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The effect of Bt toxins Bt GMO on the soil András Székács H-1537 Budapest POB 393 tel: +36 1 355-8991 FAX: +36 1 212-9853 E-mail: Director Agro-Environmental Research Institute National Agricultural Research and Innovation Centre Budapest Hungary

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Outline • Bacillus thuringiensis toxin microbial vs. plant-expressed • toxin production by Bt crop Cry1Ab in MON 810 • Bt toxin entry to soil root root exudates foliage in stubble • effects on soil-borne organisms soil biota and microbiota

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Sources • experiments in Hungary maize production the scientific basis of moratorium on MON 810 maize • data from the scientific literature Turrini et al 2015 Belowground environmental effects of transgenic crops: a soil microbial perspective. Res. Microbiol. 166 121-131. Hilbeck and Otto 2015 Specificity and combinatorial effects of Bacillus thuringiensis Cry toxins in the context of GMO Environmental Risk Assessment. Front. Environ. Sci. 3 7118 pages. Székács and Darvas 2012 Comparative aspects of Cry toxin usage in insect control. In: Advanced Technologies for Managing Insect Pests Ishaaya Palli Horowitz Eds. Springer Berlin pp. 195-230.

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 biopesticide application  GM application spraying/dusting plants with spores of bacterium production of transgenic Bt toxins in plants Bacillus thuringiensis Bt environmental analysis / ecotoxicology biopesticide with Bt-toxins as avtive ingredients cultivated plants modified for Bt-based insect resistance with gene technology  active ingredient / application  decomposition dynamics

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Comparative aspects • active ingredient • form of application • environmental fate • compatibility with agrotechnologies

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3.2 WP bacterial spores bacterium cell crystalline Cry toxin proteins parasporal bodies Cry1 toxins  32 g protein/kg DIPEL Bacillus thuringiensis Active ingredient Cry2 toxins toxin crystals require solubilization and hydrolytic activation for biological activity protoxin in crystals

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Cry1Ab preactivated toxin protein 8-17 mg protein/kg maize leaf 1.5-2 mg protein/kg maize stem 2-5.5 mg protein/kg maize root Active ingredient a single transgenic toxin protein A. Székács J. Juracsek L.A. Polgár B. Darvas 2005 FEBS J. 272 Suppl.1.: 508. A. Székács É. Lauber J. Juracsek B. Darvas 2010 Environ. Toxicol. Chem. 29 1: 182-190.

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Biopesticide vs. GM plant Bacillus thuringiensis MON 810 maize bacterial Cry1Ab crystal bipyramidal GM maize crystal structure stabilized by disulfide bond genetically modified plant DNA containing cry1Ab transgene expression of cry1Ab transgene in plant solubilization of Cry1Ab molecules by lysis of disulfide bonds pH 9-12. ME DTT Cry1Ab protoxin 131 kDa preactivated Cry1Ab toxin 91 kDa activated Cry1Ab toxin 63-65 kDa Cry1Ab protoxin trypsin-cleaved Cry1Ab toxin 131 kDa 65 kDa Cry1Ab toxin in MON 810 genetic event 91 kDa Cry1Ab toxin forms SDS PAGE 4-12 gradient gel reducing environ. type protoxin activated toxin Cry1 131 63-65 Cry3 73 55

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Cry1Ab preactivated toxin protein Active ingredient a single transgenic toxin protein  registration issues  resistance development fast resistance occurring cross-resistance with other Cry toxins substance toxicology analytical standard quantitative analytical method 8-17 mg protein/kg maize leaf 1.5-2 mg protein/kg maize stem 2-5.5 mg protein/kg maize root A. Székács J. Juracsek L.A. Polgár B. Darvas 2005 FEBS J. 272 Suppl.1.: 508. A. Székács É. Lauber J. Juracsek B. Darvas 2010 Environ. Toxicol. Chem. 29 1: 182-190.

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Form of application single or repeated applications application as justified by pest population density / damage The European corn borer Ostrinia nubilalis is a minor pest in Hungary Pesticide application is necessary only in every 7-10 years

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Form of application continuous uneven production of the transgenic toxin protein throughout the vegetation period A. Székács É. Lauber J. Juracsek B. Darvas 2010 Environ. Toxicol. Chem. 29 1: 182-190. A. Székács É. Lauber E. Takács B. Darvas 2010 Anal. Bioanal. Chem. 396 6: 2203-2211 leaf root stem

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DT 50 clay loam pH 7.3 25 o C PF2 10 days Environmental fate DT 50 better nutriment status pF3 250 days DT 50 with UV exposure 10 hours Source: The Pesticide Manual 12th Ed. 2000 Tomlin CDS Ed. The British Crop Protection Council Brighton UK. Consequent disadvantages: poor stability non-systemic effect high specificity

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Environmental fate Cry1Ab toxin production: toxin load ratio A. Székács B. Darvas 2012 Comparative Aspects of Cry Toxin Usage in Insect Control. In: Advanced Technologies for Managing Insect Pests Ishaaya Palli Horowitz Eds. Springer Berlin pp. 195-230. Cry1Ab toxin content bioavailable bioaccessible DIPEL 20.6 g/ha 0.085-8.16 g/ha MON 810 147-456 g/ha fold 7.1-22.1 10.3-260 fertilization b.m. 1.8-1.85 Cry 1.5-2.0 3.7 26.4-81.9 38.1-964 l.maturation b.m. 1.9-3.2 DK-818 Cry 1.8 5.8 152-472 219-1789 stacked 2 304-944 439-11100 Bruns and Abel 2003 2004 Ma and Subedi 2005 Fearing et al. 1997 Baumgarte and Tebbe 2005 Nguyen and Jehle 2007 Székács et al. 2010 European Food Safety Authority 2005

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Environmental fate decomposition in stubble: shredding harvest „traditional” harvest 45 biomass in stubble 2-4 toxin content in stubble after 12 months Cry1Ab: 1.5-4 g/ha 21 biomass in stubble 1-2 toxin content in stubble after 12 months Cry1Ab: 0.15-0.35 g/ha technology-dependent environmental toxin load A. Székács B. Darvas 2007 Mezõgazdasági géntechnológia Magyar Országgyűlés Mezőgazdasági Bizottság Budapest pp. 27-30.

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Ecological risks • integrated pest management IPM it is not to be attempted to eliminate the entire pest population - damage threshold – pest population threshold pest population dynamics have to be monitored intervention is justified only if pest population exceeds the threshold level • GM plant produces Cry toxin during the entire vegetation period – irrespectively from its necessity temporally and in given plant organs the effect cannot be limited to the damage period

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Summary • Bt biopesticide application • GM plant singular continuous toxin content several related toxins single toxin active ingredient protoxin preactivated toxin technology compatibility IPM non-compatible

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Toxin load on soil stubble 40-50 of Bt-maize production enter the soil on 9.1 million ha worldwide 2006 protected from rapid degradation in plant tissue additional release through root exudate

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Toxin fate in soil root Cry1Ab content: 1100-2500 ng/g 2000-5500 ng/g MON 810 leaf Cry1Ab content: 8000-17000 ng/g MON 810 root exudates 2.8 ng/g and up 218-413 ng/g or even „as much as in young leaves” Gupta et al. 2004. impact on soil organisms Stotzky 2004 Singh et al 2012 toxin released into soil is adsorbed and persists may impact on soil microbial diversity and metabolic functions Martina and Jeanne 2008 Tarafdar et al 2012 toxin remained active in soil against insect larvae for more than 230 days Tapp and Stotzky 1998 Gupta et al 2004 Ecological impacts of GM cotton on soil biodiversity. CSIRO Adelaide Australia pp. 18-31. Stotzky G. 2004. Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis especially from transgenic plants. Plant Soil 266 77–89. Singh et al 2012 Effects of transgenic Bt cotton on soil fertility and biology under field conditions in sub-tropical Inseptisol. Environ. Monit . Assess. 185: 485-495. Tarafdar 2012 Effect of transgenic cotton on soil biological health. Appl. Biol . Res. 1 15-23. Tapp and Stotzky 1998 Persistence of the insecticidal toxins from Bacillus thuringiensis subsp. kurstaki in soil. Soil Biol. Biochem. 30 471-476.

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Soil biota avoidance of Bt maize MON 810 by collembola Folsomia candida but not by Heteromurus nitidus and Sinella coeca effect not necessarily be related to Cry1Ab but possibly to other altered crop characteristics e.g. lignin content Bakonyi et al 2006 lower reproduction rate of F. candida feeding on Nutrient Agar/Bt spores parasporal crystals and vegetative cells than on yeast yet no significant effect on oviposition number of eggs and final body lengths of Folsomia candida by Cry1Ab/Cry1Ac from Bt cotton on adult mortality and on juvenile/adult ratio of F. candida and Xenylla grisea by Cry1Ab Cry1Ac Cry2A Cry3A on population growth and reproduction of Protaphorura armata Bakonyi et al 2006 Preference tests with collembolas on isogenic and Bt-maize. Eur. J. Soil Biol. 42 S132-S135. Broza et al 2001 The nonsusceptibility of soil Collembola to insect pathogens and their potential as scavengers of microbial pesticides Pedobiologia Jena 45 523-534. Yu et al 1997 Effects of Bacillus thuringiensis toxins in transgenic cotton and potato on Folsomia candida Collembola: Isotomidae and Oppia nitens Acari: Oribatidae J. Ecol. Entomol. 901 113-118. Sims et al 1997 Effect of the Bacillus thuringiensis insecticidal proteins CryIAb CryIAc CryIIA CryIIIA on Folsomia candida and Xenylla grisea Insecta: Collembola Pedobiologia Jena 41 412–416. Heckman et al 2006 Consequencesfor Protaphorura armata Collembola: Onychiuridae following expose to genetically modified Bacillus thuringiensis Bt maize and non-Bt maize Environ. Pollut. 142 212-216.

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Soil biota decreased growth rate in land snail Cantareus aspersus due to Cry1Ab from Bt maize Kramarz et al 2009 + combined effects with biotic stressors: motility reduction in snail Helix aspera in nematode-infected subjects Kramarz et al 2007b on the compost worm Eisenia fetida Shu et al 2015 significant differences in species composition in dung beetles  impaired ecosystem services feces removal seed dispersal edaphic aeration incorporation of organic matter Campos Hernandez 2015 Kramarz et al 2007 Increased response to cadmium and Bacillus thuringiensis maize toxicity in the snail Helix aspersa infected by the nematode Phasmarhabditis hermaphrodita..Environ. Toxicol. Chem. 26 73-79. Kramarz et al 2009 Effects of Bt-maize material on the life cycle of the land snail Cantareus aspersus. Appl. Soil Ecol. 42 236-242. Shue et al 2015 Multilevel assessment of Cry1Ab Bt-maize straw return affecting the earthworm ‘. Chemosphere 137 59-69. Campos and Hernandez 2015 Changes in the dynamics of functional groups in communities of dung beetles in Atlantic forest fragments adjacent to transgenic maize crops. Ecol. Indic. 49 216-227.

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Soil microbiota the soil microbial community is very robust no statistically significant differences in numbers of microbes enzyme activities or pH between soils with Bt and non-Bt corn although numbers and types of microbes and enzyme activities differed Cicoz et al 2008 Wang et al 2006 Shen 2006 no adverse effects on culturable bacteria or saprophytic fungi as well as earthworms nematodes or protozoa Icoz et al 2008 no significant or only slight effects of Bt maize plants on soil microbial communities plant age soil type and texture are overriding factors Turrini et al 2015 Icoz et al 2008 Microbial populations and enzyme activities in soil in situ under transgenic corn expressing cry proteins from Bacillus thuringiensis. J. Environ. Qual. 37 647-662. Wang et al 2006 Cry1Ab protein from Bt transgenic rice does not residue in rhizosphere soil. Environ. Pollut. 143 449-455. Shen et al 2006 Transgenic Bt cotton has no apparent effect on enzymatic activities or functional diversity of microbial communities in rhizosphere soil. Plant Soil 285 149-159. Turrini et al 2015 Belowground environmental effects of transgenic crops: a soil microbial perspective. Res. Microbiol. 166 121-131.

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Soil microbiota pleiotropic effects not linked to the products of the inserted genes but to transformation technology: the cultivation of Bt cotton affected soil microbial populations while the purified Bt toxin showed no effect Donegan et al 1995 purified Cry1Ab toxin did not inhibit growth of Fusarium graminearum or Trichoderma atroviride while Bt and non-Bt maize residues affected fungal growth in vitro Naef et al 2006 „Great attention should be paid when discussing data obtained in short-term experiments or single-point assessments because microbial communities living in the soil and in the rhizosphere are subject to seasonal shifts which represent further factors affecting the complex network of interactions characterizing natural and agricultural ecosystems. Time-course investigations based on large spatial and long temporal scales are needed to assess putative long-term modifications occurring in microbial community structure and composition during and after GMP cultivation.” Turrini et al 2015 Donegan et al 1995 Changes in levels species and DNA fingerprints of soil microorganisms associated with cotton expressing the Bacillus thuringiensis var. kurstaki endotoxin. Appl. Soil Ecol. 2:111-124. Naef et al 2006 Impact of transgenic Bt maize residues on the mycotoxigenic plant pathogen Fusarium graminearum and the biocontrol agent Trichoderma atroviride. J. Environ. Qual. 35 1001-109.

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2015: International Year of Soils • Soil is a key element in our well-being and survival • Soil microbiota is key element in fertility

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