Experiences with entomopathogenic nematodes for the control of overwintering codling moth larvae in Germany

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1 Experiences with entomopthogenic nemtodes for the control of overwintering codling moth lrve in Germny J. Kienzle 1, J. Zimmer 2, F.Volk 3 nd C.P.W. Zebitz Abstrct Entomopthogenic nemtodes were tested for their potentil s tool in resistnce mngement of codling moth Cydi pomonell L. in orgnic fruit growing. In two field tests, the dults emerging from stems treted with nemtodes were monitored. In one tril, 90 % of efficcy could be shown. On-frm trils with fvourble wether conditions showed n efficcy bout % on fruit infesttion by codling moth in the yer following the ppliction. On-frm trils with unfvourble wether conditions showed no results. The fvourble wether conditions for the ppliction re discussed with reference to Germn conditions. Keywords: Cydi pomonell, entomopthogenic nemtodes, Steinernem feltie, Steinernem crpocpse Introduction The codling moth Cydi pomonell L. (CM) is n importnt pest in fruit growing in whole Europe. Some yers go, CM control in orgnic fruit growing seemed to be well estblished with combined strtegy of mting disruption nd the Cydi pomonell grnulovirus CpGV. In 2004, the first cse of resistnce ginst CpGV ws proven in two orgnic orchrds in Germny (Fritsch et l., 2005). Since then, resistnce mngement becme importnt lso for orgnic frming nd new tools for combintion strtegies in CM control should be found. In the orchrds with CpGV-resistnt CM popultions the level of infesttion ws very high nd exceeded rpidly n unmngeble level (level of no control). Thus, there ws gret interest in methods for the reduction of popultions. The efficcy of entomophgous nemtodes (EPN) reported by Lcey et l. (1998, 2005, 2006) ws shown under Germn conditions first time by Elis (2006). In project finnced by Deutsche Bundesstiftung Umwelt severl methods of CM control re tested for their potentil nd should be possibly improved. In this context, the potentil of EPN s tool for resistnce mngement in CM control in orgnic fruit growing should be exmined. Mteril nd Methods To get more informtion bout overwintering sites of CM lrve, 28 trees of n orchrd with high infesttion of CM were removed. The trees were seprted into four prts: roots, stem, middle prt, treetop. Ech prt ws stored seprtely in big boxes. In spring, before htching of CM, the boxes were wrpped in drk plstic mteril nd kept in brn out of the sun. At ech side of the box, glued honey glsses were pplied to ctch the htching moth (figure 1). The number of moths were ssessed t 16. July Kienzle, Jutt, Apfelblütenweg 28, D Kernen, jutt@jutt-kienzle.de 2 Zimmer, Jürgen, DLR Rheinpflz, KoG Ahrweiler, Wolporzheimer Str. 48, D Ahrweiler 3 Volk, Frnk, Biof AG,. Rudolf Diesel Str. 2, D Münsingen 4 Zebitz, C.P.W., University of Hohenheim, Institute for Phytomedicine, D Stuttgrt

2 278 For n exct evlution of the efficcy of the EPN on the overwintering popultion of CM, two field trils with rndomised block design nd 4 repets (10 trees per repet, 1 border tree) were conducted in orchrds with high infesttion of CM t the loctions A (ner Stuttgrt) nd B (ner Lke Constnce). At loction A, the trees of cv. Elstr were grfted on M9 (c. 10 yers old) with pile of pine wood t n intertree distnce of 1.50 m. The CM infesttion in 2006 ws bout 5-7 %. At loction B, the orchrd ws rther old with cv. Boskoop. Not ll trees hd piles. The infesttion t hrvest in 2006 exceeded 10 %. In loction A, the ppliction ws done with motorised knpsck spryer (SOLO) until runoff. Only the stem until 1 m height ws treted. In loction B, herbicide spryer with two fltfn nozzles with >0.5 mm orifice ws used for the tretment of the stem (1 m). Per m tree height, 500 l of wter per h were pplied with 4 br pressure. The mount of nemtodes ws clculted per m tree height. During pplictions, the rows were pssed lwys twice in opposite direction to ensure good wetting even of the inner prt of the stem. In both trils, 0,25 l of Trifolio-S forte per h nd m tree height ws dded s wetting gent. In loction A, the EPN were pplied t The tretment ws done in the evening when the stems nd the grss were humid with dew nd the RH reched 100 %. For the following dy, rinfll ws forecsted but did not occur. The temperture during the tretment ws bout 15 C, during the night it decresed reching 12 C, the following dy it oscillted from 13 to 19 C. In loction B, the ppliction ws done t lte in the evening when fog ws setting in. The treted stems did not dry. During the night nd the morning it ws foggy, the temperture in the evening t during ppliction ws 15 C nd discended to 9,5 C t 8.00.m. t the following morning. The stems strted drying bout m. next morning, the temperture t this time ws bout 12 C. The forecsted rin did not fll on the tril site. Figure 1: Eclectors for ssessment of overwintering CM in the field trils nd in the boxes For the ssessment, t the stem of ech tree shortly before the beginning of CM flight, n eclector with drk fleece mteril nd rubber fom ws pplied (figure 1). At the top of this eclector glued honey glss ws instlled to cpture the dult moths flying ginst the light. It seemed, tht the htching ws remrkbly retrded by the eclector. Furthermore, not ll moths went into the glss. Thus, when the eclector ws removed, the stem ws exmined crefully for reminings of htched pupe. It could hppen, tht dult moth were found nd no reminings of pupe nd lso pupe nd no dult moth.

3 279 Thus, for ech moth found in the glss, the number of pupe reminings ws subtrcted for the number of moth found to void double counting. A certin mistke is possible since evidently not ll pupe were found nd not ll moths went into the glss. Nevertheless, since this mistke ws distributed eqully over ll repetitions, the dt cn be used. The eclectors were pplied on 23./24./ in loction A nd on 25./ in loction B. In both orchrds, CM flight ws monitored with pheromone trps nd first moth were found in the week following the ppliction of the eclectors. The eclectors were removed the t loction A nd t t loction B. The on-frm trils were conducted in the Germn regions Rhinelnd-Pltinte (tril 1 nd 2) nd Bden-Wuerttemberg (tril 3). Since the experimentl design requires rther big plots nd the orchrds in these regions re rther smll, repetitions were not possible. The orchrds were just divided in two prts trnsverse to the tree rows. Before hrvest in 2006, the CM infesttion ws ssessed. In ll orchrds, there were only mrginl differences in fruit infesttion between the plots used for the trils. If only the stems were treted (1 m tree height), the ppliction ws crried out with herbicide spryers with fltfn nozzles nd specil construction (figure 2). If the whole tree ws treted, norml spryer with fltfn nozzles ws used. In ll trils, 0,25 l of Trifolio-S forte per h nd m tree height ws dded s wetting gent. In 2007, in tril 1 nd 2 the min ssessment ws done during hrvest. In ech tretment, complete big box (c 500 kg) of fruits ws collected from mrked trees, which were spred when infested fruits were removed before hrvest nd CM infesttion ssessed. Thus, the economicl dmge for the grower cn be estimted very good. Figure 2: herbicide spryer with specil rrngement for tretment of stems In tril 3, the infesttion of fruits by first CM genertion ws ssessed on 1500 rndomly selected fruits per plot nd vriety. The sttisticl nlysis ws done by nlysis of vrince nd Tukey-Test (p<0.05). Results The study of the overwintering of CM presented some surprising results: From the boxes with the middle prt of the trees nothing emerged. One moth emerged from the box with the roots, 8 moth from the stems nd in the eclectors of the treetop box 15 moth were found. These results seemed surprising since the treetop seemed to present no hiding plces for the lrve. Thus, the content of the box ws exmined very crefully to find the exuvie of the pupe. It ws found, tht in the treetop severl fruits dmged by fungl diseses like Monili spec. hd remined from the yer before These fruits were used evidently by the lrve s hiding plces. It cn be excluded tht the fruits or the lrve were originry from fruits CM infested fruits stored in the boxes before since these were not used 2006.

4 280 N of codling moth htched from 10 tree stems b b b 90,9 90,9 90,9 93,0 29,8 Control SC 0,25 SF 0,25 SF 1,5 Control SF 0,75 SC 0,75 Loction A Loction B Figure 3: Number of codling moth htched nd degree of efficcy (ABBOTT) in % in the different tretments with Steinernem feltie (SF) nd S. crpocpse (SC) in the trils t the two loctions A nd B. The mount of EPN per h nd m tree height is given in billions (10 9 ). Different letters indicte significnt difference (TUKEY-test, p<0.05). Tble 1: Results of on-frm trils in the seson 2006/2007: Control of CM infested fruits in 2007 Tr. Nr Nemtode species N of EPN per h nd m tree height Tree height (prt of the tree treted) S. feltie 1,5 x m (stem) S. feltie 2 x m (whole tree) S. crpocpse Amount of wter per h Dte of ppliction 500 l l ,75 x m (stem) 500 l S. feltie 2 x m (whole tree) S. crpocpse 1800 l ,75 x m 500 l Wether conditions 14 C, 100 % RH, rin fter tretment 16 C, 80 % RH, rin before tretment C rinfll fter evening tretment wet during following period Degree of efficcy (ABBOTT) in % Gl: 68.2 Elstr: 65.8 Gl: 49.9 Elstr: 68.2 Gl: Elstr: -2.2 Gl: Elstr: 4.2 Idred: 19.4 Sommerregent: 12.7

5 281 In loction A of the field tril, ll EPN tretments showed significnt reduction of the surviving dults on the stems with high degrees of efficcy. There ws no difference between the high nd the low mount of S. feltie. In loction B, the CM popultion ws very high. Here the distribution between the different plots ws not so homogeneous nd there were some single trees tht seemed to be preferred hidewys for the dipusing lrve. There ws one tree with very high number of lrve in the control so tht the differences between the tretments were not sttisticlly significnt. Nevertheless, the tretment with S. feltie showed considerble reduction of the infesttion whilst there ws few effect shown of S. crpocpse (figure 3). In the on-frm tril 1 the results were rther good for stem nd whole tree tretment. There ws only mrginl difference between these two tretments. In tril 2 with worse wether conditions both tretment showed no efficcy. In tril 3 only stem ws treted. At the ssessment it could be observed tht the upper nd untreted prt of the trees hd eril roots nd some other hiding plces for dipusing lrve. Discussion These trils confirm in principle the findings of Elis et l (2006) nd Curto et l (2008) tht EPN ppliction on dipusing CM lrve cn reduce the fruit infesttion in the following yer bout %. The results from ssessment of the dults emerging from treted stems in the field trils suggest better efficcies. On of the resons might be tht in this cse the direct efficcy of EPN on the emerging dults ws ssessed. When the infesttion of fruits in the following yer is the ssessment criterium, orchrds with high CM popultions re chosen to chieve relible dt. If the emerging dults of very high popultion is reduced in the EPN plot, there my be less competition in this plot in comprison to the control plot. This could led to certin compenstion of the efficcy of EPN on the emergence of dults by more successful oviposition of the remining dults. Thus, even high efficcies of EPN on dult emergence my result in medium efficcies in fruit dmge in the orchrd. This effect could reverse if the popultion is not s high s in these trils nd in the yer fter the tretment mting disruption is pplied. In this cse, the EPN tretment cn reduce the popultion to level where mting disruption is more efficient. Thus, in orchrds with medium or rther low popultion levels, EPN ppliction should lso tested. In combintion with mting disruption it might prove to be n even more vluble tool in mintining low popultion levels thn it seems to be in the reduction of high popultions. The on-frm tril 3 showed no result lthough the wether conditions were fvourble. A possible reson might be in this cse, tht the upper prt of the tree remined untreted whilst it presented hiding plces for dipusing lrve. In tril 1, with different nd younger trees, the stem nd the whole tree-pplictions showed no difference even if the mount of EPN pplied per m tree height is not relly comprble. This indictes, tht the decision if only stem or whole tree is treted depends of the orchrd nd must be decided in ech specil conditions. More reserch should be done to find out the overwintering plces of the dipusing lrve. Our findings with the Moniliinfested fruits show, tht surprises cnnot be excluded nd tht n orchrd hs to be exmined very crefully before deciding to tret only the stem. The difference in efficcy between the on-frm tril 1 nd 2 cn be explined by less fvourble wether conditions in tril 2. On the other hnd, in the field trils in both loctions, there were considerble results even if the EPN were pplied without rinfll before or fter the tretment, just using the humidity of dewfll during the night. Elis lso hd good efficcy with lower umidity, pplying very high mount of wter.

6 282 Minimum Temperture in C xx Figure 4: Number of dys with fvourble wether conditions for EPN tretments (stems humid or wet, rin or RH > 95 % during lmost 9 h, minimum temperture > 8 (S. feltie) resp. 12 C (S. crpocpse); when the temperture discended under the minimum nd the humidity remined high, the period ws not considered s interrupted) In the tril in loction A, the lower efficcy of S. crpocpse might be due to the lower tempertures which were not lwys higher thn 12 C during the period fter ppliction. For most regions in Germny it must be considered tht frmers hve no possibility of irrigtion. Min experiences with EPN derive from Lcey et l. (1998, 2005, 2006). In his country it is possible to irrigte, nd the optiml conditions re esy to chieve with preppliction irrigtion. In our conditions, the wether conditions necessry for good efficcy of the tretment turn out to be key question for the successful use of EPN. Since there is evidence for efficcy problems in some cses of not relly fvourble wether conditions, the recommendtion for prctice ctully must be to tret only with relly fvourble conditions. There is certin consense tht EPN should not be pplied just t hrvest time of the fruits. In most orgnic orchrds there is lso need of mechnicl soil cultivtion to remove weeds from the bottom prt of the stem before EPN re pplied. This cn be done only fter hrvest when no fruits re dmged by pssing with the mchine. Thus, the period when EPN cn be pplied strts in mid of September (often lso mid of October depending of the end of hrvest). It is ssumed tht in Jnury the dipusing lrve strt to reinforce their cell for puption nd then it might be more difficult for EPN to penetrte the web. Results with spring pplictions of EPN indicte low efficcy (Curto et l., 2008) so tht it seems justified to fix the end of the possible period for EPN ppliction t the end of December. In figure 4 for the yers in loction ner Lke Constnce the dys re indicted when good wether conditions for tretments were given for the two EPN species S. feltie nd S. crpocpse. Especilly fter October there re few possible ppliction dtes, still fewer if S. crpocpse is used which requires higher minimum temperture (12 C, (Grewl et l., 1994). This sitution is not relly comfortble for n ppliction. Unfortuntely, none of the lbortory nd semi-field trils crried out in 2006 nd 2007 in the frme of this project to clrify the climtic conditions necessry for relible nd repetble efficcy gve relly sustinble results (Kienzle, Jung et l., unpublished results). Since the conditions refer not only to the surfce of the stem but minly to the hiding plces of the lrve they re very difficult to define.

7 283 Further reserch is necessry but it is possible tht to ensure efficcy prctice must lwys rely on the wether conditions mentioned bove. Actully, ppliction technique, which cn be improved since it is cler tht higher pressures cn be used ((Wright et l., 2005)) nd dditives re tested with the im to improve efficcy, especilly with not so fvourble wether conditions. Nonetheless the questions still to solve EPN seem to hve the potentil to become vluble tool for resistnce mngement in CM control in orgnic fruit growing. If they become prt of the common strtegy, it is importnt to understnd lso the effects on the ecosystem in the orchrd. It is well known tht EPN re hrmless for the common erwig (Forficul uriculri L.) which is most exposed to the stem pplictions (Nchtigll, 1991; Georgis et l., 1991, Grewl et l., 1993). A potentil effect on the whole rthropod system is difficult to monitor. In ech cse, the treted plots should be crefully observed during the introduction of the EPN in prctice. Acknowledgements We re indebted to the Deutsche Bundesstiftung Umwelt for finncil support of the project (Az 23940), to Andermtt Biocontrol for providing most nemtodes nd especilly to the stff of Andermtt Biocontrol for providing so mny CM lrve for the semilb trils we do not explin here. Furthermore, we re indebted to the growers for prticiption in the trils. References Elis, E.: Apfelwickler Regulierung durch sogennnte 'entomopthogene' Nemtoden. Oeko Obstbu 2006/1: Curto, G, Reggini, A., Vergnni, A., Cruso, S. Ldurner, E. (2008): Effectiveness of entomopthogenic nemtodes in the control of Cydi pomonell lrve in Northern Itly. In: Proceedings of the 13th Interntionl Conference on Cultivtion Technique nd Phytopthologicl Problems in Orgnic Fruit-Growing, in press Fritsch, E.; Undorf-Sphn, K.; Kienzle, J.; Zebitz, C.P.W.; Huber, J. (2004): Apfelwickler- Grnulovirus: Erste Hinweise uf Unterschiede in der Empfindlichkeit lokler Apfelwickler- Popultionen. Nchrichtenbltt Deut. Pflnzenschutzdienst, 57 (2),: Lcey L.A., Unruh T.R. (1998): Entomopthogenic nemtodes for control of codling moth: effects of nemtode species, dosge, temperture nd humidity under lbortory nd simulted field conditions. Biologicl Control 13: Lcey L.A., Unruh T.R. (2005): Biologicl control of codling moth (Cydi pomonell, Tortricide: Lepidopter) nd its role in integrted pest mngement, with emphsis on entomopthogens. Vedli 12: Lcey L.A., Arthurs S.P., Unruh T.R., Hedrick H., Fritts R. Jr. (2006): Entomopthogenic nemtodes for control of codling moth (Lepidopter: Tortricide) in pple nd per orchrds: effect of nemtode species nd sesonl tempertures, djuvnts, ppliction equipment nd post-ppliction irrigtion. Biologicl Control 37: Grewl, P.S., Selvn, S. & Gugler, R Therml dpttion of entomopthogenic nemtodes: niche bredth for infection, estblishment, nd reproduction. Journl of Therml Biology 19, Wright, D.J., Peters, A., Schroer, S. & Fife, J.P Appliction Technology. CAB Interntionl. Nchtigll, G. (1991). Untersuchungen zur Bekämpfung kryptisch lebender Insekten mit entomopthogenen Nemtoden unter besonderer Berücksichtigung von Synnthedon myopeformis Borkh. (Lep., Sesiide). Diss., TU Drmstdt, 1991, 145 S. Georgis et l. (1991). Effect of steinernemtid nd heterorhbditid nemtodes (Rhbditid: Steinermtide nd Heterorhbditide) on nontrget rthropods. Environmentl Entomology 20, Grewl et l. (1993). Infectivitiy of the entomopthogenic nemtode Steinernem scpterisci (Nemtod: Steinernemtide). Journl of Invertebrte Pthology 62,