ࡱ> hjefg7 bjbjUU .7|7|ASlZZZZZZZnL<n7`````;J5555555$U8 u:5Z;;5]+ZZ``Y7]+]+]+|Z`Z`5]+5]+]+-/r4TZZ5`T d $nn^-*.55o707<5V:-*0:5]+nnZZZZZlatko Korunia, Vlatka Rozmanb aDiatom Research and Consulting Inc.,Guelph, Canada bPoljoprivredni fakultet, Osijek Fumigacija eteri nim uljem cineol in vitro Sa~etak U ovom radu opisana su laboratorijska istra~ivanja fumigantne aktivnosti cineola, kako bi se pojasnilo da li su eteri na ulja potencijalni izvor novih fumiganata, te da li bi njihova proizvodnja i fumigacija u tom smislu, mogla biti ekonomski opravdana u praksi. Provedena su tri razli ita pokusa sa cineolom. U prvom pokusu istra~ivana je djelotvornost cineola na razli ite razvojne stadije ri~inog ~i~ka (Sitophilus oryzae L.), ~itnog kukulji ara (Rhyzopertha dominica F.) i hrastog braanara (Cryptolestes ferrugineus L. u zrnu paenice. U drugom pokusu istra~ivana je djelotvorna koncentracija cineola u prostoru 50% ispunjenom paenicom na ri~inog ~i~ka, ~itnog kukulji ara, i kestenjastog braanara (Tribolium castaneum Herbst.). Cineol je primjenjen u rasponu koncentracija od 50, 100, 150, 200, 250 l/L ili g/m3. U treem pokusu istra~ivana je djelotvorna koncentracija cineola u prostorima razli ito ispunjenim paenicom (prazni prostor, 50% i 95% ispunjen paenicom). Razvojni stadiji hrastog braanara u zrnu paenice bili su osjetljiviji na cineol u usporedbi s ostalim testiranim vrstama. Nakon 21 dan, na paenici tretiranoj cineolom razvilo se dva puta manje ~ivih imaga u odnosu na kontrolu. Iako je brojnost odraslih insekata ~itnog ~iaka i ~itnog kukulji ara fumigirana sa 50 l/L ekspozicijom od 48 sati, bila neato ni~a od njihove brojnosti na kontroli, o ito je da primjenjena doza nije dovoljno visoka za uspjeano suzbijanje ranijih razvojnih stadija ove dvije vrste insekata pa i hrastog braanara na paenici. Testiranjem 5 koncentracija cineola (50, 100, 150, 200, 250 l/L ili g/m3) tijekom 48 sati u prostoru 50% ispunjenom psenicom, 100%-tna smrtnost hrastog braanara dobivena je sa 50 l/L (najni~a koncentracija primjenjena u pokusu). Medutim, 100% smrtnost ~itnog kukulji ara dobivena je s koncentracijom od 150 l/L, a ~itnog ~iaka i kestenjastog braanara tek pri koncentraciji od 250 l/L. Koncentracija cineola od 50l/L ili 50g/m3 u praznom prostoru izazvala je gotovo 100%-tnu smrtnost kod sve etiri vrste testiranih insekata. Medutim, fumigacija u 50% ispunjenom prostoru u potpunosti je bila uspjeana kod hrastog braanara, dok su ~itni ~i~ak i ~itni kukulji ar suzbijeni samo oko 50% do 60%, a kestenjasti braanar samo 11%. U prostoru 95% ispunjenom paenicom smrtnost hrastog brasnara je bila 88%, ri~inog ~iaka 34%, ~itnog kukulji ara 64% i kestenjastog braanara svega 4.5%. Iako cineol posjeduje odreena fumigantna svojstva, za dobivanje zadovoljavajuih rezultata potrebno je primjeniti visoke koncentracije (doze) u usporedbi s fosforovodikom ili metil bromidom. Pribli~no ista djelotvornost na insekte dobivena je s dozom fosforovodika od 0.03 g/m3, metil bromida od 1 g/m3, a cineola od oko 40 g/ m3. Cijena kotanja 1 kg peleta fosforovodika je priblizno 28.4 EUR-a, dok je cijena kotanja 1 kg cineola u pakiranju od 100 grama 162 EUR-a. Kad se primjeni najvia doza, cijena fosforovodika za fumigaciju tone zrnene robe iznosi pribli~no oko 0.5 EUR-a, dok cijena cineola iznosi pribli~no od 16 do 40 EUR-a. Smatramo da je cijena koaanja cineola, a i drugih eteri ih ulja, uz ostale karakteristike (miris, sorpcija, prozra ivanje itd.) veliki ograni vajui faktor za prakti u primjenju prirodnih eteri ih ulja. Klju ne rije i: eteri na ulja, cineol, fumigacija, skladiani insekti, ekonomi nost Uvod Eteri na ulja predstavljaju sekundarne metabolite aromati nih biljaka. To je kompleksna mjeaavina organskih tvari, koji daju osobit miris i okus biljkama. Imaju zaatitnu ulogu u biljci ili slu~e za primamljivanje insekata tijekom cvatnje. Mnoge studije potvrdile su da eteri na ulja i njihovi sastojci mogu biti i va~an izvor botani kih insekticida (Singh i Upadhyay, 1993). U tom pravcu, istra~ivana je i fumigantnost pojedinih komponenti izoliranih iz eteri nih ulja, koje bi predstavljale potencijalnu alternativu za metil bromid. Prednost je ato pokazuju vrlo nisku otrovnost za toplokrvne ~ivotinje, visoku ishlapljivost, a fumigantnu otrovnost za skladiane atetnike (Shaaya i sur., 1991, 1997; Regnault-Roger i sur., 1993, Rozman, 2003). Primjerice, dokazana je fumigantna i kontaktna toksi nost glavnog monoterpena eukaliptusa, 1,8-cineola (Lee i sur., 2000), kod vrste surinamskog braanara (Oryzaephilus surinamensis L.) rezistentne na klorpirifos-metil. Eteri na ulja lovora, lavande, ru~marina i timijana s naaeg podru ja, takoer sadr~e ovaj monoterpen koji se pokazao toksi nim i za vrste kao ato su ~itni kukulji ar (Rhyzopertha dominica F.), paeni ni ~i~ak (Sitophilus granarius L.), ri~in ~i~ak (S. oryzae L.), kukuruzni ~i~ak (S. zeamais Motsch.), kestenjasti braanar (Tribolium castaneum Herbst.), hrasti braanar (Cryptolestes ferrugineus Steph.) (Rozman, 2003; Rozman i sur., 2006; 2006a; Rozman i sur., 2007; 2007a). Openito, aromati ne biljke sadr~e eteri na ulja u koncentracijama od 1-3% (akir, 1992). Stoga, velike koli ine biljnog materijala treba preraditi, da bi se dobila eteri na ulja u koli inama dovoljnim za testove komercijalnih skala. Meutim, neki sastojci mogu ispoljiti mnogo vee djelovanje nego eteri no ulje u cijelosti. Ovo bi trebalo potaknuti uzgoj biljnih varijeteta koji stvaraju takve sastojke u veim koli inama ili pak znatno jeftinije sintetski proizvoditi eteri na ulja. Da bi se bolje shvatila reakcija atetnika u svim razvojnim stadijima na eteri na ulja, a posebno na njihove pare i sastojke, potrebna su sustavna istra~ivanja u inaka razli itih koncentracija, vremena izlo~enosti, te usporedba s konvencionalnim fumigantima. Cilj rada je utvrivanje djelotvorne koncentracije cineola u suzbijanju odraslih insekata u masi paenice, u utvrivanju fumigantnog u inka cineola na insekte u praznom prostoru, u 50% ispunjenom prostoru i u 95% ispunjenom prostoru paenicom, te u utvrivanju djelotvornosti cineola na razvojne stadije insekata u zrnu paenice. Takoder, razmatra se ekonomi nost uporabe cineola u odnosu na fosforovodik. Postupak istra~ivanja Istra~ivanja su provedena u laboratoriju Diatom Research and Consulting Inc. u Kanadi, s ciljem dokazivanja fumigantnog djelovanja cineola (eucalyptola) na skladiane atetnike. U istra~ivanjima se koristilo eteri no ulje cineole 99% (proizvod  Sigme , Njema ka). Istra~ivanje je provedeno u kontroliranim laboratorijskim uvjetima pri temperaturi 301oC, 705% rel. vlage, u mraku. Postavljena su 3 laboratorijska pokusa: 1. pokus - testiranje fumigantnog efekta cineola na razli ite razvojne stadije (jajaaca, li inke, kukuljice i imaga) pri skrivenoj zarazi zrna s 3 vrste atetnika: ri~in ~i~ak (S. oryzae), ~itni kukulji ar (R. dominica) i hrasti braanar (Cryptolestes ferrugineus L.). Iz zara~ene paenice prosijavanjem su uklonjeni svi razvojni stadiji insekata izvan zrna. Zara~ena paenica je podijeljena u 200-gramske uzorke koji su stavljeni u staklene posude od 450 ml volumena (50% ispunjen prostor). Zara~eni zorci paenice od 200g/450 ml vol. u staklenim posudama tretirani su s cineolom nakapanim na filter papir (50 l/L ili 50 g/m3) koji je stavljen na povrainu zrna i hermeti ki su zatvoreni. Isti broj uzoraka postavljen je i kao kontrola bez aplikacije. Nakon 7. dana uzorci su otvoreni radi procesa prozra ivanja i prve ocjene rezultata. Pokus je postavljen u 4 ponavljanja, a rezultati razvijenih imaga iz zrna o itavani su joa dva puta nakon daljnih 13 i 21 dan. Ukupno je postavljeno 24 uzorka. 2. pokus - testiranje fumigantnog efekta razli itih koncentracija cineola na odraslim stadijima etiri vrste atetnika: ri~in ~i~ak (S. oryzae), ~itni kukulji ar (R. dominica), hrasti braanar (C. ferrugineus) i kestenjasti braanar (T. castaneum). Svaki uzorak sadr~avao je 200g paenice/450 ml vol. s uba enih 100 imaga starosti 2 do 4 tjedna za svaku vrstu. Testirano je 5 koncentracija cineola (50, 100, 150, 200, 250 l/L ili g/m3) nakapanih na filter papir, te kontrola bez aplikacije. Pokus je postavljenu u 4 ponavljanja, a broj ~ivih i uginulih imaga o itan je nakon 48 sati. Ukupno je postavljeno 96 uzoraka. 3. pokus - testiran je fumigantni u inak cineola (50 l/L) u praznom prostoru, u 50% ispunjenom prostoru paenicom, te u 95% ispunjenom prostoru paenicom, na etiri vrste imaga starosti 2-4 tjedna: ri~in ~i~ak (S. oryzae), ~itni kukulji ar (R. dominica), hrasti braanar (C. ferrugineus) i kestenjasti braanar (T. castaneum). Uzorci za test fumigaciju praznog prostora sadr~avali su posudu 450 ml volumena sa 0,5g braana i 10 zrna paenice, te uba enih 100 imaga. Uzorci za test s 50% ispunjenog prostora sadr~avali su 200 g paenice/450 ml volumena s uba enih 100 imaga, a za test s 95% ispunjenog prostora, sadr~avali su 360 g paenice/450 ml volumena s uba enih 100 imaga. U svaki uzorak na dno posude (prazni prostor) i na povrainu paenice (50% i 95% ispunjen prostor) dodan je filter papir s nakapanim cineolom (50 l/L). Na isti na in je postavljena i kontrola bez aplikacije. Pokus je postavljen u 4 ponavljanja, a broj ~ivih i uginulih imaga o itan je nakon 48 sati. Ukupno je postavljeno 96 uzoraka. Rezultati rada i diskusija Rezultati 1. pokusa  fumigacija cineolom skrivene zaraze zrna Rezultati pre~ivaljavanja atetnika kod skrivene zaraze zrna nakon 48 sati aplikacije cineola (50l/L ili 50g/m3) razlikovali su se po testiranim vrstama atetnika. Usporeujemo li Grafikone 1., 2. i 3. mo~e se primjetiti da je najbolji efekat postignut kod vrste C. ferrugineus, gdje se nakon 21 dan u tretmanu s cineolom razvilo 2 x manje ~ivih imaga u odnosu na netretirane uzorke, a ukupna brojnost razvijene populacije u odnosu na ostale dvije testirane vrste je zna ajno ni~a. O ito je da se kod S. oryzae i R. dominica u periodu od 13. do 21. dana enormno poveala ~iva populacija, s tima da je u tretiranim uzorcima njihova brojnost ipak bila neato ni~a od kontrole. Grafikon 1. C. ferrugineus  prosje an broj razvijenih imaga nakon 7-dnevne ekspozicije paenice cineolu (50l/L ili 50g/m3)  Grafikon 2. S. oryzae  prosje an broj razvijenih imaga nakon nakon 7-dnevne ekspozicije paenice cineolu (50l/L ili 50g/m3)  Grafikon 3. R. dominica  prosje an broj razvijenih imaga nakon nakon 7-dnevne ekspozicije paenice cineolu (50l/L ili 50g/m3)  Rezultati 2. pokusa  fumigacija s razli itim koncentracijama cineola Rezultati testiranja 5 koncentracija cineola ((50, 100, 150, 200, 250 l/L ili g/m3) tijekom 48 sati, vidljivi su iz Grafikona 4., 5., 6. i 7. Za vrstu C. ferrugineus 100% smrtnost je ostvarena ve s najni~om koncentracijom (50 l/L), dok je za S. oryzae i T. castaneum trebala najviaa (250 l/L), a za R. dominica srednja koncentracija cineola (150 l/L). Grafikon 4. C. ferrugineus  prosje an mortalitet (%) nakon 48 sati fumigacije s 5 razli itih koncentracija cineola  Grafikon 5. S. oryzae  prosje an mortalitet (%) nakon 48 sati fumigacije s 5 razli itih koncentracija cineola  Grafikon 6. R. dominica  prosje an mortalitet (%) nakon 48 sati fumigacije s 5 razli itih koncentracija cineola  Grafikon 7. T. castaneum  prosje an mortalitet (%) nakon 48 sati fumigacije s 5 razli itih koncentracija cineola  Rezultati 3. pokusa  fumigacija s cineolom praznog, 50% i 95% prostora ispunjenog psenicom Uspjeanost fumigacije s cineolom u koncentraciji (50l/L ili 50g/m3) u praznom prostoru gotovo je 100% kod sve etiri vrste insekata (Grafikoni 8., 9., 10. i 11.). Fumigacija u 50% ispunjenom prostoru u potpunosti je uspjeana kod C. ferrugineus, dok su za S. oryzae i R. dominica rezultati polovi ni, a najslabiji za T. castaneum. Vrsta C. ferrugineus vrlo dobro reagira i u 95% ispunjenom prostoru (88% mortalitet), dok je znatno slabiji rezultat kod S. oryzae (34%), polovi an kod R. dominica (64%), a neuspjeaan kod T. castaneum (4,5%). Grafikon 8. C. ferrugineus - prosje an broj ~ivih i uginulih imaga (%) nakon 48 sati fumigacije s cineolom (50l/L ili 50g/m3) u praznom, 50% i 95% ispunjenom prostoru  Grafikon 9. S. oryzae - prosje an broj ~ivih i uginulih imaga (%) nakon 48 sati fumigacije s cineolom (50l/L ili 50g/m3) u praznom, 50% i 95% ispunjenom prostoru  Grafikon 10. R. dominica - prosje an broj ~ivih i uginulih imaga (%) nakon 48 sati fumigacije s cineolom (50l/L ili 50g/m3) u praznom, 50% i 95% ispunjenom prostoru  Grafikon 11. T. castaneum - prosje an broj ~ivih i uginulih imaga (%) nakon 48 sati fumigacije s cineolom (50l/L ili 50g/m3) u praznom, 50% i 95% ispunjenom prostoru  Generalno, usporeujui sva tri pokusa, vidljivo je da cineol posjeduje odreen fumigantni efekat posebice izra~en kod vrste C. ferrugineus, dok je kod vrste T. castaneum njegova u inkovitost prakti no neopravdana. Vrlo dobri rezultati su kod fumigacije praznog prostora dok rezultati fumigacije prostora ispunjenog paenicom (50% i 95%) nisu uspjeani i prihvatljivi. Vjerojatni razlog tome mogao bi biti u zna ajnoj sorpciji cineola na zrna paenice, slaboj prodornosti para cineola u meuzrneni prostor kao i u samo zrno paenice, ato jako umanjuje fumigantni efekt. Odnosno, da bi se ostvario sli an uspjeh kao i s primjenom fosfina (PH3) ili metil bromida (CH3Br), koncentracije cineola bi trebale biti od 200-250 l/L ili g/m3, kako bi se suzbile sve testirane vrste. Prema Champ and Dyte (1976), da se postigne LD50 (smrtonosna doza koja ubija 50% organizama u pokusu) testiranih insekata dovoljno je u hermetiziranom prostoru upotrijebiti dozu fosforovodika od 0.03g/m3 i metil bromida od 1 g/m3. Lee et al. (2004) navode dozu cineola od 42 g/m3 za LD95 za insekte S. oryzae, R. dominica i T. castaneum (doze cineola jako sli ne dozama koje smo mi utvrdili naaim pokusima). Medutim, cijena koatanja 1 kg peleta fosforovodika je priblizno 28.4 EUR-a dok je cijena koatanja 1 kg cineola u pakiranju od 100 grama 162 EUR-a. Sa 1 kg fosfin peleta, kad se primjeni najviaa doza od 30 peleta po toni paenice, mo~e se fumigirati oko 55 tona paenice ato znaci da je trosak fumiganta po toni prosjecno oko 0.5 EUR-a. Medutim sa 1 kg cineola, prema naaim rezultatima, mogue je fumigirati samo oko 4 tone paenice, odnosno oko desetak tona prema rezultaima Lee et al. (2004) ato zna i da je troaak cineola oko 16.2 do 40 EUR-a po toni fumigirane paenice. Ako se ne promijene cijene cineola, ova usporedba jasno pokazuje da je cijena koatanja cineola, a i drugih eteri nih ulja, uz ostale karakteristike (miris, sorpcija, prozra ivanje itd.) veliki ograni avajui faktor za prakti nu primjenju prirodnih eteri nih ulja. Zaklju ak Iako eteri na ulja, poput cineola, posjeduju odreenu fumigantnu otrovnost za skladiane atetnike, i teoretski gledano mogu predstavljati alternativni oblik fumigacije, za njihovu uporabu u praksi i dobivanje zadovoljavajuih rezultata potrebno je primjeniti visoke koncentracije usporeujui s fosforovodikom ili metil bromidom. Takoer, sadaanja cijena koatanja cineola u usporedbi s fosforovodikom i metil bromidom ograni ava moguu primjenu prirodnih eteri nih ulja kao fumiganata u praksi. Fumigation with cineole essential oil in vitro Summary The paper describes laboratory research of fumigant activity of cineole to find out whether essential oils can act as potential source of new fumigants, as well as whether they can be produced and used in fumigation in economically justified manner. Three different experiments with cineole were carried out: first, to test efficacy of cineole against different developmental stages of rice weevil (Sitophilus oryzae L.), lesser grain borer (Rhyzopertha dominica F.) and rusty grain beetle (Cryptolestes ferrugineus L. in wheat grain; second, to test effective concentration of cineole against rice weevil, lesser grain borer and red flour beetle (Tribolium castaneum Herbst.) in space 50% filled up with wheat applying cineole in the concentration range of 50, 100, 150, 200, 250 or l/L g/m3; and third, to test effective concentration of cineole in spaces differently filled up with wheat (empty space, 50% and 95% filled up). Developmental stages of rusty grain beetle in wheat grain were more sensitive to cineole in comparison to the remaining tested species. Twenty one (21) days later on the cineole treated wheat twice as fewer alive adults were developed in comparison to the control. Though the number of rice weevil and lesser grain borer adults being fumigated with 50 l/L with exposure of 48 hours was somewhat lower than their number in the control, apparently, applied dose was not sufficient for effective control of younger developmental stages of the two species, and rusty grain beetle on wheat, as well. By testing 5 concentrations of cineole (50, 100, 150, 200, 250 l/L or g/m3) during 48 hours in space 50% filled up with wheat, 100% mortality of rusty grain beetle was obtained with 50 l/L (lowest applied concentration). However, 100% mortality of lesser grain borer was obtained with concentration of 150 l/L, with rice weevil and red flour beetle at concentration of 250 l/L. Concentration of cineole of 50l/L or 50g/m3 in empty space induced nearly 100% mortality in all four tested insect species. However, fumigation in space 50% filled up with wheat was absolutely effective against rusty grain beetle only, with 50% to 60% efficacy against rice weevil and lesser grain borer, and only 11% against red flour beetle. In space 95% filled up with wheat mortality of rusty grain beetle was 88%, rice weevil 34%, lesser grain borer 64% and red fluor beetle only 4.5%. Though cineole exhibited certain fumigant characteristics it would be needed to apply high concentrations (doses) relative to phosphine or methyl bromide to gain satisfactory results. Nearly equal efficacy against the insects was obtained with dose of phosphine of 0.03 g/m3, methyl bromide of 1 g/m3 and cineole of about 40 g/ m3. One (1) kg of phosphine pellets costs about 28.4 EUR, whilst 1 kg of cineole in packages of 100g reaches 162 EUR. We find such a high price for cineole, and for other essential oils, considering the remaining characteristics (scent, sorption, aeration etc.), as serious limiting factor for application of natural essential oils in practice. Key words: essential oils, cineole, fumigation, stored pests, cost/effectiveness Literatura: akir, C., (1992): Investigations on the fungitoxic potentials of some plants occurring in Antalya. M.Sc. Thesis, Akdeniz University, Antalya (in Turkish). Champ B.R., Dyte C.E. (1976): Report of FAO global survey of pesticide susceptibility of stored grain pests. FAO Plant Production and Protection Series No. %, FAO, Rome. Lee, B. H., Annis, P. C., Tumaalii F. (2004): Fumigant toxicity of essential oils from the Myrtaceae family and 1,8-cineole against 3 major stored-grain insects. Journal of Stored Products Research, 40:553-564. Lee, S. E., Choi, W. S., Lee, H. S., Park, B. S. (2000): Cross-resistance of a chlorpyrifos-metyl resistant strain of Oryzaephilus surinamensis (Coleoptera: Cucujidae) to fumigant toxicity of essential oil extracted from Eucalyptus globulus and its major monoterpene, 1,8-cineole. Journal of Stored Products Research, 36: 383-389. Regnault-Roger, C., Hamraoui, A., Holeman, M., Theron, E., Pinel, R., (1993): Insecticidal effect of essential oils from Mediterranean plants upon A. obtectus (Say) (Coleoptera: Bruchidae) a pest of kidney bean (Phaseolus vulgaris L.). Journal of Chemical Ecology 19: 1231-1242. Rozman, V. (2003): Eteri na ulja kao fumiganti u zaatiti uskladiatenih ~itarica. Doktorska disertacija, Poljoprivredni fakultet u Osijeku, 2003.Str:1-90. Rozman, V., Kalinovi, I., Koruni, Z. (2007): Toxicity of naturally occurring compounds of Lamiaceae and Lauraceae to three stored-product insects. Journal of Stored Products Research, Vol. 43. (4) pp:349-355. Rozman, V., Kalinovi, I., Liaka, A. (2006): Bioactivity of 1,8-cineole, camphor and carvacrol against rusty grain beetle (Cryptolestes ferrugineus Steph.) on stored wheat. Proceedings of the 9th International Working Conference on Stored Product Protection, Campinas, Brazil, 2006: 687-694. Rozman, V., Kalinovi, I., Liaka, A. (2006a): Insecticidal activity of some aromatic plants from Croatia against granary weevil (Sitophilus granarius L.) on stored wheat. Cereal research Communications, Proceedings of the V. Alps-adria Scientific Workshop, Vol. 34 No. 1, str: 705-708. Rozman, V., Kalinovi, I., Liaka, A., Koruni, Z., Bali evi, R. (2007a): Toxicity of naturally occurring compounds of Dalmatian (Croatia) Lamiaceae and Lauraceae to maize weevil (Sitophilus zeamais Motsch.). Cereal research Communications, Proceedings of the VI. Alps-adria Scientific Workshop, Vol. 35 No. 2, str: 1005-1008. Shaaya, E., Kostjukovsky, M., Eilberg, J., Sukprakarn, C. (1997): Plant oils as fumigants and contact insecticides for control of stored-product insects. Journal of Stored Products Research, 33: 7-15. Shaaya, E., Ravid, U., Paster, N., Juven, B., Zisman, U., Pisarrev, V., (1991): Fumigant toxicity of essential oils against four major stored-product insects. Journal of Chemical Ecology 17: 499-504. Singh, G., Upadhyay, R. K., (1993): Essential oils a potent source of natural pesticides. Journal of Scientific & Industrial Research, 52: 676-683. <>@B2DV   8 h 23KNdefg,N,.BDRTZ\jprtxzmHsHmH sH \mH sH 6 6mH sH 556CJaJ 5CJaJH*T@DFVf HX:,.$*-/33D3F3$a$$`a$$a$   !! ! !!!!!(!*!:!T!X!Z!^!`!j!l!x!z!!!!!!!!!! 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