Instituto Ncionl de Investigción y Tecnologí Agrri y Alimentri (INIA) Spnish Journl of Agriculturl Reserch 28 6(3), 434-44 Avilble online t www.ini.es/sjr ISSN: 1695-971-X Abstrct ontrolled in vivo infesttion of mndrin fruit with ertitis cpitt for development of qurntine tretments J. A. Jcs 1 *, L. Plou 2, F. Beiti 3 nd M. A. del Río 2 1 Unitt Associd d Entomologi Aplicd UJI-IVIA. Universitt Jume I (UJI). Deprtment de iènces Agràries i del Medi Nturl. mpus del Riu Sec. 1271 stelló de l Pln. Spin 2 entre de Tecnologi Postcollit. Institut Vlencià d Investigcions Agràries (IVIA). tr. Montcd-Nàquer, km 4,5. 46113 Montcd (Vlènci). Spin 3 Unitt Associd d Entomologi Aplicd UJI-IVIA. entre de Protecció Vegetl i Biotecnologi. Institut Vlencià d Investigcions Agràries (IVIA). tr. Montcd-Nàquer, km 4,5. 46113 Montcd (Vlènci). Spin Movement of citrus fruit from ertitis cpitt-infested res requires mndtory qurntine tretments. Development of such tretments requires the use of infested fruit. The in vivo pproch is the most relistic wy to obtin these fruit. However, it requires previous studies to determine the optiml fruit:fly rtio to minimize the number of decyed fruit nd to mximize the number of flies per fruit obtined. In this study, the optiml fruit:fly rtio for the in vivo infesttion of mndrin fruit ws investigted. The effect of different fruit:fly rtios from 1:5 to 1:5 for n exposure time of up to 3 dys on the number of both decyed fruit nd pupri per fruit ws determined. Provided tht n dequte fungicide tretment is pplied before the infesttion to void fruit decy, the use of 1:1 fruit:fly rtio for 48 h is enough to obtin lmost 2 helthy pupri per fruit. These results llow the use of the in vivo pproch to develop qurntine tretments ginst. cpitt in mndrins. Additionl key words: citrus, Mediterrnen fruit fly, posthrvest. Resumen Infestción in vivo controld de mndrins con ertitis cpitt pr el desrrollo de trtmientos curentenrios L frut producid en áres infestds por ertitis cpitt está sujet trtmientos curentenrios cuyo desrrollo requiere l utilizción de frut infestd. L mner más relist de obtener est frut es medinte técnics de infestción in vivo. Sin embrgo, previmente hy que relizr estudios pr determinr l proporción óptim frutos:mosc utilizr, de mner que se minimicen ls pérdids de frut por pudrición, y se mximice el número de moscs obtenido por fruto infestdo. En este estudio se h investigdo est proporción pr mndrins. Se h determindo el efecto de distints proporciones frutos:mosc, entre 1:5 y 1:5, durnte un periodo de hst 3 dís, tnto sobre el número de frutos podridos como sobre el de puprios obtenidos por fruto. Siempre que se plique un trtmiento fungicid fuerte previmente l infestción pr evitr pudriciones, el uso de un proporción frutos:mosc de 1:1 durnte 48 h es suficiente pr obtener csi 2 puprios snos por fruto. Estos resultdos permiten utilizr l infestción in vivo en el desrrollo de trtmientos curentenrios contr. cpitt en mndrins. Plbrs clve dicionles: cítricos, mosc mediterráne de l frut, post-recolección. * orresponding uthor: jcs@cmn.uji.es Received: 3-1-7; Accepted: 27-6-8.
. cpitt citrus infesttion for qurntine tretments 435 Introduction Among multitude of insect pests of qurntine importnce, fruit flies of the fmily Tephritide re probbly the most importnt group worldwide. The Mediterrnen fruit fly, ertitis cpitt (Wiedemnn), hs become the single most importnt pest species in the fmily becuse of its worldwide distribution. In Mediterrnen countries, it is prticulrly dmging on citrus nd peches. ertitis cpitt is thought to hve originted in the Pleotropicl region from where it spred to the Mediterrnen bsin nd prts of entrl nd South Americ nd Austrli (EPPO, 27). However there re some res in Asi, Oceni nd Americ which remin free of. cpitt (EPPO, 27). When host fruit such s citrus from. cpittinfested res re shipped to these pest-free res where the fly could become estblished, fruit must be subject to qurntine tretment ensuring tht no vible insects re present t destintion. The most widely used posthrvest disinfesttion tretment for citrus ginst this fruit fly involves exposure of the fruit to nerfreezing tempertures. In the cse of the USA, the U.S. Deprtment of Agriculture estblished minimum exposure during overses trnsit of 14-18 dys t 1.1-2.2 (USDA, 22). Extensive reserch is currently focused on the development of lterntive or complementry qurntine tretments, especilly for cold sensitive commodities such s citrus. As consequence lterntive or dditionl tretments re under development for Spnish citrus exports (Alonso et l., 25, 27; Plou et l., 27, 28). Although qurntine tretment idelly would be devised using ferl insects infesting the fruit nturlly, this pproch is rrely considered becuse of the difficulty in obtining the sufficient number of insects of the correct stge in good condition (Hllmn, 24). Therefore, fruit fly lrve for development of qurntine tretments hve been usully obtined following one of the following procedures (Hllmn nd Lohrnu, 22): () in vitro with or without rering medium (e.g. Block et l., 1963; Benschoter, 1987; Shrp nd hew, 1987; Mnsour nd Frnz, 1996), (b) rered on one medium followed by insertion into fresh fruit (e.g. Kmburov, 1972; Alonso et l., 22,b, 25b; Plou et l., 27, 28), nd (c) in vivo rered in rtificilly infested fruit (e.g. Seo et l., 1973; Windeguth nd Gould, 199; Hllmn nd Worley, 1999; Hllmn nd Mrtínez, 21; Agnello et l., 22). The ltter pproch is priori the most relistic cse, but requires studies to determine the optiml fruit to fly rtio (fruit:fly rtio henceforth) during infesttion nd the durtion of the exposure to minimize the number of fruit where the life cycle of. cpitt will not be completed. An importnt spect of this study is to counter fruit decy which cn significntly ffect fruit fly survivl. It is importnt to hve sufficiently high number of flies per fruit to llow robust sttisticl nlyses. Hllmn nd Worley (1999) nd Hllmn nd Mrtínez (21) in their studies with grpefruit (itrus prdisi Mcf.) nd the Mexicn fruit fly, Anstreph ludens Loew., used fruit:fly rtio rnging from 1:5 to 1:1 for period of 24 to 48 h. However these uthors did not provide ny dt to estimte the number of decyed fruit, or the men number of flies obtined per fruit. Spnish citrus exports to. cpitt-free countries consist mostly of mndrins, especilly clementines (76 1 3 Mg during the 26-7 seson; Font de Mor, 27), such s lemenules or Mrisol cultivrs. Recent mrket ccess nd posthrvest studies hve focused on these citrus cultivrs (Alonso et l., 22b, 25b, 27; Plou et l., 27, 28) nd did not use the in vivo pproch. Before such n pproch could be used it is necessry to define n efficient infesttion procedure. Therefore the objective of this reserch ws to estblish convenient fruit:fly rtio nd pproprite exposure times for the development of qurntine tretments in mndrins. Mteril nd Methods ertitis cpitt Insects used in this ssy originted from lbortory colony estblished in 21 t the Institut Vlencià d Investigcions Agràries (IVIA). This colony hs been periodiclly supplemented with the introduction of wild flies from nturlly-occurring infested fruit during summer nd fll. Adult. cpitt were rered in controlled environment cbinet t 25 ± 1 nd 75 ± 5% reltive humidity under illumintion by fluo- Abbrevitions used: i (ctive ingredient), I (color index), EPPO (Europen Plnt Protection Orgniztion), IVIA (Institut Vlencià d Investigcions Agràries), MAPA (Ministerio de Agricultur, Pesc y Alimentción), SE (stndrd error), USDA (United Sttes Deprtment of Agriculture)
436 J. A. Jcs et l. / Spn J Agric Res (28) 6(3), 434-44 rescent tubes (Sylvni F-18W/Grolux; 16 h dy -1 ; 2,5 lux). Unless otherwise stted, the sme environmentl conditions were pplied to the ll ssys reported in this study. Flies were kept in perspex cges (4 4 3 cm) with density of 2, flies per cge. These cges hd two round holes (8 cm in dimeter) on the upper side covered by mesh nd the front covered by guze used by femles for oviposition. Wter nd diet consisting of mixture of enzymtic utolyzed brewer s yest nd sugr (1:4, w:w) were supplied to the flies. Eggs were lid through the guze nd fell into dish contining wter from which they were collected dily by filtering (Jcs nd Viñuel, 1994). Immture stges were rered on n rtificil diet contining 4 g of whet brn, 112 g of sugr, 58 g of brewer s yest, 4.5 g of methyl prben, 4.5 g of propyl prben, 4 g of benzoic cid, nd 9 ml of wter, using density of 4 eggs g -1 diet (Alonso et l., 22). Mture pupri were used in ll ssys. Infesttion Infesttion took plce in plstic cges (2 2 3 cm) with lid consisting of guze hold in plce by frme. A glss vil (5 cm high nd 1 cm in dimeter) contining (control) to 5 pupri of. cpitt ws fixed with tpe to one of the corners of the cge. Wter ws supplied in 5 ml glss Erlenmeyer with wick. The sme diet s described for the rering ws spred on top of the lid for dults to feed d libitum. Five petri dishes ech contining 1 pupri were ssembled to ssess dult emergence nd sex rtio. Upon fly emergence, cges were checked dily until newly lid eggs were observed outside the guze. Two dys lter, both cges nd fruit were prepred for the infesttion. Fruit from orchrds in the Vlenci re were hrvested t commercil mturity nd trnsferred to the IVIA posthrvest fcilities where they were sorted, rndomized, wshed with tp wter nd dipped in fungicide solution for 1.5 min (s described in Tble 1). After the fruit hd ir dried, groups of 1 fruits were introduced in the cges which hd been previously cooled t 12 for 1 h to prevent flies from flying wy during mnipultion. After the introduction of the fruit t 25, the cges were left undisturbed for 24 to 72 h depending on the ssy. After this time, fruit were removed from the cge nd individully plced in 1-L cloth covered plstic boxes where both decyed fruit nd the number of pupri were scored fter 2 dys. From these dt, the percentge of decyed fruit per cge nd the number of pupri per fruit were clculted. Three seprte experiments were conducted, ech one using the mndrin cultivr tht ws commercilly vilble t tht time (Tble 1). The first experiment used clementine mndrins (itrus reticult Blnco) cv. Mrisol previously treted with the fungicide imzlil sulphte t concentrtion of.5% (Fecundl-S 7.5, Jnssen Phrmceutic N.V., Beerse, Belgium) nd were exposed to either, 5, 1, 2, 3 or 5 flies per fruit for one dy. Three cges per fruit:fly rtio were used. The second experiment used hybrid mndrins cv. Nov [. reticult (. prdisi. reticult)] treted with mixture of the fungicides imzlil sulphte t 2.5% (Fecundl-S 7.5), guztine cette t.8% (Textr 2G, Tecnidex S.A., Ptern, Vlenci, Spin) nd thibendzole t 1.5% (Tebezet-45, Fomes Fruitech S.L., Vlenci, Spin) nd exposed to either, 5, 1, 2 or 3 flies per fruit for either one, two or three dys. One cge per ech combintion dose/ exposure ws used. The third experiment used clementine mndrins cv. lemenules which received fungi- Tble 1. Assys performed: posthrvest fungicide dip tretments pplied before rtificil infesttion, men emergence (percentge ± SE) nd sex rtio (percentge femles ± SE) of the flies used, nd physicl chrcteristics of infested fruit. Dt followed by the sme letter were not significntly different ccording to Duncn s multiple comprison procedure (P.5) Fruit Emergence Sex rtio Rind width Rind color Assy cultivr Fungicide tretment (%.i.) (%) (% femles) (mm) index 1 tested (n = 5) (n = 5) (n = 1) (n = 35) 1 Mrisol Imzlil sulphte (.5%) 94. ± 6. 52.6 ± 5.6 2.19 ±.7b 7.5 ± 1.65 2 Nov Imzlil sulphte (2.5%) + guztine cette (.8%) + thibendzole (1.5%) 88. ± 8. 51.8 ± 3.5 2.78 ±.21 22. ± 1.5 3 lemenules Imzlil sulphte (2.5%) + guztine cette (.8%) + thibendzole (1.5%) 96. ± 2.4 5. ± 3.2 2.1 ±.13b 16.56 ±.5 1 I = 1/L*b (Hunter prmeters).
. cpitt citrus infesttion for qurntine tretments 437 cide tretment s in the previous ssy nd exposed to either, 5, 1, 2, 3 or 5 flies per fruit for two dys. Three cges per fruit:fly rtio were used. Ech cge ws considered s single replicte for decyed fruit nlysis wheres ech fruit constituted replicte for the nlysis of the number of pupri per fruit. Prior to infesttion, rind color of 35 fruit ws mesured s Hunter prmeters (L,, b) using colorimeter (Minolt, Model R-3). For ech fruit, three mesurements long the equtoril re were performed. The specific color index (I) for citrus ws clculted s I = 1/L*b (Jiménez-uest et l., 1981). The rind width ws mesured using digitl Vernier cliper, where 1 fruits were equtorilly divided in two hlves nd one mesurement ws conducted on ech hlf. Sttisticl nlyses Dt were subjected to either one- or two-wy nlyses of vrince (ANOVA) nd, where pproprite, mens were seprted by Duncn s multiple comprison procedure (P.5). If necessry, dt were rcsinetrnsformed before nlyses to prevent the violtion of the ssumptions underlying ANOVA (Sokl nd Rohlf, 2). Anlyses were performed using the Sttgrphics Plus 4.1 softwre pckge (Mnugistics Group Inc., Rockville, MD, USA). Results Both rind width nd color of the fruit used in these ssys depended on the vriety used (Tble 1). Rind width rnged from 2. to 2.8 nd Nov mndrins were significntly the thickest ones. Rind color depended on the vriety used nd rnged from 7.5 to 22. for Mrisol nd Nov mndrins, respectively. The dult emergence nd sex rtio of the flies used to infest those fruit re shown in Tble 1. Emergence rnged from 88. to 94.% nd round 5% of these flies were femles. These vlues fit within the vlues usully obtined under lbortory conditions. Bsed on the percentges of dult emergence obtined, the ctul fruit: fly rtios chieved in our ssys were slightly lower thn desired (from 4.4 to 48 flies per fruit insted of 5 to 5 flies per fruit, respectively). The percentge of decyed fruit ws very high in the first experiment (Fig. 1) nd this level of decy ws dependent on the fruit:fly rtio used for infesttion (F = 15.66; df = 5, 12; P <.1). There ws no decy Rottenn fruit (%) 1 8 6 4 2 b cd A A A A 5 1 2 3 5 Flies per cge (#) Rotten fruit in the control fruit but it significntly incresed from 3.% to 63.3% for 1:5 nd 1:5 rtios, respectively. Such high percentge of decy drsticlly reduced the number of fruit were. cpitt lrve could complete their life cycle. As consequence, the men number of pupri obtined per fruit ws very low (.55 individuls) irrespective of the fruit:fly infesttion rtio used (F = 1.16; df = 3, 5; P =.418). Becuse of the high percentge of decyed fruit obtined in the first ssy, fruit used in subsequent ssys were subjected to more intense fungicide regime (Tble 1). This new tretment drmticlly reduced the percentge of decyed fruit (Fig. 2 nd 3) nd s consequence, the number of lrve tht could complete their development incresed (Fig. 4). This depended on both the fruit:fly rtio used (F = 6.92; df = 4, 135; P <.1) nd the length of exposure (F = 6.16; df =2, 135; P <.1) but there ws no interction between these two fctors (F = 1.77; df = 8, 135; P =.88). The number of pupri per fruit ws the lowest for the 1:5 rtio (1.43 pupri per fruit) nd the highest for the 1:3 rtio (39.7 pupri per fruit). Rtios of 1:1 nd 1:2 were not significntly different from ech other nd yielded men of 16.35 pupri per fruit. A one dy exposure resulted in men of 5. pupri per fruit nd this vlue ws significntly lower thn tht obtined when exposure lsted either two or three dys (men of 18.3 pupri per fruit). Therefore two bc d A Pupri Figure 1. Percentge of decyed fruit nd number of pupri obtined per fruit (men ± SE) fter infesttion of Mrisol clementine mndrins using different fly densities (-5 flies per cge) during 1 dy. Mens bsed on three cges contining 1 fruits for ech fly density tested. Brs or points exhibiting the sme letter were not significntly different ccording to Duncn s multiple comprison procedure (P.5). d A 25 2 15 1 5 Pupri (#)
438 J. A. Jcs et l. / Spn J Agric Res (28) 6(3), 434-44 Rotten fruit (%) 1 75 5 25 5 1 2 Flies per cge (#) 3 3 d 2 d 1 d Exposure dys (#) Figure 2. Percentge of decyed fruit obtined fter infesttion of Nov mndrins using different fly densities ( to 3 flies per cge) during either 1, 2 or 3 dys. Vlues bsed on one cge contining 1 fruits for ech combintion of fly density nd exposure time tested. dys ws the only exposure time selected for the third experiment. The percentge of decyed fruit in the third experiment did not depend on the fruit:fly rtio (F = 1.6; df = 5, 17; P =.2336) nd rnged from 3.3 to 1.% (Fig. 3). However this rtio significntly ffected the number of pupri obtined per fruit (F = 4.59; df =5, 174; P =.6, Fig. 3), which ws significntly lower for the 1:5 rtio (9.7 pupri per fruit) thn for the rest of rtios tested (men of 18.38 pupri per fruit). Rotten fruit (%) 1 8 6 4 2 A AB 5 1 2 3 5 Flies per cge (#) Rotten fruit Pupri Figure 3. Percentge of decyed fruit nd number of pupri obtined per fruit (men ± SE) fter infesttion of lemenules clementine mndrins using different fly densities (-5 flies per cge) during 2 dys. Mens bsed on three cges contining 1 fruits for ech fly density tested. Brs or points exhibiting the sme letter were not significntly different ccording to Duncn s multiple comprison procedure (P.5). 25 2 15 1 5 Pupri (#) Pupri (#) 7 6 5 4 3 2 1 Discussion 5 1 2 3 Flies per cge (#) 1 d 2 d 3 d Figure 4. Number of pupri obtined per fruit (men ± SE) fter infesttion of Nov mndrins using different fly densities ( to 3 flies per cge) during either 1, 2 or 3 dys. Vlues bsed on one cge contining 1 fruits for ech combintion of fly density nd exposure time tested. Three different mndrin cultivrs were used in these infesttion experiments. Therefore differentil sensitivity to decy nd differentil resistnce to medfly infesttion could hmper the vlidity of the results obtined. As observed in the first experiment with Mrisol mndrins, the percentge of decyed fruit depended on the fruit:fly rtio used nd ws nil for uninfested fruit. Becuse our im ws to develop protocol tht could be pplied to infested fruit, worst cse scenrio ws chosen whereby posthrvest fungicide tretment strong enough to prevent fungl decy in the punctures mde by dult femles during oviposition. onsequently ny cultivr difference in reltion to decy becme irrelevnt. A differentil resistnce to medfly infesttion could pper from the occurrence of either ntixenotic or ntibiotic mechnisms (Wisemn, 1999) differentilly occurring in the mndrin cultivrs tested. Antixenosis describes sitution in which the insect is either repelled from or not ttrcted to its norml host plnt. All cultivrs were t commercil mturity when tested (Tble 1). Although both rind color nd width (which cn ffect oviposition nd therefore infesttion) were not the sme between cultivrs, nd presumbly other chemicl chrcteristics of fruit, these fruit would be ccepted in these no-choice tests becuse the only condition of cceptnce is fruit mturity. Antibiosis describes sitution in which the insect s norml reltionship with
. cpitt citrus infesttion for qurntine tretments 439 host plnt cuses physiologicl or developmentl detriment to the insect. Previous work with these nd other cultivrs hs been vi infesting fruit by inserting lrve tht hd been grown in n rtificil medium into the fruit (Alonso et l., 22,b, 25b; Plou et l., 27). The observtions nd results from these studies hve shown no ntibiotic phenomenon. The results of these experiments clerly showed the importnce of the fungicide tretment to prevent fruit decy. The use of posthrvest solution contining 5% imzlil sulphte ws not suitble to keep the infested Mrisol fruit helthy enough to llow the lrve of. cpitt to complete their development (Fig. 1). Only the use of higher doses of this fungicide in combintion with guztine cette nd thibendzole (second nd third experiments) kept infested fruit in good enough condition to llow. cpitt lrve to pop out from the fruit nd sfely pupte. These posthrvest fungicide ppliction doses re higher thn those uthorized for prcticl use (MAPA, 27) nd therefore cn not be pplied for commercil purposes. The use of this fungicide mixture resulted in percentge of decyed fruit higher thn 5% only when the highest fruit:fly rtio (1:3) ws pplied during three dys to Nov mndrins (Fig. 2). For ny other rtio, this percentge infrequently reched 25%, nd ws lmost nil for rtios below 1:2 when fruit ws exposed to flies for up to two dys. Under these conditions, the number of pupe obtined per fruit incresed with both decresing the fruit:fly rtio nd incresing exposure time (Fig. 4).. cpitt flies exploit fruit wounds (including preexisting oviposition punctures) s oviposition sites (Ppj et l., 1989). Therefore femles tend to ly more eggs into lredy wounded fruit thn into unpunctured fruit. The occurrence of this synergistic phenomenon could explin why the number of pupri obtined per fruit did not increse linerly with either the fruit:fly rtio or exposure time (Fig. 4). When period of two dys ws used s exposure time, the number of pupri obtined per lemenules fruit did not chnge for fruit:fly rtios between 1:1 nd 1:5 (Fig. 4). Any of these rtios llowed the collection of lmost 2 pupri per fruit. Previous ssys using in vivo infesttion techniques for citrus (Hllmn nd Worley, 1999; Hllmn nd Mrtínez, 21) did not provide dt on the number of decyed fruit, or the men number of flies per fruit obtined. Hence, it is not possible to copre those results to those obtined in this work. Working with mngoes (Mngifer indic L), Torres- Rivero nd Hllmn (27) obtined bout 45. cpitt lrve per fruit when using fruit:fly rtios of 1:21 to 1:275. These rtios re 4 to 2-fold those reported here nd resulted in 2-fold increse in the number of pupe. Previous studies for the infesttion of mndrins consist of inserting 1 third instr lrve rered on rtificil diet into fresh fruit (Alonso et l., 22b, 25b, 27; Plou et l., 27, 28). These studies yielded less pupri per fruit thn those tht could be obtined in lemenules mndrins using the in vivo infesttion fruit:fly rtio of 1:1 or higher for two dys. These results will therefore improve the current infesttion prctices involving. cpitt nd mndrins. Provided tht n dequte fungicide tretment is pplied before the infesttion to stisfctorily void fruit decy, the use of 1:1 fruit:fly rtio for two dys gurntees the collection of lmost 2 helthy pupri per fruit. This in vivo pproch cn now be used to develop qurntine tretments ginst. cpitt in mndrins. Acknowledgments The uthors thnk John Golding (Gosford Horticulturl Institute, NSW Deprtment of Primry Industries, Austrli) for criticl comments on n erlier version of this mnuscript. This work ws prtilly funded by the Spnish «Instituto Ncionl de Investigción Agrri y Alimentri (INIA)» nd the Europen Union FEDER progrm (project RTA3-13-6-1). References AGNELLO A.M., SPANGLER S.M., MILTON E.S., HARRIS T., KAIN D.P., 22. Effect of high-crbon dioxide tmospheres on infesttions of pple mggot (Dipter: Tephritide) in pples. J Econ Entomol 95, 52-526. ALONSO M., DEL RÍO M.A., JAAS J.A., 22. Ionizción con electrones celerdos como trtmiento de curenten contr ertitis cpitt (Wiedemnn) (Dipter: Tephritide) en cítricos. Bol Sn Veg Plgs 28, 419-426. [In Spnish]. ALONSO M., DEL RÍO M.A., JAAS J.A., 22b. Respuest del híbrido de mndrin «Ellendle» infestdo con ertitis cpitt (Wiedemnn) (Dipter: Tephritide) l trtmiento con tmósfer insecticid y clor. Bol Sn Veg Plgs 28, 427-433. [In Spnish]. ALONSO M., PALOU L., JAAS J.A., DEL RÍO M.A., 25. Effect of short-term exposure to O 2 -enriched tmospheres on Vlenci ornges qulity. Act Hort 682, 177-182.
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