monas fluorescens 2P24, although other biocontrol properties arediminished when the gene encoding the AHL synthase is inacti-vated (48). Likewise, the biocontrol strain P. fluorescens CHA0produces very high levels of HCN but does not possess any knownAHL-based quorum-sensing system.

Although bacterial cyanogenesis is considered to be damag-ing to animals (10), cyanide production by rhizosphere bacteriais traditionally regarded as a plant-growth-promoting trait.This is mainly due to the antifungal activity cyanogenesis con-fers to P. fluorescens CHA0 (47) and other strains used inbiocontrol against phytopathogenic fungi. However, early re-ports proposed that the observed phytotoxicity of P. aeruginosastrains might be due to HCN production (1, 3, 4, 9, 23, 37).More recently, Rudrappa et al. (35) analyzed the effect ofcyanogenic pseudomonads on A. thaliana seedlings. These au-thors observed reduced root growth in the presence of twoPseudomonas strains (P. aeruginosa PAO1 and P. fluorescens

* Corresponding author. Mailing address: Department of Microbi-ology, Institute of Plant Biology, University of Zurich, Zollikerstrasse107, CH-8008 Zurich, Switzerland. Phone: 41 44 634 82 41. Fax: 41 44634 82 04. E-mail: lweisskopf@access.uzh.ch.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 29 November 2010.

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CHA0) but normal growth in the presence of the respectiveHCN-defective mutants. This was a further indication thatbacterial cyanogenesis might be a key factor responsible for theplant-killing effects observed by us and others (43, 46) whengrowing Arabidopsis plants in contact with bacterial volatiles.

To test this hypothesis, we used a collection of environmen-tal strains including Chromobacterium, Serratia, and Pseudomo-nas species and assessed both HCN production and volatile-mediated effects on A. thaliana. In addition, we investigatedthe strain specificity of HCN production, and its consequenceson plant growth. We compared various P. aeruginosa strains,including clinical strains and the environmental plant-growth-promoting strain PUPa3 (21). Since HCN production is re-ported to be controlled by quorum sensing in P. aeruginosa PAO1and C. violaceum CV0, we hypothesized that the quorum-sensing-impaired mutants of these strains would show reduced volatile-mediated toxicity on A. thaliana. Moreover, we investigated theeffect of quorum sensing in the control of cyanogenesis in C.violaceum CV0, in two P. aeruginosa strains, and in Pseudomonaschlororaphis ATCC 13985, a strain closely related to the biocon-trol strain P. chlororaphis subsp. aureofaciens 30-84. To obtain firstinsights into the mode of action of the deleterious volatiles onplant growth, we tested the volatile-mediated effects of our cya-nogenic strains on Arabidopsis mutants with altered expressionlevels of the alternative oxidase AOX1a (45).

MATERIALS AND METHODS

Plant accession numbers and bacterial strains. The bacterial strains used in thepresent study are listed in Table 1. A. thaliana Col-0 was used for all plant experi-ments. Transgenic lines in which the AOX1a gene was overexpressed or silencedwere obtained from the European Arabidopsis stock center NASC (Table 2).

Chemicals, culture media, and growth conditions. Chemicals were purchasedfrom Sigma-Aldrich, Buchs AG, Switzerland, unless specified otherwise. Bacteriawere routinely cultured on LB (per liter: 10 g of Bacto tryptone, 5 g of Bactoyeast extract, 4 g of NaCl [AppliChem/Axon], pH adjusted to 7.4 and supple-mented with 16 g agar [European Bacteriological Agar, Chemie Brunschwig]when needed). When required, N-acyl homoserine lactones (AHLs) were addedto the medium to a final concentration of 200 nM. The AHLs used were:hexanoyl homoserine lactone (HHL) for C. violaceum CV026, oxo-dodecanoylhomoserine lactone (OdDHL) for P. aeruginosa lasI mutants and butanoyl ho-moserine lactone (BHL) for P. aeruginosa rhlI mutants. Pseudomonas isolationagar (PIA) medium contained (per liter) 45 g of Difco PIA supplemented with20 ml of glycerol and 3 g of agar. ABM soft agar contained (per liter) 2 g of(NH4)2SO4, 6 g of Na2HPO4, 3 g of KH2PO4, 3 g of NaCl, 1 ml of 0.1 M CaCl2,1 ml of 1 M MgCl2, 1 ml of 0.003 M FeCl3, 0.2% glucose (wt/vol), and 7 g of agar.Half-strength MS agar contained (per liter) 2.2 g of MS basal medium, 15 g ofsucrose, and 8 g of agar; the pH was adjusted to 5.7. When required, ascorbatewas added as sodium ascorbate to the MS medium to a final concentration of 0.1mM. The antibiotics kanamycin, chloramphenicol, spectinomycin, and tetracy-cline were used at concentrations of 50, 10, 100, and 5 �g ml�1, respectively.Bacterial strains were stored at �80°C in LB broth containing 16% glycerol.

Plant-bacterial dual growth experiments. Experiments were performed inthree-compartment dishes (Greiner) with three 6-day-old seedlings placed in onecompartment containing half-strength MS agar. The second compartment con-tained LB agar, and the third compartment was left empty in all experimentsdescribed in the present study. Three (instead of two)-compartment dishes were

TABLE 1. Strains used in this study

Species Strain name Strain no. Genotype and/orplasmid Origina Source or

reference

Agrobacteriumtumefaciens

A136 pCF218 � pCF372 56

Escherichia coli S17-1 pMLBAD-aiiA-Gmr 24HB101 pRK600 Laboratory strain

Pseudomonas chlororaphissubsp. aureofaciens

ATCC 13985 Wild type 30pMLBAD-aiiA-Gmr This study

Pseudomonas aeruginosa PA01a Wild type Wound 7PA01b MH340 Wild type Wound East Carolina

UniversityMH694 �lasI 15MH698 �rhlI 15MH710 �lasI/�rhlI 15

TB Wild type CF patient 39TBCF10839 Wild type CF patient 34PA14 Wild type Burn wound 51PUPa3 Wild type Rice rhizosphere soil 21

�lasI 41�rhlI 41�lasI/�rhlI 41�rhlR/�lasR 41

Pseudomonas fluorescens CHA0 Wild type Suppressive soil 42CHA77 �hcnABC 22

Chromobacteriumviolaceum

CV0 ATCC 31532 Wild type Soil 49CV026 AHL-negative strain 44

Serratia marcescens MG-1 Wild type Liquefied plant tissues 11Serratia plymuthica IC14 Wild type Soil 19

a CF, cystic fibrosis.

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chosen in case they would be needed at some point of the study (e.g., supplyingactive charcoal or other trapping material). Arabidopsis seeds were sterilized in1.5-ml Eppendorf tubes by adding 1 ml of 70% ethanol and shaking them for 2min on an IKA Vortex Genius 3 with an adapter at force 4. The supernatant wasremoved after centrifuging for 1 min at 6,000 rpm and 1 ml of a 1% NaOCl(Fluka) solution containing 0.03% Triton X-100 (Fluka) was added and shakenfor 20 min as described above. After centrifugation, the supernatant was re-moved, and the pellet was washed four times with sterile MilliQ water. The seedswere stratified in a sterile 0.15% agarose solution at 4°C overnight in the darkand then plated on 12-cm square petri dishes containing half-strength MS agar.The plates were incubated in a climate chamber with a 12-h/12-h day/nightalternation at 20°C, 50% relative humidity, and 100 �mol m�2 s�1 light for 6days. After 6 days, three seedlings were transferred to one compartment of thedivided petri dishes containing half-strength MS agar. In the next compartment,20 �l of a liquid culture of each strain to be investigated (grown overnight at 30°Cunder shaking) were spotted on LB agar. Sterile LB broth was used as a control.The plates containing both the plants and the bacterial inoculum (or an unin-oculated control) were sealed with Parafilm and incubated in the plant growthchamber described above. Pictures were taken after 14 and 21 days, and theplants were harvested after 21 days. Shoots were cut and weighed. The resultswere expressed as a percentage of plant biomass (fresh weight) relative to thebiomass of control plants (growing next to uninoculated LB). All experimentswere performed using four replicates.

Chemical exposure of plants to HCN. In this experiment, one petri dishcompartment contained three 6-day-old seedlings on half-strength MS agar, andthe next contained on one side a 20-�l drop of a 0.09 M NaOH solution with 0.02,0.05, 0.1, 0.2, 0.5, 1, 2, 5, or 10 �mol of KCN and on the other side a 20-�l dropof 1 M HCl. The plates were sealed with Parafilm and tapped to make the twodrops meet, thereby releasing HCN. After 21 days, pictures were taken, and theplant’s shoots were weighed.

Collection and measurement of HCN. HCN produced by bacteria was mea-sured used a method modified from that of Guilbaul and Kramer (13). Bacteriawere grown in three-compartment dishes on LB agar as described above. HCNwas trapped in 1 ml of 4 M NaOH dropped in an empty compartment, and thepetri dish was sealed with Parafilm. After incubation, a sample was taken anddiluted to a concentration of 0.09 M NaOH. Additional dilutions of the samplesin 0.09 M NaOH were made if needed to keep the cyanide concentration inthe linear range of the measuring method (0.5 to 10 �M). We prepared 0.1M o-dinitrobenzene (Fluka) and 0.2 M p-nitrobenzaldehyde solutions in 2-methoxyethanol; for each measurement, a fresh 1:1 mixture of these solutionswas made. Then, 23-�l portions of the diluted sample were added to 77 �l of thismixture, followed by incubation for 30 min at room temperature. Next, 900 �l of2-methoxyethanol was added, and the optical density was measured at 578 nm.Concentrations were obtained by comparison with a calibration curve computedusing serial dilutions of a KCN stock solution.

Statistical analyses. Differences between treatments were compared by usinga two-sided Student t test (n � 3 to 4, P � 0.05).

RESULTS

Phytotoxicity of bacterial volatiles is dependent on cyano-genesis. In order to assess the impact of bacterial cyanogenesison the growth of A. thaliana, we selected 11 bacterial strainspreviously observed to have volatile-mediated deleterious ef-fects on A. thaliana (Table 1). We then quantified their HCNproduction after different incubation times (see Fig. S1 in the

supplemental material). In parallel, we grew A. thaliana in acompartmental petri dish in the presence of each strain andcompared the biomass after 3 weeks to the biomass obtained inuninoculated control petri dishes. We observed a strong neg-ative correlation between plant biomass and the total levels ofHCN produced by the strains (Fig. 1). P. fluorescens CHA0 andP. chlororaphis subsp. aureofaciens both produced extremelyhigh quantities of HCN (17 and 12 �mol, respectively) and ledto plant death (CHA0) or to a drastic growth reduction (P.chlororaphis). The P. aeruginosa strains tested showed similarHCN production (2 to 4 �mol), causing a reduction of plantgrowth to values ranging from 5 to 18% relative to the unin-oculated control. The environmental P. aeruginosa isolate,

FIG. 1. Plant growth inhibition and cyanogenesis by different bac-terial strains. White bars indicate plant biomass (fresh weight) after 3weeks of exposure to bacterial volatiles, expressed as a percentage ofcontrol plant biomass (not exposed to volatiles). Black bars indicatetotal HCN production (�mol) over 3 weeks. P.fluo: Pseudomonasfluorescens CHA0; P.chloro, Pseudomonas chlororaphis subsp. aureo-faciens; C.viol, Chromobacterium violaceum CV0; S.plym, Serratiaplymuthica IC14; S.marc, Serratia marcescens MG1. Averages of threeto four replicates and standard errors are shown. Different lowercaseletters (a to g) indicate significant differences (Student t test, P � 0.05).

TABLE 2. Arabidopsis thaliana cell lines used in this study

Background ID no. Genotype Reference

Columbia Col-0 Wild typeColumbia N6590 Empty-vector (pBI1.4t) transformed control line for stocks CS6591 to CS6599 and CS6707 45Columbia N6595 Overexpressor of AOX1a (At3g22370) with Cys-127 mutated to Glu to confer activity

without the activator pyruvate and to prevent formation of the intersubunit disulfidebond; CaMV 35S promoter-driven expression

45

Columbia N6599 Antisense line with transgene expression construct; exhibits decreased AOX1a(At3g22370) protein in mitochondria due to presence of antisense AOX1a constructunder CaMV 35S promoter control

45

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PUPa3, produced less HCN (�2 �mol) and was less virulentthan most of the other P. aeruginosa strains (30% of the controlplant biomass). C. violaceum CV0 showed similar levels ofHCN production and phytotoxicity. Finally, Serratia plymuthicaIC14 was found to produce HCN, albeit in very low amounts(ca. 90 nmol), whereas Serratia marcescens MG1 did not pro-duce any detectable HCN but resulted in a similar inhibition ofplant growth.

An HCN-negative mutant is attenuated in A. thaliana viru-lence. The observed correlation between HCN production andplant killing supported our hypothesis that HCN constitutes amajor factor in the observed volatile-mediated deleterious ef-fects of bacteria on plants. In order to test this hypothesis, weinvestigated the volatile-mediated effect of a HCN-negativemutant of P. fluorescens CHA0 on A. thaliana. As expected, theHCN-negative mutant CHA77 exhibited drastically reducedtoxicity to A. thaliana compared to the wild-type CHA0 (Fig.2). Although the plant biomass upon exposure to CHA77 vola-tiles was variable and in average only ca. 40% of the uninocu-lated control biomass (suggesting that HCN is not the onlydeleterious volatile produced by CHA0), plants were still alive

and growing, in contrast to the plants exposed to the volatilesfrom the wild-type strain (Fig. 2, pictures).

The plant growth inhibitory effect of bacterial volatiles canbe mimicked by supplying HCN. If HCN production by bac-teria accounts to a large extent for the observed inhibitoryeffects of bacterial volatiles, exposing plants to HCN shouldgive rise to similar symptoms. The observed effects werestrongly dose-dependent: no effect was observed when the sup-

FIG. 3. Plant growth inhibition by chemical addition of HCN.(A) Plant biomass (fresh weight) after 3 weeks of exposure to differentamounts of HCN, expressed as a percentage of control plant biomass(not exposed to HCN). Averages of three to four replicates and stan-dard errors are shown. Different lowercase letters (a to c) indicatesignificant differences (Student t test, P � 0.05). (B) Representativepictures of plants grown for 3 weeks after treatment with differentamounts of HCN.

FIG. 2. Plant growth inhibition and cyanogenesis by P. fluorescenswild type (CHA0) and an HCN-negative mutant (CHA77). White barsindicate the plant biomass (fresh weight) after 3 weeks of exposure tobacterial volatiles produced by CHA0 or CHA77, expressed as a per-centage of control plant biomass (not exposed to volatiles). Black barsindicate the total HCN production (�mol) over 3 weeks. CHA0, P.fluorescens wild type; CHA77, P. fluorescens hcnABC mutant. Theaverages of three to four replicates and standard errors are shown. Thepictures are representative examples of control plants (upper picture),CHA0-treated plants (middle picture), and CHA77-treated plants(lower picture) after 3 weeks of incubation.

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plied quantities were below 1 �mol, but above this threshold,plant growth was severely inhibited: to 30% of the control with1 �mol and 6% with 2 �mol. Plant death occurred very rapidlywhen 10 �mol of HCN was supplied (Fig. 3).

Role of quorum-sensing in HCN-mediated plant killing. Inagreement with previous studies, we noticed that the QS-defi-cient strain C. violaceum CV026 produced greatly reducedlevels of HCN (Fig. 4A) (43). We concomitantly observed anincrease in plant biomass from 30% biomass reduction to160% biomass increase relative to the untreated control. Thissuggests that not only toxic substances, but also other, plant-growth-promoting, volatiles, whose effects may be masked byHCN in the wild-type, are produced by the strain. This is in linewith the results described above (see Fig. 1): despite similarHCN production rates, the volatile-mediated virulence to A.thaliana was less severe for C. violaceum than for most P.aeruginosa strains. This is also corroborated by our observationthat C. violaceum significantly promoted plant growth whencultured on MR-VP medium, which does not sustain HCNproduction (data not shown). Supplementing the mutant withC6-AHL restored both cyanogenesis and volatile-mediatedtoxicity. To assess whether HCN production was also under thecontrol of quorum sensing in P. chlororaphis subsp. aureofa-ciens, we applied a quorum-quenching approach. To this end,a lactonase that degrades AHLs was introduced into the wild-type strain, resulting in strong decrease of AHL production(see Fig. S2 in the supplemental material). In contrast to thetwo P. aeruginosa strains and to C. violaceum, inactivation ofquorum sensing increased the rate of HCN production, al-though the total amount produced did not differ significantly(Fig. 4B). No significant difference in plant killing was ob-served between the wild-type strain and the AHL-deficienttransconjugant. In P. aeruginosa PAO1, single gene deletionmutants of the AHL synthase genes (lasI or rhlI) showed re-duced levels of HCN production compared to the wild type,and inactivating both quorum-sensing systems was necessary tocompletely abolish cyanogenesis (Fig. 4C). The double mutantgave rise to significantly higher plant biomass compared toplants treated with the wild-type strain. Supplementing thedouble-knockout mutants with the corresponding AHL signalmolecules partially restored cyanogenesis and also restoredplant toxicity to wild-type levels. In the environmental strain P.aeruginosa PUPa3, simultaneous inactivation of lasI and rhlIwas also required to abolish HCN production and reduce phy-totoxicity (Fig. 4D). However, single gene deletion mutants(lasI or rhlI) produced higher levels of HCN (relative to thewild type) and were consequently more toxic to plants. Sup-plementing the two single mutants with their respective AHLsresulted in partial recovery of the wild-type phenotypes, whilethe supplemented double-knockout mutant recovered HCNproduction and phytotoxicity to a higher level than the wildtype.

Bacterial volatiles induce oxidative stress in A. thaliana. Inorder to investigate whether the alternative oxidase (AOX) isinvolved in plant tolerance to bacterial cyanogenesis, we grewlines of A. thaliana in which the AOX1a gene was either si-lenced or overexpressed (Table 2) in the presence of C. viola-ceum CV0, P. aeruginosa PAO1, P. aeruginosa PUPa3, and P.fluorescens CHA0. These four strains differ both in the totallevels of HCN produced and in the kinetics of cyanogenesis.

Furthermore, the effect of ascorbate addition in counteractingputative oxidative stress was assessed in the different plantlines. The addition of ascorbate alone did not improve plantgrowth; neither did overexpression of AOX1a (Fig. 5). How-

FIG. 4. Effect of quorum sensing on cyanogenesis and phytotox-icity. White bars indicate the plant biomass (fresh weight) after 3weeks of exposure to bacterial volatiles, expressed as a percentageof control plant biomass (not exposed to volatiles). Black bars givethe total HCN production (�mol) over 3 weeks. (A) Chromobacte-rium violaceum CV0; (B) Pseudomonas chlororaphis subsp. aureo-faciens; (C) Pseudomonas aeruginosa PAO1b; (D) Pseudomonasaeruginosa PUPa3. See Table 1 for more details. sup., supplementedwith AHLs. Averages of three to four replicates and standard errorsare shown. Different lowercase (a to e) letters indicate significantdifferences (Student t test, P � 0.05).

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ever, a combination of supplying ascorbate and overexpressingAOX1a restored plant growth in the presence of the two P.aeruginosa strains. Surprisingly, plant growth reduction causedby C. violaceum CV0 was not alleviated by this combinedtreatment. Silencing of AOX1a led to higher susceptibility tobacterial volatiles in all four strains tested, including C. viola-ceum CV0. Overexpressing AOX1a and supplying ascorbatedid not significantly change the growth of the plants whenexposed to the volatiles of the HCN-deficient mutant CHA77(data not shown). Interestingly, when plants were challengedwith 2 �mol of chemical HCN, overexpression of the AOX1aalone (without ascorbate addition) was sufficient to restoreplant growth from 2% (empty vector) to 98% of the unexposedAOX1-expressing plant (data not shown).

DISCUSSION

The volatile-mediated impact of bacteria on plants hasgained increasing attention over the last few years (14, 16, 33,46, 50, 54). Despite the magnitude of the effects observed inboth positive (14, 36) and negative cases (16, 43, 46), thecompounds responsible for the effects and the mechanismsinvolved in plant growth alteration are still poorly understood.We have presented evidence for one candidate deleteriousvolatile, hydrogen cyanide (HCN). HCN inhibits several metal-containing enzymes, most significantly cytochrome c oxidase,one of the key enzymes of the respiratory electron transportchain. Cyanogenesis occurs in few bacteria genera, mainlyPseudomonas, Chromobacterium, Rhizobium (31), and, as weshow here for the first time, Serratia. S. plymuthica IC14 pro-duced approximately 100 nmol of HCN, while cyanogenesiswas not detected in S. marcescens MG1. We observed large

differences in HCN production between the strains tested (Fig.1): the highest levels of HCN were observed for P. fluorescensCHA0 and P. chlororaphis subsp. aureofaciens, two strains ofenvironmental origin, followed by the P. aeruginosa strains andC. violaceum CV0. Interestingly, the two PAO1 strains testeddiffered significantly in the amounts of HCN produced. It wasrecently reported that an originally identical PAO1 strain keptfor decades in different laboratories displayed significantgenomic variability (20). It is thus not surprising that the twoPAO1 strains we analyzed, which were obtained from differentsources, also displayed phenotypic variability. In addition tothe different P. aeruginosa strains of clinical origin, we assessedcyanogenesis and volatile-mediated impact of the closely re-lated environmental strain P. aeruginosa PUPa3 on plants.PUPa3 was notably the least deleterious of all P. aeruginosastrains tested, even though it produced similar quantities ofHCN to the three strains of clinical origin: PA14, TB, andTBCF10839. It should be noted that P. aeruginosa PUPa3, firstdescribed in 2005 as an antifungal bacterium isolated from therhizosphere of rice, was reported to be noncyanogenic (21).However, even if PUPa3 produces less HCN than some clinicalstrains, we show here that it is definitely cyanogenic. Differ-ences in the sensitivity of the detection method (qualitativeversus quantitative), as well as in the time and temperature ofincubation, might account for this discrepancy. Under our ex-perimental conditions, PUPa3 only started to produce signifi-cant amounts of HCN after 1 week.

The observed differences in cyanogenesis between the 11strains tested were correlated with the volatile-mediated effectsof these strains on A. thaliana (Fig. 1), from rapid plant deathwhen growing next to P. fluorescens CHA0 (�17 �mol) to areduction to 30% of the control plant biomass when growing

FIG. 5. Effect of ascorbate addition and alternative oxidase (AOX) overexpression on A. thaliana’s tolerance to deleterious bacterial volatiles.Black bars, no ascorbate addition; white bars, addition of 0.1 mM ascorbate as sodium ascorbate to the plant’s culture medium. ctrl., empty vectorplants (N6590); AOX�, plants overexpressing AOX (N6595); AOX�, plants silencing AOX (N6599). See Table 2 for more details. Averages ofthree to four replicates and standard errors are shown. Within each group (bacterial strain), different letters indicate significant differences(Student t test, P � 0.05).

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next to P. aeruginosa PUPa3 (�2 �mol). While the productionof other deleterious volatiles is obvious from the plant growthinhibition observed upon exposure to the HCN-negative S.marcescens MG1 (30% of the control) or to the HCN-deficientmutant P. fluorescens CHA77 (40%), the difference betweenreduced growth and death is likely due to cyanogenesis in P.fluorescens CHA0 (Fig. 2, pictures). Moreover, the symptomsobserved upon contact with the volatiles of cyanogenic bacteria(chlorosis, reduced growth, or even death) were mimickedwhen plants were challenged with pure HCN. Interestingly, atsimilar doses, plants were more severely affected by the chem-ical supply of HCN than by bacterial HCN (Fig. 1 and 3): 2�mol of chemically supplied HCN resulted in 6% of growthcompared to the control, but when plants were grown in thepresence of strains producing a similar quantity of HCN(PUPa3 and CV0), plant biomass was five times higher (30%of the control biomass). Similar observations were made in thefield, where external application of HCN proved much moreplant growth inhibitory than cyanogenesis by inoculated rhizo-sphere pseudomonad populations (53). In addition to the pu-tative production of growth promoting volatiles by the bacte-rial strains, which could counteract the negative effect of HCN,the different timing dynamics of application is likely to accountin great part for the differences observed in plant responses.While chemical HCN was supplied all at once to young seed-lings, bacterial HCN was supplied to plants continuously asthey grew for 3 weeks, and in most cases, started only once acritical population density (the so-called “quorum”) had beenreached.

Quorum sensing has been shown to regulate many pheno-types associated with virulence (reviewed in references 2 and8). However, until now, only a single study by Muller et al. (27)investigated the involvement of quorum sensing in volatile-mediated virulence and showed that the volatiles produced bya quenched strain of S. plymuthica HRO-C48 were more ef-fective in limiting the growth of two phytopathogenic fungithan the volatiles of the wild type. We were therefore inter-ested in assessing whether volatile-mediated phytotoxicitywould also be quorum sensing dependent. In the well-studiedmodel strain PAO1, which possesses two quorum-sensing sys-tems, the RhlI/R and LasI/R systems, quorum sensing plays arole in cyanogenesis: the involvement of the RhlI/R quorum-sensing system in the regulation of HCN biosynthesis was firstreported by Winson et al. in 1995 (52), while it became clear 5years later that both AHL-based quorum-sensing systems wererequired for HCN production (31). If the main compoundresponsible for volatile-mediated phytotoxicity were indeedHCN, we would expect the quorum-sensing mutants to losetheir volatile-mediated virulence to A. thaliana. This is pre-cisely what we observed with P. aeruginosa PAO1 (Fig. 4C):deleting both AHL synthase genes led to loss of cyanogenesisand resulted in the restoration of plant growth to levels similarto those observed in control plants. In contrast to P. aeruginosaPAO1, regulation of cyanogenesis in the environmental P.aeruginosa strain PUPa3 had not yet been investigated. Weshow that in PUPa3, disruption of both quorum-sensing sys-tems was also necessary to abolish cyanogenesis and to reducephytotoxicity. This is consistent with previous findings, wherethe virulence of the PUPa3 lasI/rhlI double mutant was shownto be significantly reduced in both animal and fungal models

(41). Although the mechanisms underlying virulence have notyet been assessed, it is tempting to speculate that reducedvirulence might be due, at least in part, to the loss of cyano-genesis in the PUPa3 double mutant. Both the lasI and the rhlIsingle mutants of PUPa3 produced more HCN, which led to anincreased virulence on plants. This contrasts with observationsmade in PAO1, where single mutants showed reduced cyano-genesis relative to the wild type. Another difference comparedto PAO1 is that in the absence of HCN production, plantsreached only 50% of their control biomass, suggesting thatadditional, as-yet-unidentified toxic compounds are present inthe volatiles of PUPa3. In C. violaceum CV0, cyanogenesis hasalso been reported to be under the control of quorum sensing(44). We provide here quantitative data showing that inactiva-tion of the AHL synthase in C. violaceum abolished both cya-nogenesis and virulence on A. thaliana. Surprisingly, the quo-rum-sensing mutant CV026 promoted plant growth, suggestingthe production of plant-growth-promoting volatiles, whosebeneficial effects on plants were masked by HCN in the wildtype. We are currently investigating the chemical nature ofthese volatiles. Finally, we discovered that P. chlororaphissubsp. aureofaciens ATCC 13985, a close relative of P. chloro-raphis subsp. aureofaciens 30-84 (30), produced very high levelsof HCN, similar to those observed with P. fluorescens CHA0. P.chlororaphis 30-84 is a well-studied biocontrol strain that pro-duces phenazines in addition to HCN. Two quorum-sensingsystems have been identified in P. chlororaphis 30-84, thePhzI/R system, which is responsible for phenazine biosynthesis,and the CsaI/R system, which controls biofilm formation. Bothsystems are needed for proteolytic activity and rhizospherecompetence (32, 55). Cyanogenesis in this organism has beenreported to be independent of quorum sensing (55), a findingthat was supported by the results of the quorum-quenchingexperiment performed on a closely related strain in the presentstudy.

Besides the inhibitory effect of HCN when applied exog-enously in high amounts to plants, recent studies suggest rolesfor HCN in planta, far beyond its being solely a by-product ofethylene biosynthesis (29): in addition to its function in plantdefense against herbivory (through the wound-induced hydro-lysis of cyanogenic glucosides), HCN has been shown to breakseed dormancy in various species and to be involved in inducedresistance against viruses (12, 28, 38). However, it is not clearwhether HCN itself is the signaling molecule, or rather theburst of reactive oxygen species (ROS) that follows treatmentwith HCN. In sunflower (28) and apple (12) seeds, stimulationof seed germination by application of KCN was mediated byROS. It has further been shown that the gene expressionchanges occurring upon treatment with either KCN or with theROS-generating methyl viologen were very similar (6). Wetherefore hypothesized that the phytotoxicity of bacterial vola-tiles might be linked to oxidative stress. The results we ob-tained upon overexpression of the alternative oxidase AOX1and concomitant supplementation of ascorbate as a ROS-trap-ping compound confirmed this hypothesis, and indicated thatoxidative stress is a key process in the volatile-mediated neg-ative impact of cyanogenic bacteria on plants. Interestingly, ina study investigating the impact of truffle volatiles on plantgrowth, deleterious fungal volatiles (other than HCN) wereshown to induce oxidative stress in Arabidopsis (40). This sug-

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gests that oxidative stress might be a general response of plantsto detection of microbial volatiles (25, 26). In summary, wepresent evidence that HCN, when produced in high amountsby bacteria, can kill plants. We suggest that this accounts to agreat extent for the deleterious impact of bacterial volatilesobserved in previous studies using cocultivation of Arabidopsisand bacterial strains. Moreover, we report for the first timethat cyanogenesis is not restricted to members of the Pseudo-monas, Chromobacterium, and Rhizobium genera but that Ser-ratia species, e.g., Serratia plymuthica IC14, can also produceHCN. We further demonstrate that the volatile-mediated phy-totoxicity of C. violaceum CV0, P. aeruginosa PAO1 and P.aeruginosa PUPa3 is quorum sensing regulated. The environ-mental strain PUPa3 showed less phytotoxicity and lower cya-nogenesis than the clinical isolate PAO1, but in both strains,cyanogenesis was dependent on functional quorum-sensingsystems. In contrast, quorum-sensing was not required forHCN production in P. chlororaphis subsp. aureofaciens. Ourdata also provide initial insights into the mechanism of actionof these bacterial volatiles leading to growth reduction or plantdeath: supplying ascorbate to alternative oxidase overexpress-ing lines of Arabidopsis increased their tolerance to cyanogenicpseudomonads drastically, while silenced lines showed highersusceptibility. This indicates that volatile-mediated phytotoxic-ity involves oxidative stress and that the perception of delete-rious bacterial volatiles causes, like many other biotic andabiotic stresses, an oxidative burst in the plant. Future studiesare required for a more detailed understanding of the meta-bolic changes occurring in plants upon exposure to toxic mi-crobial volatiles.

ACKNOWLEDGMENTS

This study was part of the Zurich-Basel Plant Science Center-Syn-genta Graduate Research Fellowship and was funded by Syngenta.

We are grateful to Dieter Haas for supplying Pseudomonas fluores-cens CHA0 and CHA77 and to Vittorio Venturi for providing the P.aeruginosa PUPa3 strains. We thank Thomas Boller for constructivediscussions, Thomas Kost for his help in setting up the cyanide mea-surement methodology, and Kirsty Agnoli for English corrections.

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