Emission of volatile organic compounds by apple trees ... - CREAF


Emission of volatile organic compounds by apple trees ... - CREAF

Experimental and Applied Acarology 25: 65–77, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.Emission of volatile organic compounds by apple treesunder spider mite attack and attraction of predatorymitesJ. LLUSIÀ ∗ and J. PEÑUELASUnitat Ecofisiologia CSIC, CREAF (Centre de Recerca Ecològica i Aplicacions Forestals),Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona),Catalonia, Spain(Received 29 June 2000; accepted 26 January 2001)Abstract. Emission rates of volatile organic compounds (VOCs) from Pirus malus L. subsp.mitis (Wallr.) var. Golden Delicious and var. Starking attacked by the phytophagous mite Panonychusulmi Koch, and their attractiveness to the predatory mites Amblyseius andersoni Chantand Amblyseius californicus McGregor, were studied during three years. A large variabilitywas found in the emission of individual VOCs depending on the infestation, the apple treevariety and the date. There were larger total VOC emission rates and larger total VOC leafconcentrations in apple trees attacked by phytophagous mites, especially in the var. Starking.In infested trees of this variety, there were also more predatory mites. An olfactometer assayshowed that predatory mites preferentially chose branches infested by Panonychus ulmi (85%went to infested branches vs 15% to uninfested control branches) indicating that volatiles maybe used as cues to find their prey.Key words: Amblyseius andersoni, Amblyseius californicus, Panonychus ulmi, phytophagousmites, predatory mites, VOC emissionIntroductionSome plants have been found to produce and emit volatile organic compounds(VOCs) such as ethylene, isoprene, mono and sesquiterpenes, alkanes, alcohols,aldehydes, organic acids, ketones and others in response to an attack oran injury by external agents (Langenheim, 1994; Peñuelas et al., 1995). Thesecompounds represent an arsenal of defences ranging from chemical toxinsto feeding deterrents (Langenheim, 1994). Some of these compounds emittedduring phytophagous attack may be used in another defensive strategynamely the attraction and recruitment of the herbivores’ natural enemies:predators and parasites (Dicke et al., 1990; Turlings et al., 1990; Pallini et al.,∗ Author for correspondence: Fax: 34.3.581 13 12; E-mail: J.Llusia@CREAF.uab.es

661997). Moreover, not only the infested leaves but the whole plants, and evenneighbouring uninfested plants, have been shown to emit chemical signals inresponse to phytophagous attack due to systemic responses in infested plants(Dicke et al., 1990; Turlings and Tumlinson, 1992; Röse et al., 1996), andchemical signalling between plants (Bruin et al., 1995).Among phytophagous animals, mites attack a large number of plants, andseveral studies have demonstrated that plants infested by spider mites initiatethe release of VOCs that are attractive to predatory mites (Dicke, 1988; Dickeet al., 1990; Bruin et al., 1995; Koveos et al., 1995). This is a widely studiedphenomenon for its great ecological interest and its possible application toagricultural pest control. Apple trees are frequently attacked by spider mites(Monetti and Fernandez, 1996; Croft and Slone, 1997; Stanyard et al., 1997).Here we aimed to test whether apple trees (Pirus malus L.) attacked byphytophagous spider mites (Panonychus ulmi (Koch) (Acari: Tetranychidae))quantitatively and/or qualitatively change the content and emission of VOCs,and whether there is an increased attraction of predatory mites, Amblyseiusandersoni Chant and A. californicus McGregor (Acari: Phytoseiidae), to treesinfested by Panonychus ulmi (Koch).Materials and MethodsExperimental field conditions and mite samplingVOC emissions were measured from six-year-old P. malus var. GoldenDelicious and var. Starking cultivated in Masbadia experimental station atTorroella (North-East of Catalonia, Spain). Two levels of spider mite attack(absence and about 60 individuals per leaf) were obtained by treating theabsence plots with pesticides (METI acaricides and Propargite) about 2 weeksbefore VOC and leaf sampling. The number of individuals was determinedafter leaf collection. VOC emission was measured in five apple trees pertreatment. Measurements of VOC emission were conducted during three consecutiveyears, on August 7 1995, on June 14 1996, and on July 8 1997between 12:00 and 15:00 h solar time. Temperatures ranged between 28 and30 ◦ C. After VOC sampling, 100 leaves per treatment were sampled to countboth the predatory mites Amblyseius andersonii and A. californicus and thephytophagous spider mite Panonychus ulmi.VOC sampling and analysisFive apple trees were sampled for VOC emissions each year. Sampling ofVOC emission was conducted by enclosing a branch with 8–12 fullydeveloped leaves attached to a tree within 2 L cylindrical (30 cm long)

polyethylene teraftalate chambers connected to peristaltic pumps (A.P.BUCK, INC. Orlando, Florida). Sampled branches were the same size (about25 cm long), with 7–9 leaves and in the same stage of development. Moreover,to obtain comparable results, the emission rates were expressed on a per-leafdry weight basis, and the sampling was conducted always during midday, atthe same time of the day for both infested and uninfested trees. Air comingout of the chamber passed through a glass tube (11.5 cm long and 0.4 cm internaldiameter) filled with Carbotrap C (300 mg), Carbotrap B (200 mg) andCarbosieve S-III (125 mg) adsorbents from Supelco (Bellefonte, PA, USA)separated by plugs of quartz wool. These tubes were previously conditionedfor3minat350 ◦ C with a stream of purified helium. The sampling time was5 min and the flow varied between 100 and 200 ml min −1 depending on theadsorbent package of each tube (the flow rate was measured with a bubbleflowmeter every time the tube was used). The trapping and desorption efficiencyof liquid and volatilized standards such as hexanal, heptanal, heptane,nonanal, undecane, decanal, α-pinene, β-pinene or limonene was practically100%. Controls of 5-min air sampling without enclosing any twig were carriedout immediately before and after each measurement. The glass tubes(with trapped VOC) were stored in a portable refrigerator at 4 ◦ C, and takento the adjacent laboratory. At the laboratory, the glass tubes were stored at−30 ◦ C before analysis, for no longer than 24–48 h.For VOC analysis, a GC-MS (Hewlett Packard HP59822B, Palo Alto, CA,USA) was used. Trapped emitted VOCs were desorbed (Thermal DesorptionUnit, Model 890/891; Supelco, INC, Bellefonte, PA, USA) at 320 ◦ C during3 min into a 30-m × 0.25-mm × 0.25-µm film thickness capillary column(Supelco HP-5, non-polar column Crosslinked 5% pH Me Silicone). Aftersample injection, the initial temperature was increased from 46 to 70 ◦ Cat30 ◦ Cmin −1 , and thereafter at 10 ◦ Cmin −1 up to 150 ◦ C, then held at thattemperature for an additional 5 min. Helium flow was 1 ml min −1 .Theidentityof the VOCs was confirmed by comparison with standards from Fluka(Chemie AG, Buchs, Switzerland) and with spectra from the literature andGCD Chemstation G1074A HP. Frequent calibration (once every three analyses)with external standards (dodecane, α-pinene, 3-carene, p-cymene, limonene,heptanal, hexanal, and heptane) were used for quantification. Detectionlimit was about 0.6 ng. Calibration curves were always highly significant(r 2 > 0.99). After analysis, blank VOCs (background or impurity peaks) weresubstracted from VOCs found in branch sampling.Leaf VOC concentrations were analysed in 1995, 1996 and 1997. For extractionof leaf VOCs, five trees were sampled each year. For each extraction,4–5 fully developed leaves were submerged in liquid N 2 after VOC emissionsampling and taken to the laboratory, where they were homogenised in Teflon67

68tubes with a Teflon pestle and with 2 ml of pentane. The extracts were storedat −30 ◦ C until analysis in the GC-MS as described above by injecting 2 µlofthe extract (not longer than 48–72 h later). An internal standard (dodecane)was used to quantify recovery of extracted volatiles (recovery was alwayslarger than 90%). The leaf pellet dry weight was determined after drying at60 ◦ C until constant mass was reached.Olfactometer assaysThe fork-tube olfactometer used in this study was similar to the olfactometerdescribed by Steinberg and Cohen (1992). The odour source was adetached uninfested branch of var. Golden Delicious (treated with acaricides)and collected in the field the day before. It was infested with 30 individualsof Panonychus ulmi per leaf (eight leaves per branch), that were released inthis chamber 24 h before releasing predatory mites. This infested branch wasplaced in one PET (Polyethylene Teraftalate) chamber (9 × 9 × 30 cm) (Figure1). Other two identical PET parallel chambers were used. One containeda control-uninfested branch and the other one was used as blank without anyplant material in it (Figure 1). The three chambers were connected with teflontubes to a chamber containing predatory mites. A fan pushed air throughthe system. The air flow was set at 170 ml min −1 for each arm. Predatorymites were released in sets of 100 individuals and allowed 4 h to chooseone of the chambers. In each replication of the experiment, chambers andconnections were rotated among treatments to rule out the effect of asymmetricalbias in the olfactometer or its surroundings. Tests were conductedin the lab at 24–25 ◦ C starting at 15 h, and were replicated five times withdifferent branches. Phytophagous mites were Panonychus ulmi (Koch) andpredatory mites were Amblyseius andersoni (Chant) and Amblyseius californicus(McGregor). Volatiles were collected from the olfactometer chambersby trapping them in adsorbent glass tubes as described above just 5 min afterthe predatory mites were released in the bioassay. VOCs were analysed usingthe same method previously described.Tray assaysTwo leaves of uninfested apple trees var. Golden Delicious were placed atopposite ends of a tray as shown in Figure 1 the morning after being detachedfrom the tree (15 h earlier). Leaves were kept fresh by keeping their petiole ina small glass bottle sealed with a rubber cap. At 0800 h, one leaf was infestedwith 100 individuals of phytophagous mites (Panonychus ulmi) and the otherleaf remained uninfested, as control. Predatory mites (Amblyseius andersoniand Amblyseius californicus) were liberated at the middle of the tray half

70as dependent variables and treatment (infested vs. uninfested) and year asindependent variables were conducted using STATISTICA 5.0 for Windows(StatSoft, Inc. Tulsa, OK, USA, 1996). Data were log or arcsin transformedwhen necessary to accomplish normality requirements.ResultsField studiesVOC emissionsApple trees infested by phytophagous mites emitted larger amounts of VOCsthan uninfested trees (P < 0.01, ANOVA). However, when considering separateyears or separate apple tree varieties, this difference was statisticallysignificant only for the var. Starking (P < 0.01, ANOVA). This variety alsopresented much higher absolute emission rates than var. Golden Delicious(Figure 2).There were not only enhanced emission rates, but also qualitative differences.Regarding particular VOCs, acetic acid, α-pinene, 2-methyl-4-bromo-1-butene, and dodecane were emitted by infested var. Golden Delicious treesand they were not detected in uninfested trees. And in infested trees of var.Starking there were significant (P < 0.01, ANOVA) increases of emissionrates of dodecane, 2,4,4-trimethyl-1-hexene, tetradecane and 1-decene(Figure 3).In parallel to these increased VOC emissions, more predatory mites werefound on infested (3 per leaf ± 0.2) than in non-infested trees (0 per leaf)across the studied years. Also in accordance with the larger emission rates invar. Starking, more predatory mites were found on infested Starking trees (4per leaf ± 0.2) than on infested Golden Delicious trees (1.75 per leaf ± 0.5)(P < 0.001, ANOVA).Leaf VOC concentrationsLeaf VOC concentrations were also larger in infested trees (P < 0.05, AN-OVA), but they did not present significant differences when consideredseparately by variety or year (Figure 4). Regarding particular VOCs, therewas a larger (P < 0.05, ANOVA) concentration of some compounds, suchas (E)-2-hexenal, 3-hexen-1-ol acetate, butanoic acid 3-hexenyl ester, (Z, E)-3, 7, 11-trimethyl-1, 3, 6, 10-dodecatetraene and 3-hexen-1-ol benzoate ininfested trees (Figure 4). There was therefore no correspondence betweenthe most abundant volatile compounds and the most abundantly extractedcompounds.

Figure 2. Histograms of total VOC emission rates by Panonychus ulmi-infested and uninfested field apple trees. Results for three years and twovarieties (Golden Delicious and Starking) of apple trees (Pyrus malus ssp. mitis). Notice the different emission scale for the two varieties ( ∗ p

72Figure 3. Histograms of individual VOC emission rates by Panonychus ulmi-infested anduninfested apple trees. Results for two years and two varieties (Golden Delicious and Starking)of apple trees (Pyrus malus ssp. mitis). Bars are SE (n = 5). Depicted individual VOCs arethose that presented significant increases (P < 0.05, ANOVA) or that were only emitted ininfested trees.Laboratory studiesOlfactometer and tray assaysIn the fork-olfactometer tests, the percentages of predators reaching the applebranch infested by spider mite Panonychus ulmi Koch were significantlygreater (ANOVA, P < 0.001, n = 5, with 100 predatory mites for eachreplicate) than those reaching the control uninfested branch (85 ± 5.4% vs15 ± 5.4%) (Figure 5). None went to the air control.In the tray assays, 64 ± 6% predatory mites went to the leaf infested withphytophagous mites whereas 36 ± 6% predatory mites went to the non-

73Figure 4. Histograms of individual VOC concentrations in leaves of Panonychus ulmi-infestedand uninfested apple trees in two years and two varieties (Golden Delicious and Starking) ofapple trees (Pyrus malus ssp. mitis). Bars are SE (n = 5). Total VOCs refer to all the detectedvolatile organic compounds with the method explained in Materials and methods. Depictedindividual VOCs are those that presented significant increases (P < 0.05, ANOVA) or thatwere only emitted in infested trees. A was not identified.infested leaf (ANOVA, P < 0.01, n = 5, with 30 predatory mites for eachreplicate) (Figure 5).DiscussionLarger VOC concentrations and emission rates were found in apple treesinfested by the herbivore Panonychus ulmi than in uninfested apple trees(Figure 2). Some compounds such as dodecane, 2,4,4-trimethyl-1-hexene,tetradecane or 1-decene were released in increased amounts by var. Starking

74Figure 5. Histograms representing the percentage of predatory mites (Amblyseius andersoniand A. californicus) choosing for Panonychus ulmi-infested or uninfested apple branches inthe olfactometer (n = 5 replicates with 100 predatory individuals each) and apple leaves in thetray assays (n = 5 replicates with 30 predatory individuals each) ( ∗∗∗ p

indicate certain independence between VOC emission and content in responseto infestation.There was, thus, a large variability in emission of individual VOCs and ontheir responses to infestation between apple tree varieties and among the differentdates of the different studied years. In fact, infochemical VOCs are reportedto vary with herbivore species (Dicke, 1988; Takabayashi et al., 1991),plant species and even plant cultivar (Takabayashi and Dicke, 1993), leaf age(Takabayashi et al., 1994b) and abiotic conditions (light intensity, time ofthe year, water stress) (Takabayashi et al., 1994a). Temperature can also playan important role in emission rates (Röse et al., 1996), but in this case temperaturewas not very different among treatments and years. Thus, genotypictraits and phenology could explain part of the variability as the measurementswere conducted with different plant varieties and during different months indifferent years.There were more predatory mites in the infested Starking trees measuredin 1997 than in the infested Golden Delicious trees measured in 1995 and1996, which is in accordance with their larger total VOC emission rates. However,as the field data did not allow to test whether predatory mites were moreattracted to infested trees than to non-infested ones, because of the acaricidetreatment, we conducted the fork-olfactometer assay. Predatory mites wentpreferentially to branches infested by P. ulmi in the fork-olfactometer assay.Arrestment response rather than attraction can not be totally precluded speciallyfor the tray assay and to a lesser extent for the olfactometer experiment;however, the olfactometer construction partly blocked the way back of predatorymites once in the chamber, and moreover, no movement of predatorymites from one chamber to the other was observed. This likely attraction is inagreement with previous reports on predatory mites responding to qualitativeand quantitative genotypic and phenological variations in particular VOCemission rates (Takabayashi et al., 1994a). The spider mites themselves donot seem to be attractive (Sabelis et al., 1984) and neither seems mechanicaldamage alone (Takabayashi et al., 1994a). Recently, more mechanisticand biochemical details of these signal emission phenomena have been reported.An elicitor molecule (volicitin) in beet armyworm oral secretionsseems to trigger plant release of a terpenoid mixture, which attracts a parasiticwasp, Cotesia marginiventris (Turlings and Tumlinson, 1992; Alborn et al.,1997).In summary, plants infested by the phytophagous mite P. ulmi emittedlarger amounts of VOCs than non-infested plants, and the predatory mitesvery likely used these volatiles as cues to find their preys as indicated bythe olfactometer assays. A higher number of predatory mites were found inthe var. Starking that emitted more VOCs than the var. Golden Delicious.75

76However, further studies including experiments involving the VOCs in theabsence of the phytophagous mites and tests for the separate effects of individualcompounds and of the complete blends are necessary to clarify thistritrophic communication.AcknowledgementsThis research was made possible by a grant CICYT CLI97-0344. We gratefullythank technical assistance from Dr. Marià Vilageliu from experimentalstation of Mas Badia (IRTA). We gratefully acknowledge an F.P.I. (Spain)fellowship to J. Llusià and Fundació Territori i Paisatge (Caixa de Catalunya)and IMMPACTE (DURSI-DMA) for continuous support to this research.ReferencesAlborn, H.T., Turlings, T.C.J., Jones, T.H., Stenhagen, G., Loughrin, J.H. and Tumlinson,J.H. 1997. An elicitor of plant volatiles from beet armyworm oral secretion. Science 276:945–949.Bruin, J., Sabelis, M.W. and Dicke, M. 1995. Do plants tap SOS signals from their infestedneighbours? Trends Ecol. Evol. 4: 167–170.Croft, B.A. and Slone, D.H. 1997. Equilibrium densities of European red mite (Acari: Tetranychidae)after exposure to three levels of predaceous mite diversity on apple. Environ.Entomol. 2: 391–399.Dicke, M. 1988. Prey preference of the phytoseiid mite Typhlodromus pyri. I. Response tovolatile kairomones. Exp. Appl. Acarol. 3: 1–13.Dicke, M., Sabelis, M.W., Takabayashi, J., Bruin, J. and Posthumus, M.A. 1990. Plantstrategies of manipulating predator-prey interactions through allelochemicals: prospectsfor application in pest control. J. Chem. Ecol. 11: 3091–3118.Koveos, D.S., Kouloussis, N.A. and Broufas, G.D. 1995. Olfactory responses of the predatorymite Amblyseius andersoni Chant (Acari, Phytoseiidae) to bean plants infested bythe spider mite Tetranychus urticae Koch (Acari, Tetranychidae). J. Appl. Entomol. 9:615–619.Langenheim, J.H. 1994. Higher plant terpenoids: A phytocentric overview of their ecologicalroles. J. Chem. Ecol. 6: 1223–1280.Llusià, J., Estiarte, M. and Peñuelas, J. 1997. Terpenoids and plant communication, Butll. Inst.Cat. Hist. Nat. 64: 125–133.Monetti, L.N. and Fernandez, N.A. 1996. Differences in European red mite infestation (Panonychusulmi) in three apple tree varieties of a sprayed apple orchard. Acarologia 3:181–188.Pallini, A., Janssen, A. and Sabelis, M.W. 1997. Odour-mediated responses of phytophagousmites to conspecific and heterospecific competitors. Oecologia 110: 179–185.Peñuelas, J., Llusià, J. and Estiarte, M. 1995. Terpenoids: A plant language. Trends Ecol. Evol.7, 289.

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