How maize root volatiles affect the efficacy of entomopathogenic nematodes in controlling the western corn rootworm?

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1 1 How mize root voltiles ffect the efficcy of entomopthogenic nemtodes in controlling the western corn rootworm? Ivn Hiltpold Stefn Toepfer Ulrich Kuhlmnn Ted C. J. Turlings Abstrct Becuse the ferocious mize pest Dibrotic virgifer virgifer LeConte cn dpt to ll currently used control strtegies, focus hs turned to the development of novel, more sustinble control methods, such s biologicl control using entomopthogenic nemtodes (EPN). A good understnding of the biology nd behviour of these potentil control gents is essentil for their successful deployment. Root systems of mny mize vrieties emit (E)-b-cryophyllene (EbC) in response to feeding by lrve of the beetle D. v. virgifer. This sesquiterpene hs been shown to ttrct certin species of EPN, thereby enhncing their control potentil. In this study, we tested the effect of this root-produced voltile on the field efficcy of the three EPN Heterorhbditis bcteriophor, Heterorhbditis megidis nd Steinernem feltie ginst D. v. virgifer lrve in southern Hungry. By compring beetle emergence nd root dmge for two mize vrieties, one tht emits EbC nd one tht does not, it ws found tht root protection by H. megidis nd S. feltie ws higher on the emitting vriety, but this ws not the cse for H. bcteriophor. Overll, ll three nemtode species showed good control potentil. We conclude tht, if properly pplied nd I. Hiltpold T. C. J. Turlings (&) FARCE Lbortory, University of Neuchâtel, Emile-Argnd 11, cp158, 2009 Neuchâtel, Switzerlnd e-mil: ted.turlings@unine.ch S. Toepfer CABI Europe, c/o Plnt Helth Service, Rrosi ut 110, 6800 Hodmezovsrhely, Hungry U. Kuhlmnn CABI Europe-Switzerlnd, Rue des Grillons 1, 2800 Delémont, Switzerlnd in combintion with the right mize vriety, the relese of these nemtodes cn be s effective s other control methods. Keywords Heterorhbditis megidis Heterorhbditis bcteriophor Steinernem feltie Dibrotic virgifer virgifer Ze mys Tritrophic interction Crop protection Biologicl control Root exudtes Nemtode ttrction Introduction Since the domestiction of mize, Ze mys (L.), bout 5,000 7,000 yers go (Piperno nd Flnnery 2001; Sluyter nd Dominguez 2006), this crop hs been trgeted by vriety of rthropod pests, often cusing tremendous yield losses (Oerke 2006). In nture, plnts hve evolved vrious defence strtegies to fend off their herbivorous ttckers either directly (Bldwin nd Preston 1999; Agrwl 1998; Dicke et l. 2003; Krbn et l. 1997; Krbn nd Bldwin 1997; Schoonhoven et l. 1998) or indirectly (Agrwl 1998; Dicke nd Sbelis 1998; Dicke et l. 2003; Turlings nd Wäckers 2004). Direct defence trits of plnts comprise physicl or chemicl brriers, wheres indirect defences consist of the ttrction nd mintennce of the herbivore s nturl enemies by providing shelter nd/or food (Jnzen 1966; Stpley 1998) nd/or the emission of inducible voltile orgnic compounds (Dicke et l. 2003; Turlings nd Benrey 1998; Turlings nd Wäckers 2004). For mize, the ttrctiveness of such herbivore-induced plnt voltiles to nturl enemies of herbivores hs been demonstrted in both lbortory nd field experiments (Turlings et l. 1990; Bernsconi et l. 1998; Hobllh nd Turlings 2005). For instnce, green lef voltiles, s well s

2 2 terpenoids such s monoterpenes, sesquiterpenes nd homoterpenes, hve been found to ttrct prsitoids boveground (D Alessndro nd Turlings 2005; Hobllh nd Turlings 2005; Schnee et l. 2006). Recently it ws found tht roots lso re ble to recruit belowground enemies of soil dwelling herbivorous insects by relesing voltile signls. These voltiles cn ttrct entomopthogenic nemtodes (EPN) (vn Tol et l. 2001; Boff et l. 2001; Bertin et l. 2003; Rsmnn et l. 2005; Rsmnn nd Turlings 2008; Degenhrdt et l. 2009), predtory mites (Artchige et l. 2004) nd even prsitoids (Neveu et l. 2002). Mize roots fed upon by lrve of Dibrotic virgifer virgifer LeConte (western corn rootworm, WCR, Coleopter: Chrysomelide), one of the most destructive mize pests worldwide (Miller et l. 2005; Vidl et l. 2005), relese the sesquiterpene (E)-b-cryophyllene (EbC). EbC diffuses well in soil (Hiltpold nd Turlings 2008) nd plys n importnt role in the recruitment of the EPN Heterorhbditis megidis Poinr (Rhbditid: Heterorhbditide) (Rsmnn et l. 2005), which is highly virulent to WCR lrve (Kurtz et l. 2009). Two other species of EPNs, Heterorhbditis bcteriophor Poinr nd Steinernem feltie Filipjev, re lso promising cndidtes s biologicl control gents ginst WCR lrve (Kurtz et l. 2009; Toepfer et l. 2005), but it is unknown if their host finding bility is lso improved by ttrction to belowground signls. The im of the current study ws to determine the reltive importnce of EbC emission by WCR-dmged mize roots for the efficcy of H. bcteriophor, H. megidis, nd S. feltie in controlling WCR lrve under field conditions. For this purpose, the three nemtode species were relesed t two different time points in seprte, WCRinfested mize plots in Hungry. The results of the field study prompted us to lso conduct dditionl lbortory ssys to test the pprent lck of ttrction of H. bcteriophor towrds EbC. We discuss the control potentil of the tested nemtode species nd the importnce of choosing the right mize vriety to fully exploit this potentil. Mterils nd methods Field sites nd mize vrieties The study ws crried out in four mize fields (referred to s fields A to D) in Csongrd County in southern Hungry in 2005 nd 2006 (Tble 1). All fields contined n experimentl section tht hd been plnted with non-host plnts of WCR the yer before to ensure the initil bsence of this pest in the experimentl plots. Experimentl fields were divided in two plots, the first plnted with the vriety Mgister (UFA Semences, Bussigny, Switzerlnd) tht emits EbC fter WCR feeding (Hiltpold 2008) nd the second with the vriety Pctol (Syngent, Budpest, Hungry) tht does not emit EbC (Rsmnn et l. 2005). The seeds were sown between lte April nd erly My (Tble 1). All mize seeds were sown in rows with plnt spcing of 15 cm nd row spcing of 75 cm. The fields were treted once with 0.16 l of the herbicide Merlin SC (75% Izoxflutol, Byer Crop Science) per hectre when mize ws t the 3 5 leves stge. No insecticides were pplied. Tble 1 Chrcteristics of the study fields in southern Hungry nd the timing of EPN ppliction Field A B C D Loction Northwest of Hodmezovsrhely North of Sztymz North of Sztymz Hodmezo-vsrhely Coordintes N N N N E E E E Elevtion (m) Size (h) Soil bulk density (g/cm 3 ) 1.04 ± ± ± ± 0.13 Soil moisture (wt%)* 17.2 ± ± ± ± 2.1 Snd content (%) Lom content (%) Cly content (%) ph (H 2 O) Mize sown 25 April My My April 2005 EPN pplictions 25 April My My April June June June June 2005 Significnce is indicted by sterisks

3 3 Entomopthogenic nemtodes Three EPN species were used in this study: (1) cross of Europen nd US strins of Heterorhbditis bcteriophor Poinr provided from liquid culture by e-nem GmbH (Risdorf, DE), (2) the NL-HW79 strin of H. megidis Poinr, Jckson & Klein from The Netherlnds, re-isolted from Swiss soils nd provided from semi-liquid culture by Andermtt Biocontrol AG (CH), nd (3) cross of Europen strins of Steinernem feltie Filipjev provided from liquid culture by e-nem GmbH. H. bcteriophor nd S. feltie were shipped in cly from the producer to the experimentl sites, nd H. megidis ws shipped in vermiculite. All EPNs were stored in their shipping mteril t 7 9 C in drkness until use. About 2 3 h prior to ppliction, EPNs together with the crrier mteril were diluted in tp wter. Before ppliction, liquots of EPNs were tken to determine the qulity of the shipment btches. For this purpose Glleri mellonell L. (Lepidopter: Pyrlide) lrve were exposed to nemtodes in plstic cups (40 mm dimeter, 60 mm height). Ech cup ws filled with 200 g of 10% moist sterilised snd to which five lrve nd 100 infective juvenile nemtodes were dded. Three replictes per EPN shipment btch were used for this ssy. After 1 week in drkness t 22 C, mortlity of % ws found for ll EPN btches, which ws considered sufficient for use. Dibrotic virgifer virgifer WCR eggs were obtined from eggs lid by field-collected beetles from southern Hungry (for procedures see Singh nd Moore 1985). Eggs were kept in dipuse in moist snd t 6 8 C. The dipuse of WCR eggs ws broken in erly April by trnsferring them to climte chmber t 25 C for 3 weeks. The snd ws sieved through 250 lm mesh to recover the eggs. The eggs were then mixed into solution of wter nd 0.15% gr in order to obtin n egg suspension of 38 eggs/ml. Mize plnts of ech field were infested in erly My (1 3 lef stge) with the suspension of vible nd redy-to-htch eggs. Using stndrd pipette (Eppendorf Compny, Hmburg, Germny), 2 ml of the egg suspension ws pplied into ech of two 12 cm deep holes t distnce of 5 8 cm from either side of the mize plnt (*150 eggs/plnt). The lrve were expected to htch by mid-to-lte My nd to rech the second lrvl instr in June (Toepfer nd Kuhlmnn 2006). Experimentl setup nd EPN ppliction Ech of the four fields contined two plots of t lest 14 rows of Mgister nd Pctol plnts. Seven groups of six to seven mize plnts were rndomly selected from either the 3rd, 6th, 9th or 12th row of ech section, thereby ensuring buffer rows between experimentl groups. The three different entomopthogenic nemtode species were pplied t two different times (erly: during sowing in April/My; lte: in June, see Tble 1). Thus six groups, ech treted with one nemtode species t one prticulr dte, were distributed over one experimentl plot. The seventh group served s control nd ws not treted with nemtodes. The EPN suspensions were poured by hnd in continuous strem into 10 cm deep groove dug into the soil directly long ech row. When pplied t the erlier dte in April/My, this ws done t the sme time s mize ws hnd-sown. Suspended in 0.2 l of wter, ± 0.07 SD infective juvenile nemtodes were pplied per metre. At the lter EPN ppliction dte in June, they were suspended in 0.2 l of wter nd ± 0.07 SD infective juvenile nemtodes were pplied per metre. All pplictions were crried out in the evening or during cloudy fternoons to void hrmful UV rdition. Effects of EPN ppliction, EPN species, nd mize vriety on WCR dult emergence Ech of the 14 experimentl groups (6 7 plnts) of fields A to C (becuse of technicl problems, emergence ws not ssessed in field D) ws covered with fine-mesh screen cge (1.3 m height m width m length, mize plnts hd been cut to height of 1 m). WCR dult emergence within these cges ws recorded weekly between 20 June nd 16 August 2005 nd between 27 June nd 16 August Totl dult emergence ws normlised to 100 eggs per plnt. Nemtode efficcy ws clculted s percentge reduction in WCR dults compred to their untreted controls (corrected efficcy% = (1 - WCR in treted plots/wcr in the control) 9 100) (Abbott 1925). Effect of ppliction time, nemtode species, nd mize vriety on root dmge by D. v. virgifer In mid-september, fter dult emergence ws completed, field cges were removed nd ll plnts of ech group were dug up. Plnts from field D were lso used for this prt of the experiment. Soil nd other prticles were removed from the roots using high-pressure wter spryer. Dmge ws rted ccording to Oleson s Node Injury Scle from 0.00 to 3.00 with 0.00 being no dmge nd 3.00 being three or more dmged root nodes (Oleson et l. 2005). The efficcy of EPNs ws clculted s percentge reduction in root dmge compred to the respective control groups tht did not receive ny nemtodes (corrected efficcy % = (1 - root dmge in treted plots/root dmge in the control) 9 100) (Abbott 1925).

4 4 Olfctometer ssys Following the methodology developed by Rsmnn et l. (2005), ttrction of H. bcteriophor ws ssessed in six belowground olfctometers filled with moist snd. EPNs hd to choose between Pctol mize plnt dmged by four WCR lrve, helthy Pctol mize plnt nd four empty control pots. After 1 dy of exposure, the olfctometers were disssembled, the snd from ech of the six connectors ws plced in Bermnn extrctor (Hss et l. 1999), nd the next dy, nemtodes were counted under microscope on counting plte. Sttisticl nlyses % of reduction of WCR dults emergence A B mgister pctol mgister pctol mgister pctol H. bcteriophor H. megidis S. feltie Mize vrieties nd EPN species b B The effect of the tested prmeter (EPN species, ppliction periods nd mize vrieties) on reduction of WCR emergence nd root dmge ws nlysed using three-wy ANOVA. Then EPN species, mize vrieties nd ppliction periods were compred using Tukey s post hoc tests. All sttisticl tests of field dt were performed using SAS 9.1 with three-wy ANOVA (GLM procedure) with EPN species, ppliction period, mize vriety, EPN species 9 ppliction period, EPN species 9 mize vriety, ppliction period 9 mize vriety nd EPN species 9 ppliction period 9 mize vriety s independent vribles nd WCR emergence (reltive to control) nd node injury rte (reltive to control) s dependent vribles. Differences were nlysed using LSMEANS with Tukey Krmer djustments for the P vlues (SAS 9.1). The nemtodes behviourl responses in the six-rm olfctometer were tested with log-liner model. The entity computing repetition in the sttisticl nlysis corresponds to the response of group of 2,000 nemtodes relesed, which ws shown to follow multinomil distribution. As the dt did not conform to simple vrince ssumptions implied in using the multinomil distribution, we used qusilikelihood functions to compenste for the over dispersion of nemtodes within the olfctometer (Turlings et l. 2004). The model ws fitted by mximum qusi-likelihood estimtion in the softwre pckge R ( nd its dequcy ws ssessed through likelihood rtio sttistics nd exmintion of residuls (Turlings et l. 2004). Results Effect of EPN ppliction, EPN species, nd mize vriety on WCR dult emergence All tested EPN species significntly reduced the percentge of emerging D. v. virgifer, nd the time of ppliction hd no mjor effect on their respective efficcies (Fig. 1; Tble 2). Fig. 1 Comprison of the reduction of WCR emergence reltive to the untreted controls in mize fields with the EbC-emitting Mgister vriety nd the non-emitting Pctol vriety (pooled dt for the two relese dtes). Uppercse letters indicte sttisticl differences between the three EPN species (Tukey post hoc test, H. bcteriophor vs. H. megidis P \ 0.001, H. bcteriophor vs. S. feltie P \ nd H. megidis vs. S. feltie P = 0.70). Lowercse letters bove brs indicte sttisticl differences between mize vrieties within ech EPN species (Tukey post hoc test, H. bcteriophor Mgister vs. Pctol P = 0.08, H. megidis Mgister vs. Pctol P \ 0.001, S. feltie Mgister vs. Pctol P = 0.12). Error brs represent the stndrd error of men Overll, WCR emergence ws significntly different between the two mize vrieties (Fig. 1; Tble 2) with lower emergence from rows with the EbC-emitting Mgister thn from rows with the non-emitting Pctol. H. megidis reduced WCR emergence 2.5-fold more in Mgister plots thn in Pctol plots (P \ 0.001). There ws no significnt difference in the efficcy of H. bcteriophor nd S. feltie between the two mize vrieties (Fig. 1, P = 0.08 nd P = 0.12, respectively). On verge, the reduction in WCR emergence ws higher for plots treted with H. bcteriophor thn for plots treted with either H. megidis or S. feltie (Fig. 1; Tble 2). This ws reflected in n verge WCR emergence per plnt, which ws 0.65 nd 0.75 dult WCR per 100 eggs for the Mgister rows treted with H. megidis nd S. feltie, respectively, versus 0.28 dults for rows treted with H. bcteriophor (1.13 WCR dults emerged per plnt from Mgister control rows). In Pctol rows, 0.3 WCR dults emerged when treted with H. bcteriophor, wheres on verge 0.8 WCR dults emerged from Pctol rows when treted with either H. megidis or S. feltie (1.60 WCR dults emerged per plnt from Pctol control rows). Effect of EPN ppliction, EPN species, nd mize vriety on root dmge by D. v. virgifer All tested EPN species significntly reduced root dmge cused by WCR lrve, nd the time of ppliction hd no

5 5 Tble 2 Effects of EPN species, ppliction period nd mize vriety on WCR dult emergence (% efficcy reltive to control) ccording to the three-wy ANOVA Fctor Sum of squres df Men of squres F P EPN species \0.001*** Appliction period Mize vriety ** EPN species 9 ppliction period EPN species 9 mize vriety Appliction period 9 mize vriety EPN species 9 ppliction period 9 mize vriety Significnce is indicted by sterisks % of reduction in root dmge effect on their efficcies (Fig. 2; Tble 3). However, the efficcy of the nemtodes ws different for the two mize vrieties (Fig. 2; Tble 3). This difference ws due to H. megidis nd S. feltie, which reduced root dmge to higher degree in rows with the EbC-emitting Mgister thn in rows with the non-emitting Pctol (Fig. 2; Tble 3). Olfctometer ssys A mgister pctol mgister pctol mgister pctol H.bcteriophor H. megidis S. feltie When offered choice between voltiles emitted by WCR-dmged Pctol mize plnt or helthy Pctol plnt, H. bcteriophor preferred the rm with the pest feeding on the roots (Fig. 3, ANOVA, F 2,33 = 6.6, P \ 0.001). Surprisingly, the helthy plnts were found to be repellent, evidenced by the fct tht fewer nemtodes were collected from rms connected to pots with helthy B Mize vrieties nd EPN species Fig. 2 Comprison of the reduction of root dmge reltive to the untreted controls in mize fields with the EbC-emitting Mgister vriety nd with non-emitting Pctol vriety (pooled dt for the two relese dtes). Uppercse letters indicte sttisticl differences between the three EPN species (Tukey post hoc test, H. bcteriophor vs. H. megidis P = 0.012, H. bcteriophor vs. S. feltie P = nd H. megidis vs. S. feltie P = 0.480). indictes sttisticl differences between mize vrieties within ech EPN species (Tukey post hoc test, H. bcteriophor Mgister vs. Pctol P = 1.00, H. megidis Mgister vs. Pctol P = 0.042, S. feltie Mgister vs. Pctol P = 0.024). Error brs represent the stndrd error of men b AB b plnts thn from rms connected to the control pots with snd only. Discussion The results from the field experiment confirm tht the choice of mize vriety nd/or nemtode species cn significntly ffect the control efficcy of EPNs. Kurtz et l. (2009) hd lredy compred the efficcy of the three nemtode species ginst WCR in lbortory study nd lso reported tht WCR ws most susceptible to H. bcteriophor. EPN persistence in the soil hs been shown to rpidly decrese with time (Kurtz et l. 2007). It ws, therefore, surprising to find tht there ws no difference in the efficcy of EPN between the two ppliction periods (during sowing in April/My or lter in June) (Tbles 1, 2). Apprently some erly pplied nemtodes persisted, probbly by producing new genertion on lterntive hosts tht resulted in sufficiently high bundnce to reduce the lter htching WCR popultion. For inundtive biologicl control strtegies, it remins essentil to find the optiml dose nd relese timing (Fenton et l. 2002). The choice of the right mize vriety seems prticulrly importnt for two of the three EPN species investigted, H. megidis nd S. feltie (Figs. 1, 2). H. megidis ws more effective ner the EbC-emitting Mgister vriety ws expected from the results of previous studies (Rsmnn et l. 2005; Rsmnn nd Turlings 2007). However, tht this ws lso the cse for S. feltie ws surprising, s S. feltie is considered to minly use the so-clled mbusher (nictting) forging strtegy. Although S. feltie is known to ctively move through soil (Grewl et l. 1994; Lewis 2002), it never responded to ny cues in olfctometer experiments (Rsmnn nd Turlings 2008; personl observtions), suggesting tht they were not very mobile or not responding to the compounds tested. S. feltie hs been shown to be effective ginst WCR (Kurtz et l. 2009). Yet, the trend of reduced of dult emergence nd significnt reduction of root dmge for the Mgister plnts

6 EPN per rm 6 Tble 3 Effects of EPN species, ppliction period nd mize vriety on WCR s root dmge (% efficcy reltive to control) ccording to the three-wy ANOVA Fctor Sum of squres df Men of squres F P EPN species * Appliction period Mize vrieties ** EPN species 9 ppliction period EPN species 9 mize vriety Appliction period 9 mize vriety EPN species 9 ppliction period 9 mize vriety Significnce is indicted by sterisks WCR infested helthy control pots Pctol plnt Fig. 3 H. bcteriophor is ttrcted by EbC non-emitting plnts. When offered choice between helthy, WCR dmged Pctol plnt or snd, this nemtode species is significntly ttrcted towrds the dmged plnt even if no EbC is emitted (ANOVA, F 2,33 = 6.6, P \ 0.001). The helthy plnt ppers to repel H. bcteriophor compred to the control pots filled with snd only. Letters indicte sttisticl differences. Error brs represent the stndrd error of men fter S. feltie ppliction suggests tht this nemtode lso responds to this root signl under field conditions. However, S. feltie forging efficiency might lso hve been ffected by other spects, such s WCR lrvl behviour, ffected by Mgister plnts. The combined results from these studies suggest tht S. feltie forging behviour is strongly determined by the medi in which it hs to mbush or cruise. The effectiveness of H. bcteriophor ws not ffected by EbC emission from WCR-dmged mize roots (Figs. 1, 2) suggesting rndom nd uniformed spred of the nemtode in the field. However, when offered Pctol plnt infested with WCR lrve (no emission of EbC) or helthy Pctol plnt, H. bcteriophor significntly migrted more towrd the dmged plnt (Fig. 3), implying tht it uses other chemicl cues for host loction. These cues might come from the plnts, but lso from the hosts themselves. These nd other (Rsmnn nd Turlings 2008) b c olfctometer ssys show tht helthy mize roots re repellent to H. bcteriophor. This could imply tht it uses unique nd potentilly highly efficient host loction strtegy. It remins unknown wht signls llow H. bcteriophor to mke the distinction, but it hs been shown tht it is sensitive to long-chin lcohols nd possibly other, more insect specific, voltiles (O Hllorn nd Burnell 2003). Current WCR mngement strtegies involve crop rottion nd the use of insecticides (Levine nd Oloumi- Sdeghi 1991), but WCR hs shown the bility to evolve resistnce to both these methods (Bll nd Weekmn 1962; Meinke et l. 1998; Zhou et l. 2002; Levine et l. 2002; O Nel et l. 2001). Moreover, soil insecticides tht re still effective pose environmentl nd humn helth risks. Recently, geneticlly modified mize expressing Cry3 proteins, Bcillus thuringiensis toxin ginst WCR lrve, hs become vilble on US mrket (Moellenbeck et l. 2001). Bt mize ppers to be effective ginst WCR, reducing popultions of by 80 96% in the field/lb (Siegfried et l. 2005; Storer et l. 2006; Vughn et l. 2005). This high but incomplete efficcy cn be expected to led to rpid resistnce to Bt-mize in WCR popultions. While some models estimte tht resistnce will not occur until t lest 20 yers fter frmers strt growing Bt mize with 5 10% refuge (Storer et l. 2006), others hve shown tht resistnce developed within three genertions under greenhouse conditions (Meihls et l. 2008). In the current study, we show tht the synergetic effect of using the pproprite EPN species combined with ttrctive mize vrieties cn result in control of WCR tht is lmost s effective s the use of pesticides or Bt mize (Figs. 1, 2). WCR popultions re unlikely to be ble to develop resistnces ginst EPNs. Moreover, EPNs re ble to infect nd kill ll the lrvl instrs of WCR (Jckson nd Brooks 1995, Kurtz et l. 2009; Toepfer et l. 2005), wheres trnsgenic mize seems to be efficient only ginst the first instr (Oyedirn et l. 2005). Neonte WCR lrve my survive on neighbouring weed roots nd s second instr lrve could move bck to the Bt mize

7 7 roots on which they cn survive (Moeser nd Vidl 2004, Oyedirn et l. 2005). In contrst, EPN will lso be effective ginst WCR lrve on roots of other plnts (Christen et l. 2007; Gugler nd Cmpbell 1991; Re et l. 2006; Rmos-Rodriguez et l. 2007). Conclusion In conclusion, the efficcy of the tested EPN species in controlling WCR popultions is promising. Bsed on our findings, it should be possible for frmers to mtch their crops with the most effective nemtode. Further studies re needed to tke optiml dvntge of the biology nd behviourl plsticity of EPN to mximise their persistence nd their responses to plnt-provided signls in the soil. Acknowledgments This work ws possible thnks to the hospitlity of the Plnt Helth Service in Hodmezovsrhely in Hungry, offered by Iboly Zseller, Jozsef Gvllier, Ktline Buzs, Erzsebet Dormnnsne, Pirosk Szbo, Andrs Vrg nd others. We would lso like to thnk the summer students Bobe Kovcs, Benedikt Kurtz, nd Ferenc Koncz for their help in field work, s well s Arne Peters (e-nem GmbH, Germny) nd Erich Frnk (Andermtt Biocontrol AG, Switzerlnd) for providing nemtodes. This study ws funded by the CTI Innovtion nd Technology Fund of Switzerlnd in collbortion with Lndi REBA (Bsel, Switzerlnd) nd e-nem GmbH (Risdorf, Germny). References Abbott WS (1925) A method of computing the effectiveness of n insecticide. J Econ Entomol 18: Agrwl AA (1998) Induced responses to herbivory nd incresed plnt performnce. Science 29: Artchige NS, Lesn I, Sbelis MW (2004) Below-ground plnt prts emit herbivore-induced voltiles: olfctory responses of predtory mite to tulip bulbs infested by rust mites. Exp Appl Acrol 33:21 30 Bldwin IT, Preston CA (1999) The eco-physiologicl complexity of plnt responses to insect herbivores. Plnt 208: Bll HJ, Weekmn GT (1962) Insecticide resistnce in the dult western corn rootworm in Nebrsk. J Econ Entomol 55: Bernsconi ML, Turlings TCJ, Ambrosetti L, Bssetti P, Dorn S (1998) Herbivore-induced emissions of mize voltiles repel the corn lef phid, Rhoplosiphum midis. Entomol Exp Appl 87: Bertin C, Yng X, Weston LA (2003) The role of root exudtes nd llelochemicls in the rhizosphere. Plnt Soil 256:67 83 Boff MIC, Zoon FC, Smits PH (2001) Orienttion of Heterorhbditis megidis to insect hosts nd plnt roots in Y-tube snd olfctometer. Entomol Exp Appl 98: Christen JM, Cmpbell JF, Lewis EE, Shpiro-Iln DI, Rmswmy SB (2007) Responses of the entomopthogenic nemtode, Steinernem riobrve to its insect hosts, Glleri mellonell nd Tenebrio molitor. Prsitology 134: D Alessndro M, Turlings TCJ (2005) In Situ modifiction of herbivore-induced plnt odors: novel pproch to study the ttrctiveness of voltile orgnic compounds to prsitic wsps. Chem Senses 30: Degenhrdt J, Hiltpold I, Köllner TG, Frey M, Gierl A, Gershenzon J, Hibbrd BE, Ellersieck MR, Turlings TCJ (2009) Restoring mize root signl tht ttrcts insect-killing nemtodes to control mjor pest. Proc Ntl Acd Sci USA 106: Dicke M, Sbelis MW (1998) How plnts obtin predtory mites s bodygurds. Neth J Zool 38: Dicke M, vn Poecke RMP, de Boer JG (2003) Inducible indirect defence of plnts: from mechnisms to ecologicl functions. Bsic Appl Ecol 4:27 42 Fenton A, Gwynn RL, Gupt A, Normn R, Firbirn JP, Hudson PJ (2002) Optiml ppliction strtegies for entomopthogenic nemtodes: integrting theoreticl nd empiricl pproches. J Appl Ecol 39: Gugler R, Cmpbell JF (1991) Selection for enhnced host-finding of scrb lrve (Coleopter, Scrbeide) in n entomopthogenic nemtode. Environ Entomol 20: Grewl PS, Lewis EE, Gugler R, Cmpbell JF (1994) Host finding behvior s predictor of forging strtegy in entomopthogenic nemtodes. Prsitology 108: Hss B, Griffin CT, Downes MJ (1999) Persistence of Heterorhbditis infective juveniles in soil: comprison of extrction nd infectivity mesurements. J Nemtol 31: Hiltpold I (2008) Mnipultion of tritrophic interctions: key for belowground biologicl control? Ph.D. disserttion. University of Neuchâtel, Switzerlnd Hiltpold I, Turlings TCJ (2008) Belowground chemicl signlling in mize: when simplicity rhymes with efficiency. J Chem Ecol 34: Hobllh ME, Turlings TCJ (2005) The role of fresh versus old lef dmge in the ttrction of prsitic wsps to herbivore-induced mize voltiles. J Chem Ecol 31: Jckson JJ, Brooks MA (1995) Prsitism of western corn rootworm lrve nd pupe by Steinernem crpocpse. J Nemtol 27:15 20 Jnzen DH (1966) Coevolution of mutulism between nts nd ccis in Centrl Americ. Evolution 20: Krbn R, Bldwin I (1997) Induced responses to herbivory. University Press of Chicgo, Chicgo Krbn R, Agrwl AA, Mngel M (1997) The benefits of induced defenses ginst herbivores. Ecology 78: Kurtz B, Toepfer S, Ehlers RU, Kuhlmnn U (2007) Assessment of estblishment nd persistence of entomopthogenic nemtodes for biologicl control of western corn rootworm. J Appl Entomol 131: Kurtz B, Hiltpold I, Turlings TCJ, Kuhlmnn U, Toepfer S (2009) Comprison of the infectiousness of entomopthogenic nemtodes mong lrvl instrs nd pupe of the western corn rootworm. Biocontrol 54:2 Levine E, Oloumi-Sdeghi H (1991) Mngement of Dibroticite rootworms in corn. Annu Rev Entomol 36: Levine E, Spencer JL, Isrd SA, Onstd DW, Gry ME (2002) Adpttion of the western corn rootworm, Dibrotic vigifer virgifer LeConte (Coleopter: Chrysomelide), to crop rottion: evolution of new strin in response to culturl mngement prctice. Am Entomol 48: Lewis EE (2002) Behviourl ecology. In: Gugler R (ed) Entomopthogenic nemtology, CABI Interntionl, pp Meihls LN, Higdon ML, Siegfried BD, Spencer TA, Miller NK, Sppington TW, Ellersieck MR, Hibbrd BE (2008) Incresed survivl of western corn rootworm, Dibrotic virgifer virgifer LeConte, on trnsgenic corn in three genertions of on-plnt greenhouse selection. PNAS 105: Meinke LJ, Siegfried BD, Wright RJ, Chndler LD (1998) Adult susceptibility of Nebrsk western corn rootworm (Coleopter: Chrysomelide) popultions to selected insecticides. J Econ Entomol 91:

8 8 Miller N, Estoup A, Toepfer S, Bourguet D, Lpchin L, Derridj S, Kim KS, Reynud P, Furln L, Guillemud T (2005) Multiple trnstlntic introductions of the western corn rootworm. Science 310: Moellenbeck DJ, Peters ML, Bing JW, Rouse JR, Higgins LS, Sims L, Nevsheml T, Mrshll L, Ellis RT, Bystrk PG, Lng BA, Stewrt JL, Koub K, Sondg V, Gustfson V, Nour K, Xu DP, Swenson J, Zhng J, Czpl T, Schwb G, Jyne S, Stockhoff BA, Nrv K, Schnepf HE, Stelmn SJ, Poutre C, Koziel M, Duck N (2001) Insecticidl proteins from Bcillus thuringiensis protect corn from corn rootworms. Nt Biotechnol 19: Moeser J, Vidl S (2004) Do lterntive host plnts enhnce the invsion of the mize pest Dibrotic virgifer virgifer (Coleopter: Chrysomelide, Glerucine) in Europe? Environ Entomol 33: Neveu N, Grndgirrd J, Nenon JP, Cortesero AM (2002) Systemic relese of herbivore-induced plnt voltiles by turnips infested by conceled root-feeding lrve Deli rdicum L. J Chem Ecol 28: O Hllorn DM, Burnell AM (2003) An investigtion of chemotxis in the insect prsitic nemtode Heterorhbditis bcteriophor. Prsitology 127: O Nel ME, Gry ME, Rtcliffe S, Steffey KL (2001) Predicting western corn rootworm (Coleopter: Chrysomelide) lrvl injury to rotted corn with pherocon AM trps in soybens. J Econ Entomol 94: Oerke EC (2006) Crop losses to pests. J Agric Sci 144:31 43 Oleson JD, Prk YL, Nowtzki TM, Tollefson JJ (2005) Node-injury scle to evlute root injury by corn rootworms (Coleopter: Chrysomelide). J Econ Entomol 98:1 8 Oyedirn IO, Hibbrd BE, Clrk TL (2005) Western corn rootworm (Coleopter: Chrysomelide) beetle emergence from weedy Cry3Bb1 rootworm-resistnt trnsgenic corn. J Econ Entomol 98: Piperno DR, Flnnery KV (2001) The erliest rcheologicl mize (Ze mys L.) from highlnd Mexico: new ccelertor mss spectrometry dtes nd their implictions. PNAS 98: Re RG, Robertson JF, Wilson MJ (2006) The chemotctic response of Phsmrhbditis hermphrodit (Nemtod: Rhbditid) to cues of Derocers reticultum (Mollusc: Gstropod). Nemtology 8: Rmos-Rodriguez O, Cmpbell JF, Christen JM, Shpiro-Iln DI, Lewis EE, Rmswmy SB (2007) Attrction behviour of three entomopthogenic nemtode species towrds infected nd uninfected hosts. Prsitology 134: Rsmnn S, Turlings TCJ (2007) Simultneous feeding by boveground nd belowground herbivores ttenutes plnt-medited ttrction of their respective nturl enemies. Ecol Lett 10: Rsmnn S, Turlings TCJ (2008) First insights into specificity of below ground tritrophic interctions. Oikos 117: Rsmnn S, Köllner TG, Degenhrdt J, Hiltpold I, Toepfer S, Kuhlmnn U, Gershenzon J, Turlings TCJ (2005) Recruitment of entomopthogenic nemtodes by insect-dmged mize roots. Nture 434: Schnee C, Köllner TG, Held M, Turlings TCJ, Gershenzon J, Degenhrdt J (2006) The products of single mize sesquiterpene synthse form voltile defense signl tht ttrcts nturl enemies of mize herbivores. PNAS 103: Schoonhoven LM, Jermy T, JJAv Loon (1998) Insect-plnt biology, from physiology to evolution. Chpmn & Hll, London Siegfried BD, Vughn TT, Spencer T (2005) Bseline susceptibility of western corn rootworm (Coleopter: Chrysomelide) to Cry3Bb1 Bcillus thuringiensis toxin. J Econ Entomol 98: Singh P, Moore RF (1985) Hndbook of insect rering. Elsevier, Amsterdm Sluyter A, Dominguez G (2006) Erly mize (Ze mys L.) cultivtion in Mexico: dting sedimentry pollen records nd its implictions. PNAS 103: Stpley L (1998) The interction of thorns nd symbiotic nts s n effective defence mechnism of swollen-thorn ccis. Oecologi 115: Storer NP, Bbcock JM, Edwrds JM (2006) Field mesures of western corn rootworm (Coleopter: Chrysomelide) mortlity cused by Cry34/35Ab1 proteins expressed in mize event nd implictions for trit durbility. J Econ Entomol 99: Toepfer S, Kuhlmnn U (2006) The life-tble of the invsive lien Dibrotic virgifer virgifer (Coleopter: Chrysomelide) in Centrl Europe. J Appl Entomol 103: Toepfer S, Gueldenzoph C, Ehlers RU, Kuhlmnn U (2005) Screening of entomopthogenic nemtodes for virulence ginst the invsive western corn rootworm, Dibrotic virgifer virgifer (Coleopter: Chrysomelide) in Europe. Bull Entomol Res 95: Turlings TCJ, Benrey B (1998) Effects of plnt metbolites on the behvior nd development of prsitic wsps. Ecoscience 5: Turlings TCJ, Wäckers F (2004) Recruitment of predtors nd prsitoids by herbivore injured-plnts. In: Crdé RT, Millr JG (eds) Advnces in insect chemicl ecology. Cmbridge University Press, London Turlings TCJ, Tumlinson JH, Lewis WJ (1990) Exploittion of herbivore-induced plnt odors by host-seeking prsitic wsps. Science 250: Turlings TCJ, Dvison AC, Tmò C (2004) A six-rm olfctometer permitting simultneous observtion of insect ttrction nd odour trpping. Physiol Entomol 29:45 55 vn Tol RWHM, vn der Sommen ATC, Boff MIC, vn Bezooijen J, Sbelis MW, Smits PH (2001) Plnts protect their roots by lerting the enemies of grubs. Ecol Lett 4: Vughn T, Cvto T, Brr G, Coombe T, DeGooyer T, Ford S, Groth M, Howe A, Johnson S, Kolcz K, Pilcher C, Purcell J, Romno C, English L, Pershing J (2005) A method of controlling corn rootworm feeding using Bcillus thuringiensis protein expressed in trnsgenic mize. Crop Sci 45: Vidl S, Kuhlmnn U, Edwrds R (2005) Western corn rootworm: ecology nd mngement. CABI Publishers, Wllingford Zhou XG, Schrf ME, Primi S, Meinke LJ, Wright RJ, Chndler LD, Siegfried BD (2002) Dignostic ssys bsed on estersemedited resistnce mechnisms in western corn rootworms (Coleopter: Chrysomelide). J Econ Entomol 95: