Insecticidal delta-endotoxins of Bacillus thuringiensis are among the most abundant recombinant proteins released by genetically modified (GM) crops into agricultural soils worldwide. However, there is still controversy about their degradation and accumulation in soils. In this study, 14C-labelled Cry1Ab protein was applied to soil microcosms at two concentrations (14 and 50 μg g−1 soil) to quantify the mineralization of Cry1Ab, its incorporation into the soil microbial biomass, and its persistence in two soils which strongly differed in their texture but not in silt or pH. Furthermore, ELISA was used to quantify Cry1Ab and its potential immunoreactive breakdown products in aqueous soil extracts. In both soils, 14CO2-production was initially very high and then declined during a total monitoring period of up to 135 days. A total of 16 to 23 % of the 14C activity was incorporated after 29 to 37 days into the soil microbial biomass, indicating that Cry1Ab protein was utilized by microorganisms as a growth substrate. Adsorption in the clay-rich soil was the most important factor limiting microbial degradation; as indicated by higher degradation rates in the more sandy soil, extremely low concentrations of immunoreactive Cry1Ab molecules in the soils’ aqueous extracts and a higher amount of 14C activity bound to the soil with more clay. Ecological risk assessments of Bt-crops should therefore consider that the very low concentrations of extractable Cry1Ab do not reflect the actual elimination of the protein from soils but that, on the other hand, desorbed proteins mineralize quickly due to efficient microbial degradation.
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Accinelli C, Koskinen WC, Becker JM, Sadowsky MJ (2008) Mineralization of the Bacillus thuringiensis Cry1Ac endotoxin in soil. J Agr Food Chem 56(3):1025–1028. doi:10.1021/jf073172p
Badea EM, Chelu F, Lacatusu A (2010) Results regarding the levels of Cry1Ab protein in transgenic corn tissue (MON810) and the fate of Bt protein in three soil types. Romanian Biotechnological Letters 15(1):55–62
Baumgarte S, Tebbe CC (2005) Field studies on the environmental fate of the Cry1Ab Bt-toxin produced by transgenic maize (MON810) and its effect on bacterial communities in the maize rhizosphere. Mol Ecol 14(8):2539–2551. doi:10.1111/j.1365-294X.2005.02592.x
Chevallier T, Muchaonyerwa P, Chenu C (2003) Microbial utilisation of two proteins adsorbed to a vertisol clay fraction: toxin from Bacillus thuringiensis subsp. tenebrionis and bovine serum albumin. Soil Biol Biochem 35(9):1211–1218. doi:10.1016/s0038-0717(03)00182-2
Daudu CK, Muchaonyerwa P, Mnkeni PNS (2009) Litterbag decomposition of genetically modified maize residues and their constituent Bacillus thuringiensis protein (Cry1Ab) under field conditions in the central region of the Eastern Cape, South Africa. Agr Ecosyst Environ 134(3–4):153–158. doi:10.1016/j.agee.2009.06.012
Dohrmann R (2006) Cation exchange capacity methodology II: a modified silver-thiourea method. Appl Clay Sci 34(1–4):38–46. doi:10.1016/j.clay.2006.02.009
Douville M, Gagné F, Masson L, McKay J, Blaise C (2005) Tracking the source of Bacillus thuringiensis Cry1Ab endotoxin in the environment. Biochem Syst Ecol 33(3):219–232
Feng YJ, Ling L, Fan HZ, Liu YH, Tan FX, Shu YH, Wang JW (2011) Effects of temperature, water content and pH on degradation of Cry1Ab protein released from Bt corn straw in soil. Soil Biol Biochem 43(7):1600–1606. doi:10.1016/j.soilbio.2011.04.011
Fierer N, Schimel JP (2002) Effects of drying-rewetting frequency on soil carbon and nitrogen transformations. Soil Biol Biochem 34(6):777–787. doi:10.1016/s0038-0717(02)00007-x
Fu QL, Zhang YH, Huang W, Hu HQ, Chen DQ, Yang C (2012) Remaining dynamics of Cry1Ab proteins from transgenic Bt corn in soil. J Food Agric Environ 10(1):294–298
Griffiths BS, Heckmann L, Caul S, Thompsom J, Scrimgeour C, Krogh PH (2007) Varietal effects of eight paired lines of transgenic Bt maize and near-isogenic non-Bt maize on soil microbial and nematode community structure. Plant Biotechnol J 5(1):60–68
Gruber H, Paul V, Guertler P, Spiekers H, Tichopad A, Meyer HHD, Muller M (2011) Fate of Cry1Ab protein in agricultural systems under slurry management of cows fed genetically modified maize (Zea mays L.) MON810: a quantitative assessment. J Agr Food Chem 59(13):7135–7144. doi:10.1021/jf200854n
Gruber H, Paul V, Meyer HHD, Muller M (2012) Determination of insecticidal Cry1Ab protein in soil collected in the final growing seasons of a nine-year field trial of Bt-maize MON810. Transgenic Res 21(1):77–88. doi:10.1007/s11248-011-9509-7
Helassa N, M'Charek A, Quiquampoix H, Noinville S, Dejardin P, Frutos R, Staunton S (2011) Effects of physicochemical interactions and microbial activity on the persistence of Cry1Aa Bt(Bacillus thuringiensis) toxin in soil. Soil Biol Biochem 43(5):1089–1097. doi:10.1016/j.soilbio.2011.01.030
Hopkins DW, Gregorich EG (2005) Decomposition of residues and loss of the delta-endotoxin from transgenic (Bt) corn (Zea mays L.) in soil. Can J Soil Sci 85(1):19–26
ISO (2009) Soil quality—determination of particle size distribution in meral soil material. Method by sieving and sedimentation. ISO, Geneva
James C (2014) Global status of commercialized Biotech/gm crops: 2014 ISAAA Brief. vol 49. ISAAA, Ithaca NY
Jenkinson DS, Powlson DS (1976) Effects of biocidal treatments on metabolism in soil. 5. Method for measuring soil microbial biomass. Soil Biol Biochem 8(3):209–213. doi:10.1016/0038-0717(76)90005-5
Kielland K, McFarland JW, Ruess RW, Olson K (2007) Rapid cycling of organic nitrogen in taiga forest ecosystems. Ecosystems 10(3):360–368. doi:10.1007/s10021-007-9037-8
Koskella J, Stotzky G (1997) Microbial utilization of free and clay-bound insecticidal toxins from Bacillus thuringiensis and their retention of insecticidal activity after incubation with microbes. Appl Environ Microbiol 63(9):3561–3568
Lipson DA, Schmidt SK, Monson RK (1999) Links between microbial population dynamics and nitrogen availability in an alpine ecosystem. Ecology 80(5):1623–1631. doi:10.1890/0012-9658(1999)080[1623:lbmpda]2.0.co;2
Madliger M, Sander M, Schwarzenbach RP (2010) Adsorption of transgenic insecticidal Cry1Ab protein to SiO2. 2. Patch-controlled electrostatic attraction. Environ Sci Technol 44(23):8877–8883. doi:10.1021/es103007u
Madliger M, Gasser CA, Schwarzenbach RP, Sander M (2011) Adsorption of transgenic insecticidal Cry1Ab protein to silica particles. Effects on transport and bioactivity. Environ Sci Technol 45(10):4377–4384. doi:10.1021/es200022q
Margarit E, Reggiardo MI, Permingeat HR (2008) Bt protein rhizosecreted from transgenic maize does not accumulate in soil. Electron J Biotechn 11(2) doi:3 10.2225/vol11-issue2-fulltext-3
Martens R (1985) Limitations in the application of the fumigation technique for biomass estimations in amended soils. Soil Biol Biochem 17(1):57–63. doi:10.1016/0038-0717(85)90090-2
Nguyen HT, Jehle JA (2007) Quantitative analysis of the seasonal and tissue-specific expression of Cry1Ab in transgenic maize MON810. J Plant Dis Protect 114(2):82–87
Nguyen HT, Jehle JA (2009) Expression of Cry3Bb1 in transgenic corn MON88017. J Agr Food Chem 57(21):9990–9996. doi:10.1021/jf901115m
Okumura M, Filonow AB, Waller GR (1999) Use of 14C-labeled alfalfa saponins for monitoring their fate in soil. J Chem Ecol 25(11):2575–2583. doi:10.1023/A:1020886527371
Padmaja T, Suneetha N, Sashidhar RB, Sharma HC, Deshpande V, Venkateswerlu G (2008) Degradation of the insecticidal toxin produced by Bacillus thuringiensis var. kurstaki by extracellular proteases produced by Chrysosporium sp. J Appl Microbiol 104(4):1171–1181. doi:10.1111/j.1365-2672.2007.03644.x
Quiquampoix H, Burns RG (2007) Interactions between proteins and soil mineral surfaces: environmental and health consequences. Elements 3(6):401–406. doi:10.2113/gselements.3.6.401
Romeis J, Meissle M, Bigler F (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nat Biotechnol 24(1):63–71. doi:10.1038/nbt1180
Sander M, Madliger M, Schwarzenbach RP (2010) Adsorption of transgenic insecticidal Cry1Ab protein to SiO(2). 1. Forces driving adsorption. Environ Sci Technol 44(23):8870–8876. doi:10.1021/es103008s
Sander M, Tomaszewski JE, Madliger M, Schwarzenbach RP (2012) Adsorption of insecticidal Cry1Ab protein to humic substances. 1. Experimental approach and mechanistic aspects. Environ Sci Technol 46(18):9923–9931. doi:10.1021/es3022478
Santruckova H, Bird MI, Elhottova D, Novak J, Picek T, Simek M, Tykva R (2005) Heterotrophic fixation of CO2 in soil. Microb Ecol 49(2):218–225. doi:10.1007/s00248-004-0164-x
Saxena D, Flores S, Stotzky G (2002) Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events. Soil Biol Biochem 34(1):133–137
Shan GM, Embrey SK, Herman RA, Wolt JD, Weston D, Mayer LA (2005) Biomimetic extraction of Bacillus thuringiensis insecticidal crystal proteins from soil based on invertebrate gut fluid chemistry. J Agr Food Chem 53(17):6630–6634. doi:10.1021/jf0511493
Tapp H, Calamai L, Stotzky G (1994) Adsorption and binding of the insecticidal proteins from Bacillus thuringiensis subsp. kurstaki and subsp. tenebrionis on clay-minerals. Soil Biol Biochem 26(6):663–679. doi:10.1016/0038-0717(94)90258-5
Valldor P, Miethling-Graff R, Dockhorn S, Martens R, Tebbe CC (2012) Production of the 14C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coli in small-scale batch fermentations. Appl Microbiol Biotechnol 96(1):221–229. doi:10.1007/s00253-012-4299-2
Vance ED, Brookes PC, Jenkinson DS (1987) Microbial biomass measurements in forest soils: determination of Kc values and tests of hypotheses to explain the failure of the chloroform fumigation incubation method in acid soils. Soil Biol Biochem 19(6):689–696. doi:10.1016/0038-0717(87)90050-2
Wang HY, Ye QF, Wang W, Wu LC, Wu WX (2006) Cry1Ab protein from Bt transgenic rice does not residue in rhizosphere soil. Environ Pollut 143(3):449–455. doi:10.1016/j.envpol.2005.12.006
Wang HY, Ye QF, Gan J, Wu JM (2008) Adsorption of Cry1Ab protein isolated from Bt transgenic rice on bentone, kaolin, humic acids, and soils. J Agr Food Chem 56(12):4659–4664. doi:10.1021/jf800162s
This study was supported by the excellent technical assistance of Kathrin Ahrends, which we gratefully acknowledge. We thank all collaborators of the joint research projects “Biosafety and monitoring methods of Bt-maize” and “Field release accompanying research on transgenic Bt-maize”, both coordinated by Ingolf Schuphan and Stefan Rauschen, RWTH, Aachen, Germany. The work was financially supported by the German Federal Ministry for Education and Research, project numbers 0312631E and 0313279F.
Conflict of interest
The authors declare that they have no competing interests.
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Valldor, P., Miethling-Graff, R., Martens, R. et al. Fate of the insecticidal Cry1Ab protein of GM crops in two agricultural soils as revealed by 14C-tracer studies. Appl Microbiol Biotechnol 99, 7333–7341 (2015). https://doi.org/10.1007/s00253-015-6655-5
- Protein degradation
- Cry proteins
- Genetically modified plants
- Environmental risk assessment
- Soil microbial biomass
- 14C-labelled compounds
- Soil microbiology