DEFRA Research Contract EPG 1/5/156 Environmental Impact of Bt Exudates from Roots of Genetically Modified Plants Final Report Prepared by Dr H F Evans Forest Research Alice Holt Lodge Wrecclesham Farnham, Surrey GU10 4LH [email protected] April 2002 Executive Summary......................................................................................................................................................i 1 Background and purpose of the review.............................................................................................................1 2 Bacillus thuringiensis as a microbial insecticide...............................................................................................2 2.1 Biology and mode of action of Bacillus thuringiensis................................................................................. 2 2.1.1 History of early use of Bacillus thuringiensis......................................................................................... 2 2.1.2 Historical overview of the genetic basis of Bt toxin crystal expression.................................................. 3 2.1.3 Classification of Bt Cry genes................................................................................................................. 3 2.1.4 Structure of the Bt toxin..........................................................................................................................4 2.1.5 Cry genes and the host range of Bt ......................................................................................................... 4 2.1.6 Host interactions at the gut level............................................................................................................. 7 2.1.6.1 Gut pH............................................................................................................................................ 8 2.1.6.2 Proteolysis: gut enzyme effects...................................................................................................... 9 2.1.6.3 Receptor binding: the final determinant of toxicity ..................................................................... 11 2.2 Incidence of Bacillus thuringiensis in nature............................................................................................ 15 2.2.1 Stored product facilities ........................................................................................................................ 16 2.2.2 Aerial plant surfaces ............................................................................................................................. 17 2.2.3 Soil........................................................................................................................................................ 18 2.2.3.1 Factors in the soil environment that determine long-term survival of viable bacterial spores ..... 18 2.2.3.2 The presence of Bacillus thuringiensis in soils ............................................................................ 20 3 Use as a pest management tool.........................................................................................................................23 3.1 Conventional application .......................................................................................................................... 23 3.1.1 The scale of use of conventional Bt ...................................................................................................... 23 3.1.2 Stability of sprayed applications of Bt .................................................................................................. 28 3.1.3 Application technology to deliver the dose to the target....................................................................... 28 3.2 Effects of Bt on non-target organisms....................................................................................................... 31 3.3 Application as a component of transgenic plants...................................................................................... 35 3.4 The scale of development and use of Bt crops........................................................................................... 36 3.4.1 Expression within the host plant ........................................................................................................... 37 3.4.1.1 Leaves .......................................................................................................................................... 38 3.4.1.2 Stem ............................................................................................................................................. 40 3.4.1.3 Plant reproductive tissue .............................................................................................................. 40 3.4.2 Expression in pollen.............................................................................................................................. 41 3.5 Expression in roots.................................................................................................................................... 41 3.6 Bt in root exudates .................................................................................................................................... 41 3.6.1 Background........................................................................................................................................... 41 3.6.2 Studies of Bt exudation by roots ........................................................................................................... 42 3.7 Root exudation of Bt and interaction with the rhizosphere .......................................................................48 4 Interaction effects..............................................................................................................................................52 4.1 The process of encounter between potential hosts and expressed Bt – introduction................................. 52 4.2 Encounter between target and non-target organisms and wild type and truncated Bt ............................. 53 4.2.1 The potential effects of Bt in the corn environment.............................................................................. 57 4.2.1.1 Phylloplane and stem ................................................................................................................... 57 4.2.2 Rhizosphere and surrounding soil......................................................................................................... 60 4.3 Factors affecting the likelihood of expression and retention of lethal/sub-lethal dosages of Bt............... 61 4.3.1 Expression during the life of the transgenic plant – a whole life budget for Bt (with emphasis on the relative quantities from root exudates and expression in other tissues, debris, etc.).......................................... 61 4.3.2 Accumulation and retention of activity of transgenic Bt vs. wild type Bt in soil.................................. 64 4.4 Conceptual framework for encounter between Bt toxin and putative hosts .............................................. 68 4.4.1 Risk assessment in relation to encounter between hosts and wild type or truncated Bt toxins ............. 71 4.4.1.1 Encounter in the phylloplane........................................................................................................ 71 4.4.1.2 Indirect effects on natural enemies by removal of key prey or host organisms ........................... 73 4.4.1.3 Encounter in the rhizosphere and bulk soil .................................................................................. 73 4.4.1.4 Conclusions on likelihood of encounter with Bt .......................................................................... 77 4.4.2 Survival of ingested toxin under gut conditions.................................................................................... 78 4.4.3 Likelihood of Cry toxin binding to midgut receptors ........................................................................... 81 4.4.4 Likelihood of pore formation and cell disruption – toxicity. ................................................................ 81 5 Conclusions and research needs ......................................................................................................................83 5.1 Hazard – Cry toxin effects on non-target organisms and possible development of resistance ................. 83 5.2 Risk – likelihood of hazard being realised ................................................................................................ 84 5.2.1 Risk is dependent on survival of toxin through a sequence of events................................................... 84 5.3 Key research needs ................................................................................................................................... 87 5.3.1 Relative fitness of truncated toxins....................................................................................................... 87 5.3.1.1 Environment................................................................................................................................. 87 5.3.1.2 Within the guts of putative hosts in relation to pH, enzymes and receptor binding..................... 88 5.3.1.3 Identification of vulnerable organisms in relation to risk profile................................................. 88 5.4 Ecological framework for rational risk assessment 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