Introduction

1. Effect of Cotton Plant Genetically Modified with An Antimicrobial Synthetic Peptide D4E1 On Soil Microbial Diversity and Enzyme Activity. ODOM*, L.J., R. Ankumah, C. Bonsi, J. Cary, M. Egnin, J. Jaynes, D. Mortley, L. Ogden, and K. Rajasekaran. Department of Agricultural and Environmental Sciences, Tuskegee University, Tuskegee, AL 36088. Abstract One of the least understood areas in the environmental risk assessment of genetically modified crops is their impact on soil- and plant- associated communities. Incorporation of transgenic plant products into the soil could alter the soil microbial diversity due to responses by microorganisms to novel proteins (Dunfield et al. 2004). Genetic modification has been used to confer disease resistance to the two fungal pathogens associated with Cotton Seedling Disease, in Alabama, (Rhizoctonia Solani and Pythium), a antimicrobial synthetic peptide D4E1, which has been shown in vitro and in planta to have broad spectrum antimicrobial action against many fungal orders, has been transformed into cotton seeds (Rajasekaran et al. 2005). In a completely randomized designed field trial, two 150 x150 ft test plots were assigned either one of 3 isogenic lines of cotton seed transformed with D4E1 (designated 357, 358, and 373) or a control line containing GUS marker gene. Acid and Alkaline Phosphatase enzyme assays were performed in order to determine if the enzymatic activity was affected, in any way, by the introduction of the genetically modified crop. There were no difference between the control and the three isogenic lines, however there were differences in both acid and alkaline phosphomonesterase activity sampling periods. Materials & Methods Two test fields, 150ft x 150ft, were planted (June 10, 2008 and July 10, 2008 respectively) in a Completely Randomized Design. The soil for the study is a Marvin taxonomic class: loamy sand [ Fine-loamy, kaolinitic, thermic, Typic Kanhapludults] Three Transgenic Cotton seeds Lines (designated 357, 358, and 373 also referred to as lines 1, 2, and 3) were transformed with antimicrobial synthetic peptide D4E1 seeds and were then planted. Soil samples were taken according to five phases of plant development. Week 0 (taken 1 day from planting), Week 1, True Leaf Emergence (TLE), Square Formation/Flowering (SF), and Harvest (Harv) which were taken at 7, 13, 51, and 143 days from planting, respectively. Samples were air dried and then subjected to Phosphatase Enzyme Assay as described by Tabitabai 1977. The results were statistically evaluated by means of analysis of variance using SAS statistical system. Results and Discussion • Introduction of synthetic antimicrobial peptide D4E1 did not have any statistically significant effect on phosphatase activity. (see figure 1 and 2). • Over growing season, however, there was significant differences in both acid and alkaline phosphatase activity . (see figure 1 and 2). • Across growing season, there was no correlation between acid and alkaline phosphatase activities. (see figure 3). The values differed at all times with the exception of at harvest (p<.0565). Overall, an effect of type was not detected. This is because at some times Alkaline>Acid and other time Acid>Alkaline. So on the average across times there was no detectable differences. • There was increased acid phosphomonesterase activity until day 51 which coincided with square flowering and development. (see Figure 1). Lu-Sheng et al 2005 observed a similar trend in rice, where soil acid phosphatase activity was shown to be decreased early in planting, and then along with plant development, increased gradually and then gradually decreased. • Nayekorala (1998) observed that P flux by cotton roots was greatest during early growth stages and decreased with plant maturity. • The activity of acid and alkaline phosphatases was found to correlate with organic matter in various studies (Guan 1989; Jordan and Kremer, 1994; Aon and Colaneri, 2001 Makoi and Ndakidemi 2005). • Plants have also been shown to have a marked effect on soil enzyme activity. Sarapatka et al (2004 ) observed that this effect could be due to changes in organic matter content and the microbial population. Results Introduction With the increased use of transgenic crops, concerns have been raised about the environmental risk associated with the release of transgenic crops, including the potential impact on non-target organisms, such as beneficial insects, soil bacteria, and fungi, which play a fundamental role in crop residue degradation and in biogeochemical cycles. In fact, many studies showed that soil microbes represent important key non-target organisms able to highlight unforeseen collateral effects of transgenic plants on natural and agricultural ecosystem (Castaldini et al. 2005). One of the least understood areas in the environmental risk assessment of genetically modified crops is their impact on soil- and plant- associated communities. As synthetic products are introduced into genetically modified plant products, it is essential that the impact of this new input into agricultural systems on the community is assessed as the soil microbial community is an integral component of soil quality (Motavalli et al. 2004). It is inevitable that the novel gene products will eventually come into contact with the soil microbial community. However, the question is whether these products will have any effect on soil microorganism’s function (Dunfield and Germida, 2004). The activity and diversity of soil communities are known to be directly affected by common variables of agricultural practices, including: changes to plant species, water stress, fertilization, field management, tillage, fungal disease, plants and field management, grassland improvement, nitrification, and soil depth. It is therefore expected that GM crops will have some effects on soils (Lilley et al 2006). In addition, knowledge of the complex diversity of soil microorganisms is limited since only a small portion of soil microbial populations can be cultured and identified using standard approaches, including use of molecular biological techniques, show some promise in helping to understand the impact of transgenic crops on soil microbial ecology (Angle, 1994; Bruinsma et al., 2003). • Summary • Addition of D4E1 appears to have no effect on phosphomonesterase activity. • Changes over time are possibly attributed to plant requirements. • More analysis is currently underway to determine the microbial communities involved in fluctuations of activity. • Organic matter content of each treatment is currently being evaluated to determine impact on observed trends. Figure 1: Comparison of acid phosphatase activity in traditional cotton versus three isogenic lines of cotton containing containing synthetic antimicrobial peptide D4E1 • References • Angle, J. S. (1994). Release of transgenic plants: biodiversity and population level consideration. Molecular Ecology 3:45–50. • Aon M.A. and A.C. Colaneri (2001). Temporal and spatial evolution of enzymatic activities and physico-chemical properties in an agricultural soil. Appl. Soil Ecol. 18: 255–270. • Bruinsma, M., G. A. Kowalchuk, and J. A. van Veen. (2003). Effects of genetically • modified plants on microbial communities and processes in soil. Biology Fertility Soils 37:329–337. • Castaldini, M., Turrini, A., Sbrana, C., Benedetti, A., Marchionni, M., Mocali, S., Fabiani A, Landi S.Santomassimo, F., Pietrangeli, B., Nuti, M.P., Miclaus, N., and M. Giovannetti (2005). Impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms. Applied and Environmental Microbiology, 71: 6719–6729. • Dunfield, K.E., and Germida, J.J (2004) Impact of Genetically Modified Crops on Soil- and Plant-Associated Microbial Communities. 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Trends in Biotechnology Vol.24 No.1 January 2006 • Motavalli PP, Kremer RJ, Fang M, and Means NE (2004). Impact of genetically modified crops and their management on soil microbially mediated plant nutrient transformations. Journal of • Environmental Quality, 33: 816–824. • Tabatabai M. A. (1977) Effects of trace elements on urease activity in soils.‘Soil’Biology & Biochemistry 9, 9-13. • Sarapatka, B., Dudova, L. and M. Krkova. (2004). Effect of pH and phosphate supply on acid phosphatase activity in cereal roots. Biologia, Bratislava, 59/1: 127|131, 2004. • Acknowledgements • Tuskegee University • George Washington Carver Agriculture Experiment Station (GWCAES) • Tuskegee University Farm Crew • Mr, Roger Hagerty • Dr. Wenddell Mcelhanney • Mr. Victor Kahan • Dr. Jeffery Cary and Dr. Kanniah Rajasekaran of USDA, ARS Division, New Orleans, LA. • CREATE-IGERT • ALGA Figure 2: Comparison of alkaline phosphatase activity in traditional cotton versus three isogenic lines of cotton containing containing synthetic antimicrobial peptide D4E1 Objectives Evaluate the effect that cotton seeds transformed with synthetic antimicrobial peptide D4E1 will have on phosphates enzymatic activity over several time periods. ug n-phenolg-1h-1 Figure 3: Comparison of acid and alkaline phosphatase activity in traditional cotton versus three isogenic lines of cotton containing containing synthetic antimicrobial peptide D4E1 over entire growth season