NON-TARGET EFFECTS OF GENETICALLY MODIFIED TREES Patrik Blomberg 2007 Department of Ecology and Environmental Science Umeå University SE-901 87 Umeå Sweden AKADEMISK AVHANDLING som med vederbörligt tillstånd av rektorsämbetet vid Umeå universitet för erhållande av filosofie doktorsexamen i ekologi kommer att offentligen försvaras lördagen den 6 oktober 2007, kl. 10.00 i Lilla hörsalen, KBC. Examinator: Professor Lars Ericson, Umeå universitet Opponent: Professor Jan Stenlid, Institutionen för skoglig mykologi och patologi, Sveriges lantbruksuniversitet, SLU, Uppsala ISBN: 978-91-7264-391-8 © PATRIK BLOMBERG 2007 Printed by VMC, KBC, Umeå University, Umeå 2007 ORGANISATION DOCUMENT NAME Dept. of Ecology and Environmental Science Doctoral Dissertation Umeå University DATE OF ISSUE SE-901 87 Umeå, Sweden October 2007 AUTHOR Patrik Blomberg TITLE Non-target Effects of Genetically Modified Trees ABSTRACT To date, few studies have focused on the effects of genetically modified trees (GM trees) on the environment. One concern with GM trees is that they may have unanticipated effects on non-target organisms, i.e. effects on organisms that are not direct targets of the genetically modified trait. The main objective of this thesis was to study potential non-target effects from the interaction between GM trees and natural enemies, including phytopathogens and herbivorous insects. To study this I used a system consisting of GM trees featuring changes in growth-related characteristics, and naturally occurring enemies. The GM trees used were the aspen hybrids Populus tremula x tremuloides: one unmodified wild type clone T89 (control) and transgenic lines with altered expression of gibberellin (GA 20-oxidase), sucrose (SPS) or pectin (PME); and Populus tremula x alba: one unmodified wild type clone INRA 717-1-B4 (control) and lines modified to suppress the activity of the enzymes in the lignin biosynthetic pathway, i.e. CAD, COMT, CCR or CCoAOMT. The natural enemies used were the parasitic phytopathogens Melampsora pinitorqua, M. populnea and Venturia tremulae, and the herbivorous leaf-beetle Phratora vitellinae. To address this question inoculation experiments, feeding preference experiments, analyses of secondary chemistry and field inventories were performed. The results of the studies showed that the GM trees significantly affected the interaction with the natural enemies, both in the laboratory as well as in the field. For instance, both M. pinitorqua and V. tremulae showed an altered disease incidence on the GM trees of P. tremula x tremuloides compared to the unmodified wild type T89, where all tested transgenic lines exhibited altered susceptibility to the pathogens. However, there were also differences in aggressiveness to the aspens depending on pathogen population. The results from the field inventory showed that lines within all tested transgenic construct, COMT, CAD, CCoAOMT and CCR of P. tremula x alba differed significantly from the wild type INRA 717-1-B4 in susceptibility to M. populnea. In addition, the susceptibility to the rust also differed significantly between lines carrying the same transgenic constructs. Furthermore, we found that overexpression of SPS in P. tremula x tremuloides, unintentionally induced changes in plant secondary chemistry, where the GM-line SPS33A exhibited the largest deviation from the wild type T89 in contents of plant phenolics and nitrogen, and that these changes coincide with a concurrent decrease in herbivory by P. vitellinae on this line. I argue that the altered interactions are the result of physiological changes in the trees. They can originate from direct effects i.e. altered expression of the modified trait, indirect effects of the genetic modification process e.g. pleiotropy, or effects from the transformation process e.g. position effects, to which the tested natural enemies respond. The result stresses the importance of further research on the causes and mechanisms responsible for the altered interaction between GM trees and non-target organisms, as well as evaluating the potential environmental effects of cultivation of GM trees in the field. Such research will require collaboration between researchers from different disciplines, such as plant ecology and physiology, functional genomics, proteomics and metabolomics. KEY WORDS: Genetically modified, GM trees, Populus, transgenic, non-target effects, natural enemies, plant-pathogen/herbivore interaction, environmental effects, parasitic fungi, herbivorous insect, secondary metabolism, phytochemistry LANGUAGE: English ISBN: 978-91-7264-391-8 NUMBER OF PAGES: 27 + 4 papers SIGNATURE: DATE: 2007-09-03 NON-TARGET EFFECTS OF GENETICALLY MODIFIED TREES Patrik Blomberg 2007 Department of Ecology and Environmental Science Umeå University SE-901 87 Umeå Sweden Dept. of Ecology and Environmental Science Umeå University SE-901 87 Umeå Sweden ISBN: 978-91-7264-391-8 © PATRIK BLOMBERG 2007 Printed by VMC, KBC, Umeå University, Umeå, Sweden, 2007 LIST OF PAPERS This thesis is based on the following papers, which will be referred to in the text by the corresponding Roman numerals: I Blomberg, P., Wennström, A., Hjältén, J., Lindau, A. & Ericson, L. Testing the effect of genetically modified hybrid aspen Populus tremula x tremuloides on the interaction with the non-target pathogen Melampsora pinitorqua. (Submitted manuscript) II Blomberg, P., Wennström, A., Hjältén, J., Lindau, A. & Ericson, L. Genetically engineered aspen (Populus tremula x tremuloides) alters its interactions with the non-target pathogen Venturia tremulae. (Accepted manuscript) III Hjältén, J., Lindau, A., Wennström, A., Blomberg, P., Witzell, J., Hurry, V. & Ericson, L. (2007). Unintentional changes of defence traits in GM trees can influence plant-herbivore interactions. Basic and Applied Ecology 8:434-443. IV Blomberg, P., Wennström, A., Hjältén, J., Lindau, A., Ericson, L. & Pilate, G. Field survey of genetically modified trees (Populus tremula x alba) with altered lignification unveils altered susceptibility to the non-target pathogen Melampsora populnea. (Manuscript) Paper III is reproduced with kind permission from the publisher. TABLE OF CONTENTS INTRODUCTION 7 OBJECTIVES OF THE THESIS 9 MATERIAL AND METHODS 9 Populus: a model tree 9 Study systems 10 Description of the studies 12 MAJOR RESULTS 14 DISCUSSION 15 Mechanisms responsible to altered interactions 15 Potential environmental effects 18 Future perspectives 19 ACKNOWLEDGEMENTS 20 REFERENCES 20 APPENDICES: PAPER I – IV INTRODUCTION Forestry and forest products are of great importance to the world economy, but forests are also important for biodiversity, creating potential conflicts between production and environmental goals. Forest trees have a wide range of commercial uses, providing wood for heating and cooking, timber for construction, raw materials for paper and pulp production and biofuel. Furthermore, due to increases in the world’s human population, economic development, and pressures to reduce the use of non-renewable resources, global demands for forest products are increasing (FAO, 2001). Thus, there is an increasing need to increase forest productivity and to create novel forest products while finding ways to protect biodiversity in forest ecosystem. Since improving trees by conventional breeding is heavily constrained by e.g. long reproductive cycles genetic engineering offers an attractive alternative, offering the potential to transfer specific traits into selected genotypes without affecting desirable aspects of their genetic background (Pena & Séguin, 2001). Plantation forestry combined with forest biotechnology and genetic engineering of trees is likely to become a major source for wood products in the future (Tzfira et al., 1998, Pullman et al., 1998, Sedjo, 2001, Fenning & Gershenzon, 2002, Campbell et al., 2003). Currently a number of ongoing research projects are exploring the possibilities to genetically modify forest trees for use in forestry applications. The major goals of such projects include: improving the productivity of trees by increasing their growth rates; altering wood quality and chemical parameters in desired ways for specific uses, e.g. to improve the cost efficiency of paper and pulp production; increasing their resistance to pests and herbicides; enhancing their tolerance of various kinds of abiotic stresses; controlling flowering and maturation; and optimizing their suitability for bio/phytoremediation of polluted land and water (Tzfira et al., 1998, Pena & Séguin, 2001, Bradshaw & Strauss, 2001, Campbell et al., 2003, Brunner & Nilsson, 2004). - 7 - Although genetically modified (GM) trees have great potential utility in forestry, a number of possible environmental risks associated with their release have been identified, as discussed in a number of reviews in the last decade (e.g. James et al., 1998, Mathews & Campbell, 2000, Wennström, 2004, Van Frankenhuyzen & Beardmore, 2004, Snow et al., 2005, Hoenicka & Fladung, 2006). However, little empirical information is available for assessing the probability and scale of the possible risks. In contrast to crops, on which most research concerning risks associated with GM organisms has focused, trees are essentially undomesticated, having long lifespans and defining the structure of many communities (Raffa, 2001). These features increase not only the likelihood of transgene escape and hybridization with wild relatives, but also the trees’ exposure to various abiotic and biotic stresses (Raffa, 2001, Van Frankenhuyzen & Beardmore,