Toxicity of several d-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain

1 Ti le: Toxici y o se e al -endo oxins o Bacillus hu ingiensis agains Helico e pa a mige a (Lepidop e a: Noc uidae) om Spain. Au ho s: C. A illa(1), E. Va gas-Osuna(2), J. González-Cab e a(3), J. Fe é(3), J.E. González-Zamo a(1) A ilia ions: (1) Depa amen o de Ciencias Ag o o es ales, Uni e sidad de Se illa. Ca e e a de U e a, km 1. E-41013 Se ille (Spain) (2) Depa amen o de Ciencias y Recu sos Ag ícolas y Fo es ales. Uni e sidad de Có doba. Apa ado 3048. E-14080 Có doba (Spain) (3) Depa amen de Genè ica, Facul a CC. Biològiques, Uni e si a de València. A . D . Moline , 50. E-46100 Bu jasso (Valencia, Spain) Add ess o manusc ip co espondence: J.E. González-Zamo a Escuela Uni e si a ia de Ingenie ía Técnica Ag ícola (Uni e sidad de Se illa) Ca e e a de U e a, km 1 E-41013 Se illa (Spain) E-mail: [email p o ec ed] Phone numbe : +34-954 486 459 Fax numbe : +34-954 486 436 A ailable in: Jou nal o In e eb a e Pa hology 90 (2005) 51–54 doi:10.1016/j.jip.2005.04.003 2 ABSTRACT Toxici y and la al g ow h inhibi ion o ele en insec icidal p o eins o Bacillus hu ingiensis we e e alua ed agains neona e la ae o Helico e pa a mige a , a majo pes o impo an c ops in Spain and o he coun ies, by a whole-die con amina ion me hod. The mos ac i e oxins we e C y1Ac4 and C y2Aa1, wi h LC50 alues o 3.5 and 6.3 µg/ml, espec i ely. A he concen a ions es ed, C y1Ac4, C y2Aa1, C y9Ca, C y1Fa1, C y1Ab3, C y2Ab2, C y1Da, and C y1Ja1, p oduced a signi ican g ow h inhibi ion, whe eas C y1Aa3, C y1Ca2, and C y1Ea had no e ec . Keywo ds: Bacillus hu ingiensis , Helico e pa a mige a , Mic obial insec con ol, Co on pes s, C y oxins, G ow h inhibi ion, ICP. 3 INTRODUCTION Helico e pa a mige a (Hübne ) is an impo an pes o di e en c ops (co on, oma o, among o he s) in Spain and o he coun ies. To p e en he damage ha la ae p oduce in hese c ops, a a ie y o me hods a e used o hei con ol, including he use o chemical pes icides and also mic oo ganisms. The mos impo an o he la e is Bacillus hu ingiensis (Be line ), a bac e ium which p oduces di e en p o eins (-endo oxins) oxic o la ae o di e en species o Lepidop e a and o he insec s (Schnep e al ., 1998). B. hu ingiensis ( B ) has been used since he 50’s o he 20 h cen u y by sp aying i s spo es and c ys als on he plan s. In he la e 80’s and ea ly 90’s di e en cul i a s, such as obacco, oma o, po a o, co on, and co n, among o he s, we e gene ically enginee ed o exp ess -endo oxins, which allowed hem o be ole an o insec a ack (Schnep e al ., 1998). The use o insec esis an ansgenic c ops has been adop ed in many coun ies because o he highe p o ec ion hey p o ide and he educ ion in pes icide applica ions, bu he e is conce n abou he de elopmen o esis ance in a ge pes popula ions. Di e en s a egies, such as he use o e uges, gene o a ion, and gene s acking, ha e been p oposed o a oid (o a leas delay) he de elopmen o esis ance. To da e, he only ansgenic c op wi h a B gene au ho ized in Spain is B - co n, which inco po a es a gene ha codes o he C y1Ab oxin. Howe e , i is expec ed ha o he ansgenic cul i a s be au ho ized in he nea u u e, in pa icula B - co on (which exp esses he C y1Ac oxin) because o he impo an losses ha H. 4 a mige a can p oduce in his c op. Se e al au ho s ha e s udied he e ec o B oxins on H. a mige a popula ions om China, India and Aus alia (Chak aba i e al., 1998; Gajend a Babu e al., 2002; Liao e al., 2002; Kuma e al., 2004; Fengxia e al., 2004; Jalali e al., 2004). Howe e , o ou knowledge he e is no published s udy desc ibing he ac i i y o B oxins in Eu opean popula ions. Since suscep ibili y o a gi en B oxin may a y among popula ions (González-Cab e a e al., 2001; Wu e al, 1999; Jalali e al., 2004), i is o g ea in e es o de e mine he e ec o B oxins on local popula ions o H. a mige a p io o he possible in oduc ion o ansgenic cul i a s and also o he mo e a ional use o B sp ay o mula ions. The esul s ob ained will also be use ul o sea ch o al e na i es in he case esis ance e ol es in he a ge popula ions o some o he cu en ly used B oxins. MATERIALS AND METHODS The H. a mige a colony was es ablished om la ae and egg masses collec ed in co on ields in June-July 2001-02 in Andalucia, sou he n Spain. Bioassays we e ca ied ou by inco po a ing he B oxins in o he a i icial die (Poi ou and Bues, 1974). C y1Aa3, C y1Ab3, C y1Ac4, C y1Ca2, C y1Da, C y1Ea, C y1Fa1, C y1Ja1, C y2Aa1, and C y2Ab2 we e pu i ied om ecombinan B s ains EG1273, EG7077, EG11070, EG1081, EG7300, EG11901, EG11069, EG7279 (ob ained om Ecogen Inc., Langho ne, Pa.), EG7543, and EG7699 ( om Monsan o Co., Ches e ield, Mo.), espec i ely. B s ains we e g own a 29ºC o 48 h in CCY medium (S ewa e al., 1981) supplemen ed wi h he sui able an ibio ic. Spo es and c ys als we e collec ed by cen i uga ion a 22000 × g o 10 min a 4ºC. The pelle was washed ou imes wi h 1 M NaCl/10 mM EDTA and suspended in 10 mM KCl. P o oxin inclusions we e 5 solubilized in 50 mM sodium ca bona e bu e , pH 10.5, con aining 10 mM di hio h ei ol. P o oxins we e ypsin-ac i a ed ( ypsin ype XI: om bo ine panc eas, Sigma Chemical, S . Louis, MO) a 37ºC o wo hou s (1 mg ypsin pe 10 mg p o oxin). Pu i ied and ypsin ac i a ed C y9Ca was ob ained om Je oen Van Rie (Baye BioScience, Ghen , Belgium). P o ein concen a ion was measu ed by he me hod o B ad o d (B ad o d, 1976). The die was pou ed in o 100 ml beake s and he oxin was added o inal concen a ions o 1, 2, 4, 8, and 16 µg o oxin pe ml o die . A con ol was p epa ed wi h dis illed wa e . The solidi ied die was cu in o small cylinde s and placed in o boxes o 30 mm diame e and 15 mm heigh , wi h no en ila ion hole in he lid. One neona e la a was added o each box. Th ee g oups o en boxes we e used wi h each concen a ion and he con ol. Mo ali y was de e mined a e 7 days and su i ing la ae we e also weighed. Boxes we e kep in a ea ing oom a 26±1ºC, 60±10% ela i e humidi y, and a pho ope iod o 16:8 (L:D). The esul s o bioassays we e e alua ed by p obi analysis and he 50% le hal concen a ion (LC50) alues we e es ima ed using he POLO-PC p og am (LeO a So wa e, Be keley, Cali .). The a io be ween he weigh o ea ed and un ea ed la ae a day se en was used o calcula e la al g ow h inhibi ion. The e ec i e concen a ions ha p oduce a educ ion in la al g ow h o 50 % (EC50) and 99 % (EC99) we e calcula ed by adjus ing he cu e as p oposed by Sims e al. (1996). 6 RESULTS AND DISCUSSION Among he ele en B oxins es ed, C y1Ac4 and C y2Aa1 we e he mos oxic and p oduced a conside able mo ali y on H. a mige a la ae (Table 1), wi h C y1Ac4 being almos wice as much ac i e as C y2Aa1. These esul s a e in ag eemen wi h hose o o he au ho s, who ound C y1Ac mo e o equally ac i e han C y2Aa wi h simila bioassay p o ocols (bu using p o oxins), al hough he LC50 a io be ween oxins di e s. Thus, he C y1Ac/C y2Aa LC50 a io was 6.5- old (Gajend a Babu e al., 2002) and 35- old (Chak aba i e al., 1998) in wo independen popula ions om India, bu i was non-signi ican ly di e en in a popula ion om Aus alia (Liao e al., 2002). In he ange o concen a ions es ed, he es o oxins did no gi e enough mo ali y as o allow es ima ion o LC50 alues. Howe e , mos o he oxins wi h no de ec able le hal e ec showed a clea e ec on la al g ow h (Fig. 1), wi h se e al o hem wi h an EC50 below o nea 1 μg/ml o included in he ange o concen a ions es ed (Table 1), like C y9Ca, C y1Fa1, C y1Ab3, C y2Ab2, C y1Da, and C y1Ja1. Toxins C y1Aa3, C y1Ca2, and C y1Ea did no ha e any e ec on ei he mo ali y o la al g ow h. Only wo s udies wi h H. a mige a ha e es ed he oxici y o a conside able numbe o B oxins (Chak aba i e al., 1998; Liao e al., 2002). Fo he Aus alian popula ion C y1Ac and C y2Aa we e he mos ac i e oxins, whe eas o he Indian popula ion hese wo oxins we e he mos ac i e ones oge he wi h C y1Aa, which ga e no oxici y in ou s udy. In ag eemen wi h ou esul s, a se e e e ec on la al g ow h 7 has been obse ed o C y1Ab, C y1D and C y1F in he Indian popula ion (Chak aba i e al., 1998), and C y1Ab and C y2Ab in he Aus alian popula ion (Liao e al., 2002), and no e ec (o negligible) o C y1Ca and C y1Ea in bo h popula ions. The absolu e alues o LC50 es ima ed in ou s udy o C y1Ac4 (3.5 µg/ml) and C y2Aa1 (6.3 µg/ml) a e gene ally highe han hose epo ed in p e ious pape s using he simila bioassay condi ions (die inco po a ion): 0.02 µg/ml o C y1Ac and 0.71 µg/ml o C y2Aa (Chak aba i e al., 1998), 0.14-0.18 µg/ml o C y1Ac (K an hi e al., 2000), 0.24 µg/ml o C y1Ac and 1.57 µg/ml o C y2Aa (Gajend a Babu e al., 2002), 0.17-0.54 µg/ml o C y1Ac (Fengxia e al., 2004), 0.09-91 µg/ml (wi h a mean o 1.4 µg/ml o he 24 popula ions s udied) o C y1Ac (Wu e al., 1999), and 0.11-0.71 μg/ml o C y1Ac (Jalali e al., 2004). These disc epancies migh be due o di e ences in he na u e and mode o applica ion o he oxins, he me hod employed o es ima e p o ein concen a ion, he empe a u e a which he bioassay was pe o med ( his a ies om 26 o 30ºC), and unques ionably, he o igin o he insec s. Rega ding he na u e o he oxin, ou es s we e ca ied ou wi h pu i ied and ac i a ed C y p o eins, whe eas he es o s udies used p o oxins o a mix u e o c ys als and spo es. We ha e chosen ac i a ed oxins because we ied o simula e he o m o oxin ound in ansgenic c ops and o a oid he syne gis ic e ec o spo es. T ansgenic c ops seem o be he mos widesp ead way o using B oxins in he u u e. The a ea dedica ed o hese cul i a s is s eadily g owing since 1996 and i is expec ed o keep his endency in he u u e (James, 2004). In conclusion, he epo ed esul s ep esen he i s s udy on he oxici y o B oxins o an Eu opean popula ion o H. a mige a . The Spanish popula ion was e y 8 suscep ible o C y1Ac4 and C y2Aa1, as i has been ound o popula ions o his species om o he pa s o he globe. Fu he mo e, g ow h inhibi ion was se e e wi h C y9Ca, C y1Fa1, C y1Ab3, C y2Ab2, C y1Da, and C y1Ja1. Ei he in sp ayable o mula ions o in B -c ops, he combina ion/ o a ion o oxins wi h a di e en binding si e is desi able om a esis ance managemen poin o iew. The in o ma ion on he oxici y o he di e en B oxins, along wi h ha om binding s udies o B oxins o binding si es in he midgu memb ane (Es ela e al., 2004), should allow o choose hose oxins ha , no binding o he same si es, a e he mos ac i e agains his pes . ACKNOWLEDGEMENTS This wo k was unded by he Spanish Minis y o Science and Technology (P ojec No. AGL2000-0840-C03 and AGL2003-09282-C03). The au ho s would like o hank So ia Camúñez and Sil ia Pé ez o hei echnical suppo . REFERENCES B ad o d, M.M., 1976. A apid and sensi i e me hod o he quan i a ion o mic og am quan i ies o p o ein u ilizing he p inciple o p o ein-dye binding. Anal. Biochem. 72, 248-254. Chak aba i, S.K., Mandaoka , A., Ananda Kuma , P., Sha ma, R.P., 1998. E icacy o lepidop e an speci ic del a-endo oxins o Bacillus hu ingiensis agains Helico e pa a mige a . J. 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