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Item No. 1 of 21

INVESTIGATOR: Schultz, R. C.; Isenhart, T. M.; Raich, J. W.; Simpkins, W. W.

PERFORMING INSTITUTION:
FORESTRY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

IMPROVING SOIL AND WATER QUALITY WITH A MULTI-SPECIES RIPARIAN BUFFER STRIP

OBJECTIVES: 9501579. Our specific project objectives are: 1) to demonstrate the effectiveness of a constructed multi-species riparian buffer strip system (CMRPBS) in retaining and/or transforming nitrate in vadose zone and shallow ground waters; 2) to evaluate mechanisms of nitrate retention and transformation within the CMRBS by: a) assessing rates of denitrification and the potential for denitrification as affected by vegetation and soil properties, b) assessing the importance of plant immobilization of nitrogen in different vegetation types, and c) assessing the importance and potential of microbial immobilization of nitrate. 3) to quantify soil quality parameters potentially regulating water movement, microbial activities, and overall rates of nitrate transformation, and to investigate their relationships to vegetation cover.

APPROACH: These goals will be achieved through intensive sampling and experimental approaches focused on artificial buffer strips in central Iowa. These sites have been the focus of research by the Iowa State Agroforestry Research Team for the past four years and background data on water and agricultural chemical fluxes, and on rates of plant establishment in the buffer strips, provides a valuable information base upon which we will build. We will measure rates of denitrification, potential denitrification activity, N immobilization in above-and belowground plant materials, potential rates of microbial nitrate immobilization, and soil quality characteristics (Bulk density, infiltration rates, rooting depths, organic matter contents, C:N ratios) in each of three replicates of each of three transects in each of two different types of streamside buffer strips. The CMRBs consists of fast-growing trees placed closest to the stream, then several rows of shrubs, and a wide strip of native grasses established next to the agricultural field. This 20 m-wide buffer strip is being compared to a cool season grass buffer strip of the same width. Study results will be integrated using the soil-plant ecosystem model CENTURY.

PROGRESS: 1995/09 TO 1999/08
A sixteen-meter-wide multi-species riparian buffer was planted in 1990 consisting of 5 rows of trees adjacent to the stream, 2 rows of shrubs adjacent to the trees and a 7-meter-wide strip of native warm-season grass between the shrubs and cropfield. Seven years after establishment the buffer removed 95% of the sediment, 94% of total-N, 85% of nitrate nitrogen, 91% of total-P and 80% of phosphate phosphorus from surface runoff. Total soil carbon increases since the buffer establishment were 123% under trees, 85% under switchgrass and 61% under cool-season grasses. Particulate organic matter showed similar increases. Fine root and microbial biomass were three times higher in buffer soil than in crop soil. As a result, soil respiration rates were twice as high in the buffer soil as in the crop soil. Soil infiltration rates under the buffer were four times as high as in crop soils. Nitrate in the soil water of the unsaturated zone was reduced by up to 90% as it crossed through the buffer. Four years after establishment, denitrification rates in the soil under trees were significantly higher than in crop soil, but rates under switchgrass were not different from the crop soil. After seven years the denitrification rates under both trees and switchgrass were more than four times higher than in the crop soil and not significantly different from those in soil under cool-season grass. Removal of nitrate from the groundwater depends on the hydrogeologic setting of the aquifer. In this project, 10-15% of the nitrate was removed from the groundwater, mainly near the water table. In thinner aquifers, up to 90% of the nitrate might be removed. Nitrate removal seems limited by available carbon in the groundwater, and work has been started to assess the production of dissolved organic carbon under different plant communities. People in the Bear Creek watershed 1) want clean surface and ground water, 2) perceive that multi-species riparian buffers are acceptable and functional best management practices (BMPs), and 3) are willing to pay for improvements in surface water quality. Landowners in the Bear Creek watershed rated the 1993 surface water quality at a 6.0 (on a 0 to 10 water quality ladder scale with 10 representing best quality for human use). They wanted to improve the surface water quality to 8.3. This represents an improvement of over 30%. To achieve this improvement they perceive that > 50% reductions in sediment, fertilizer and herbicide pollutants are required. In 1993, landowners (farmers and non-farmers) were willing to pay ~ $48 per year to improve surface water quality. A 1997 focus group of farmers indicated a willingness to pay (WTP) of $25 per year to improve surface water quality. Bear Creek landowners believe that a mix of upland (field) BMPs and riparian buffers will improve water quality the most. Considering the Bear Creek watershed and a 20-year WTP present value (@5% and $48 per year) of $610,000, a 10 to 20 m wide buffer could be placed along the entire stream on both sides. Such a buffer would provide additional non-market goods and services such as hunting and aesthetics, which are highly valued by landowners.

IMPACT: 1995/09 TO 1999/08
Buffers developed in this project have served as a template for the NRCS Riparian Forest Buffer Conservation Standard - 392 that is being applied along streams nationwide. The research has demonstrated that the buffers can significantly reduce non-point source pollution from cropfields and pastures. Landowners value these buffers and have shown strong support for their installation.

PUBLICATIONS: 1995/09 TO 1999/08
1. Johnston, D.A. 1998. Hydrogeology and geology of three restored riparian buffers near Roland, Iowa. M.S. Thesis. Iowa State University.
2. Andress, R.J. 1999. Fate and transport of nitrate in groundwater within a riparian buffer in the Bear Creek watershed. M.S. Thesis. Iowa State University.
3. Lee, K.H. 1999. Effectiveness of a multi-species riparian buffer system for sediment and nutrient removal. Ph.D. Dissertation. Iowa State University.
4. Lee, K.H., Isenhart, T.M., Schultz, R.C. and Mickelson, S.K. 1999. Nutrient and sediment removal by switchgrass and cool-season grass filter strips in Central Iowa, USA. Agroforestry Systems 44:121-132.
5. Marquez, C.O., Cambardella, C.A., Isenhart, T.M., and Schultz, R.C. 1999. Assessing soil quality in a riparian buffer by testing organic matter fractions in central Iowa, USA. Agroforestry Systems 44:133-140
6. Pickle, J. 1999. Microbial nitrate immobilization in a multi-species riparian buffer. M.S. Thesis. Iowa State University.
7. Szymanski, M. and Colletti, J. 1999. Combining the socio-economic-cultural implications of community owned agroforetry: the Winnebago Tribe of Nebraska. Agroforestry Systems 44:227-239.
8. Tufekcioglu, A., Raich, J.W., Isenhart, T.M., and Schultz, R.C. 1999. Root biomass, soil respiration, and root distribution in crop fields and riparian buffer zones. Agroforestry Systems 44:163-174.
9. Schultz, R.C., Isenhart, T.M., Colletti, J.P., and Marquez, C.O. 2000. Integrated riparian management systems to protect water quality. Chapter 7 in B. Rietveld and G. Garrett (eds.) Agroforestry: An integrated Science and Practice. American Society of Agronomy, Inc., Madison, WI. USA (In press).

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 2 of 21

INVESTIGATOR: Harrison, R. L.

PERFORMING INSTITUTION:
ENTOMOLOGY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

THE BASEMENT MEMBRANE AS A BARRIER TO BACULOVIRUS DISSEMINATION IN THE HOST

OBJECTIVES: 9702936. The objectives of the proposed research are (1) to evaluate the extent to which insect basement membranes inhibit the infection of host insect tissues by baculoviruses, and (2) to determine if degradation of the basement membranes will enhance the pesticidal potential of baculoviruses.

APPROACH: Recombinant Autographa californica MNPV baculoviruses will be constructed which express genes encoding proteases that degrade basement membrane proteins. To determine if enhancement of systemic infection occurs with the expression of basement membrane-degrading proteases, larvae of the tobacco budworm, Heliothis virescens, will be infected with wild-type and protease-expressing viruses and systemic infection will be measured both by in vitro and in vivo methods. I will then construct a recombinant virus which expresses both the insect-selective scorpion toxin AaIT and the protease which promotes the highest level of systemic infection in H. virescens larvae. The insecticidal efficacies of this virus and of wild-type virus and viruses that express the protease or AaIT alone will then be measured by bioassay and determination of the extent of feeding damage caused by infected larvae.

PROGRESS: 1997/08 TO 2000/12
The goal of this project was to optimize the pesticidal potential of baculoviruses by targeting the host basement membrane, which is a potential barrier to secondary infection of host tissues. We produced recombinant clones of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) that expressed one of three genes for proteases that degrade basement membrane proteins. One of these viruses (AcMLF9.ScathL) killed Heliothis virescens larvae significantly faster than wild-type AcMNPV in survival time bioassays. This virus also killed larvae significantly faster than recombinant AcMNPV expresssing the AaIT and LqhIT2 scorpion toxins. To determine if simultaneous expression of a protease and a scorpion toxin within the host would have an additive or synergistic effect on speed of kill, survival time bioassays were set up in which neonate H. virescens were infected with mixtures of protease viruses and AcMLF9.LqhIT2, a recombinant virus that expresses the LqhIT2 scorpion toxin. The mixed infections resulted in survival times that were not significantly different than that achieved with either AcMLF9.LqhIT2 by itself or (in those cases where the mixed infection included AcMLF9.ScathL) AcMLF9.ScathL by itself. Feeding damage assays were carried out to assess the reduction in feeding by virus-infected larvae caused by expression of ScathL. H. virescens neonates infected with AcMLF9.ScathL consumed significantly less lettuce than larvae infected with wild-type AcMNPV. Although AcMLF9.ScathL kills H. virescens significantly faster than AcMLF9.LqhIT2, no signicant difference in the amount of feeding by H. virescens larvae infected with these two viruses was observed. AcMLF9.ScathL caused extensive premature cuticular melanization in 5th instar Heliothis virescens and also some melanization of internal tissues. A considerable amount of fragmentation of internal tissues was also observed in larvae infected with AcMLF9.ScathL. These observations are consistent with destruction of host basement membranes by the expressed proteases, followed by the melanotic encapsulation of tissues that have a damaged or missing basement membrane (a phenonomenon previously observed in the fruit fly, Drosophila melanogaster) and the reduction of tissue integrity. Currently, recombinant baculoviruses that express scorpion toxins are considered to have greatest potential for deployment as pest control agents. The results from our studies suggest that viruses that express basement membrane-degrading proteases may function better than toxin-expressing viruses for control of insect pests. Although some observations suggest that the basement membrane is being degraded in insects infected with AcMLF9.ScathL, there is no direct evidence for this at this time. The precise mechanism by which ScathL reduces survival time of infected larvae is also currently unknown.

IMPACT: 1997/08 TO 2000/12
With the execution of the Food Quality Protection Act, growers are faced with the loss of a number of chemical insecticides. In addition, public anxiety about genetically modified crops may result in a reassessment of the use of plants engineered with insect resistance genes. Therefore, there is an urgent need for additional insect control measures. Baculoviruses are environmentally safe alternatives to chemical insecticides that can fill this need. However, the utility of baculoviruses as control agents is hindered by the amount of time after infection that is required to kill or incapacitate infected insects. The work described above is part of an effort to improve the insecticidal activity of baculoviruses by accelerating their speed of kill with a class of enzymes that break down barriers to the progression of viral infection. The results so far indicate that the use of these enzymes can significantly improve baculovirus speed of kill. The success of this effort will pave the way for the widespread use of baculoviruses to control lepidopteran pests, which in turn will reduce the growers dependence on chemical insecticides and provide a viable alternative to transgenic crops.

PUBLICATIONS: 1997/08 TO 2000/12
1. Harrison, R.L. and B.C. Bonning. 2001. Use of proteases to improve the insecticidal activity of baculoviruses. Biological Control 20, in press.
2. PATENTS/INVENTIONS: Harrison, R.L. and B.C. Bonning. 2000. Basement membrane degrading proteases as insect toxins and methods of use for same. Filed July 12, 2000. Serial No. 09/614,789.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 3 of 21

INVESTIGATOR: Thornburg, R. W.

PERFORMING INSTITUTION:
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

SELECTION OF MUTATIONS IN UNDEFINED, COMPLEX BIOCHEMICAL PATHWAYS

OBJECTIVES: 95000801. The objective of this work is to understand the mechanismsthat control the wound-response in plants. To accomplish this, we have chosen a genetic approach to characterize Arabidopsis thaliana plants unable to respond to wounding. Plants will be selected on fluorocytosine by failure to express a wound-inducible selectable marker.

APPROACH: We will examine an alternative negative selection scheme based upon cytidine deaminase. For these studies we will: A) contstruct a reverse selectable marker in which the wound-inducible pin2 promoter is linked with the E. coli codA gene (pin2-codA). B) transform Arabidopsis thaliana pin2-codA construct and select for plants which grow in the presence of fluorocytosine. C) characterize plants with respect to wound-induction and expression of the selectable marker gene.

PROGRESS: 1997/09 TO 1999/08
The cytosine deaminase coding sequence was linked with the pin2 promoter and terminator to form the construct pRT349. Arabidopsis plants were transformed. Plants with multiple copies of the transgene were selected. Plants were characterized for expression of cytosine deaminase. The pin2-codA transgene was expressed analogously to the pin2 gene in potato plants. We next developed a seedling expression system, thereby completing the goals of objective one of our previous proposal. We next demonstrated the proof of principle, that fluorocytosine is not toxic to plants, that fluorouracil is toxic to plants and that expression of cytosine deaminase in the presence of fluorocytosine is toxic to plants. Next we bulked seed from our transgenic plants and mutagenized them. M2 seed were collected and plated on selective media. Four plants survived the two rounds of screening and each had lost the wound-inducible cytosine deaminase activity, thereby completing the goals of our second experimental objective.

IMPACT: 1997/09 TO 1999/08
Every year farmers use millions of pounds of lethal insecticides in combating plant herbivores and other pests. The strategy we are using is designed to understand the molecular mechanisms that plants naturally use to successfully defend themselves against insect attack. Our strategy is designed to produce mutant plants that have lost the ability to respond to insect attack. By characterizing these mutants we expect to learn how plant defenses naturally function. The goal is to replace the use of lethal insecticides with methods to defend plants with their own natural defenses.

PUBLICATIONS: 1997/09 TO 1999/08
1. Santoso, D. and Thornburg, R.W. (1998) UMP synthase is transcriptionally regulated during pyrimidine starvation in Nicotiana plumbaginifolia. Plant Physiol. 116:815-821.
2. Zhou, L. Lacroute, F. and Thornburg, R.W. (1998) Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase. Plant Physiol. 117:245-254.
3. Weers, B. and Thornburg, R.W. (1998) Characterization of the cDNA and gene encoding the Arabidopsis thaliana. AMP kinase (AF082882). Plant Physiol. 118:711.
4. Zhou, L. and Thornburg, R.W. (1998) Site Specific mutagenesis of conserved residues in the phosphate binding loop of the Arabidopsis UMP/CMP kinase alter ATP and UMP binding. Arch. Biochem. Biophys. 358:297-302
5. Thornburg, R.W., Park, S., Caviedes, M., Santoso, D. and Zhou, L. (1998) Selection of mutants in complex, undefined biochemical pathways: Application of new technology to the wound-induction pathway in plants. Proceedings of the International Symposium on the Future and Development towards the 21st Century. Taegu University November 27-28, 1998 pp. 81-97.
6. Weers, B. and Thornburg, R.W. (1998) Characterization of the cDNA and gene encoding the Arabidopsis thaliana. GDP-mannose pyrophosphorylase (AF076484). Plant Physiol. 118:1101.
7. Zhou, L. and Thornburg, R.W. (1999) Wound-inducible genes in plants in Inducible Gene Expression in Plants. Reynolds, P, Ed. CAB International, Wallingford, UK 127-167.
8. Weers, B. and Thornburg, R.W. (1999) Characterization of the cDNA and gene encoding an Arabidopsis thaliana. Uracil Phosphoribosyltransferase (AF116860). Plant Physiol. 119:1567.
9. Kafer, C. and Thornburg, R.W. (1999) Pyrimidine Metabolism in Plants. Paths to Pyrimidines. 5:7-19.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 4 of 21

INVESTIGATOR: Brummer, E. C.

PERFORMING INSTITUTION:
AGRONOMY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

IDENTIFYING HETEROTIC REGIONS IN THE TETRAPLOID ALFALFA GENOME

OBJECTIVES: 9701471. This project will attempt to identify molecular markers associated with heterosis for forage yield and fertility in tetraploid alfalfa, to determine if marker heterozygosity per se is related to fitness, and to compare the results with previous work from mapping diploid alfalfa populations.

APPROACH: This study will use molecular markers (RFLP, RAPD, and others) to develop a genetic map of tetraploid alfalfa. Two alfalfa populations will be developed from a cross of divergent plants, one population consisting of 200 F1 individuals and the second of 300 F2 plants derived by selfing a single F1. The former population will be non-inbred and the latter partially in-bred. These populations will be clonally propagated and plant in replicated field tests assessing dry matter production. Greenhouse tests of self and cross fertility will be conducted. Phenotypic data will be used to identify heterotic regions for seed and forage yield. Segregation distortion of markers in both populations will be evaluated for the possibility that marker heterozygosity is related to the fitness of plants in the two populations differing in the level of inbreeding. Markers significantly associated with seed and forage yield will be used in further studies to identify parental plants for new synthetic populations and as a beginning point for more extensive analysis of heterotis in autotetraploid plants.

PROGRESS: 1997/09 TO 2001/09
Alfalfa (Medicago sativa L.), the most economically important forage crop in the United States, forms the basis of the dairy and hay industries and contributes to all other livestock enterprises. Improved cultivars, necessary for these farms to remain profitable, could be developed more efficiently with a better knowledge of alfalfa genetics. We are using molecular markers to develop a comprehensive genetic map of the tetraploid alfalfa genome with specific objectives (1) to identify molecular markers associated with heterosis for forage yield and fertility, (2) to determine if marker heterozygosity per se is related to fitness, and (3) to compare the results from this study on tetraploid alfalfa with previous work from mapping diploid populations. A segregating F1 population developed by crossing an elite genotype of subspecies sativa with a semi-improved genotype of subspecies falcata is being evaluated for numerous phenotypic traits and being used to develop a genetic map. The population of 200 F1 progeny was clonally propagated and planted five clones to a plot with four replications at two locations in 1998 and two in 1999. We harvested plots three times in 1999, 2000, and 2001, providing the most detailed yield information ever collected for a single alfalfa population. The population showed significant segregation for autumn growth and for winter survival, and that the traits are not genetically correlated in this population, a result that has major positive consequences for developing high yielding, persistent cultivars. For all traits, considerable transgressive segregation beyond the parents was present. For total forage yield, the population mean was above the higher parent, indicating the extreme heterosis in this cross. Some F1 progeny had yields above the higher parent by 300% at first harvest and 200% for total forage yield. Heterosis was less apparent for other traits, but the progeny exhibited wide variation. A linkage map for each parent was constructed with RFLP and AFLP markers and used to locate chromosomal regions affecting yield and other agronomic traits. Analyses of quantitative trait locus (QTL) using single factor analysis of variance have shown that we can detect QTL for all traits. Different loci were involved in biomass production at each location and in different years. Differences among years may represent environmental changes or differences in plant development, or a combination of the two. Beneficial alleles for all traits have been found from both parents, showing that the agronomically inferior subsp. falcata possesses useful alleles. Finally, several strong forage yield QTL were identified in this population providing strong support that these regions are associated with yield heterosis. Future work will examine the importance of these chromosomal regions by more detailed mapping in this population (selfed families have been developed from each F1 individual that will be phenotyped to identify loci associated with inbreeding depression) and in other alfalfa breeding populations.

IMPACT: 1997/09 TO 2001/09
Alfalfa can improve soil and water quality and limit erosion. Improving alfalfa yield and persistence will make its incorporation into cropping systems more economical. Our results suggest that we can identify genomic regions controlling yield and winter survival. Incorporating this information into a breeding program will lead to improved cultivars.

PUBLICATIONS: 1997/09 TO 2001/09
1. Brummer EC, MK Sledge, JH Bouton and G Kochert G. 2001. Molecular marker analyses in alfalfa and related species. p. 169-180. IN RL Phillips and IK Vasil (eds.) DNA-based Markers in Plants. 2nd Ed. Kluwer, Dordrecht, The Netherlands.
2. Brummer EC. 2001.The genetics of yield and winter survival in tetraploid alfalfa. Proc. 4th Intl. Workshop on Medicago truncatula. 7-9 July 2001. Madison, WI.
3. Brummer EC. 2001. Integrating genomics and breeding to improve yield and winter hardiness in alfalfa. Proc. Plant and Animal Genome IX. 13-17 January 2001, San Diego, CA.
4. Woodfield D and EC Brummer. 2001. Integrating molecular techniques to maximise the genetic potential of forage legumes. p. 51-65. IN G Spangenberg (ed.) Molecular Breeding of Forage Crops: Proc. 2nd Int'l Symp., Molecular Breeding of Forage Crops, Lorne and Hamilton, Victoria, Australia, Nov. 19-24, 2000. Kluwer, Dordrecht, The Netherlands.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 5 of 21

INVESTIGATOR: Tim, U. S.; Kanwar, R. S.; Batchelor, W.; Babcock, B. A.; Mallarino, A.

PERFORMING INSTITUTION:
AGRI & BIOSYSTEMS ENGINEERING
IOWA STATE UNIVERSITY
AMES, IOWA 50011

INTEGRATED ASSESSMENT OF ENVIRONMENTAL AND ECONOMIC IMPLICATION OF PRECISION FARMING ON CROP PRODUCTION

OBJECTIVES: 9703966. Our research goals will be evaluated by the following specific aims: to collect data for evaluating the effects of variable rate application of fertilizer and atrazine herbicide on water quality; to develop methods to quantify the spatial distribution of cup yields and the impact of variable rate application on chemical losses to surface and groundwater; to evaluate the economic consequences of precision-farming and variable rate technology so that farmers can make informed management decisions.

APPROACH: This study will collect soil and water quality data at the Northeast Research Farm in Nashua to facilitate determination of variable rate application of nitrogen (N) and atrazine on leaching and runoff losses, uptake of N, and crop yields. Methods will be developed to characterize the spatial distribution of crop yields and the quantitative assessment, the Root Zone Water Quality model will be linked with a GIS to enable characterization and display of the spatial distribution of crop yield and chemical losses under site-specific chemical management. The study will also develop trade-off frontiers and measure the economic inputs to more site-specific and precise management, net farm returns change, and the need to identify the critical amounts of variability that justify a given level of investement invariable rate technology increases. Overall, the approaches used and data collected in this project will make a significant contribution to improved understanding of the economic and egronomic, and water quality benefits of precision farming.

PROGRESS: 1997/11 TO 2002/11
In this project, a systems approach was used to evaluate the agronomic, environmental, and economic implications of precision agriculture. A comprehensive problem-solving and decision-support system that improves analyses, simulation and visualization of field-scale impacts of precision agriculture practices (e.g, variable rate nutrient and pesticide management) on environmental quality and productivity has been developed and tested. The system combines biophysical modeling provided by RZWQM and CEREES-Maize models, (RZWQM 98), S-PLUS, and ArcView GIS. Components of the system have been validated and AgLink for Windows SSToolbox and many other programs. The problem solving environment decision support system also features an economic analysis component that provides risk-based estimates and trade-offs of effects of variable rate nutrient application on productivity and profitability of the farm operations. The problem-solving environment/decision support system is used to assess different options for implementing site-specific and nutrient and pesticide management practices. The modeling environment has been used to assess different combinations of climate, landscape, and management regimes on the agronomic and environmental benefits of precision agriculture.

IMPACT: 1997/11 TO 2002/11
The significance of this research to production agriculture is threefold. First, it enables us to move beyond the lumped treatment of ecological processes in agricultural fields and creates an integrated approach needed to facilitate the evolution towards site-specific management of crop production inputs. Second, it addresses the research needs articulated in the 1997 National Research Council on the scientific basis of precision agriculture. Third, the integrated decision support system significantly improves the use of computer models and the evaluation of "What if" scenarios to elucidate the optimal combination of soil, crops chemicals, terrain, and weather that enhances farm productivity and reduces off-site environmental impacts on a site-specific basis.

PUBLICATIONS: 1997/11 TO 2002/11
1. Tim US and X Wang. 1999. Integrated Spatial Decision Support System for Precision Resource Management. Proceeding of Conference on Geosolutions: Integrated Our World, GIS '99, March 1-4, Vancouver, BC, Canada.
2. Wang X and US Tim. 2003. Mining Factor Effects on Spatial Structure of Corn Yield. Journal of Agricultural, Biological and Environmental Statistics (in press).
3. Wang X and US Tim. 2003. Evaluating the environmental and agronomic implications of variable rate nitrogen management. Transactions of the ASAE (in press).
4. Tim US. 2003. Precision Agriculture and Water Quality. Encyclopedia of Agricultural, Food and Biological Engineering. New York, NY: Marcel Dekker, Inc.
5. Warnemuende EA and RS Kanwar. 2002. Effects of swine manure application on bacterial quality of leachate from intact soil columns. Transactions of the American Society of Agricultural Engineers 45(6):1849-1857
6. Bakhsh A, RS Kanwar, TB Bailey, CA Camberdella, DL Karlen and TS Colvin. 2002. Cropping systems effects on NO3-N loss with subsurface drainage water. TRANSACTIONS of the American Society of Agricultural Engineers 45(6):1789-1797.
7. Chung S, PW Gassman, R Gu and RS Kanwar. 2002. Evaluation of EPIC for assessing tile flow and nitrogen losses for alternative agriculture management systems. Transactions of the American Society of Agricultural Engineers 45(4):1135-1146.
8. Ella VB, SW Melvin, RS Kanwar, L Jones and R Horton. 2002. Inverse three-dimensional groundwater modeling using finite difference method for recharge estimation in a glacial till aquitard. Transactions of the American Society of Agricultural Engineers 45(3):703-715.
9. Chinkuyu AJ, RS Kanwar, JC Lorimor, H Xin and TB Bailey. 2002. Effects of laying hen manure application rate on water quality. Transactions of the American Society of Agricultural Engineers 45(2):299-308.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 6 of 21

INVESTIGATOR: Isenhart, T. M.; Parkin, T. B.; Simpkins, W. W.; Schultz, R. C.

PERFORMING INSTITUTION:
NATURAL RESOURCE ECOLOGY & MANAGEMENT
IOWA STATE UNIVERSITY
AMES, IOWA 50011

ASSESSMENT AND PREDICTION OF THE FATE OF NITRATE IN RE-ESTABLISHED RIPARIAN BUFFERS

OBJECTIVES: The goal of this work is to obtain a process level understanding of the mechanisms of nitrate transport and transformation in riparian buffers re-established on previously cropped or pastured land. Specific objectives of this work are: 1) to utilize a groundwater and vadose zone monitoring network to define the spatial and temporal heterogeneity and evaluate mechanisms of nitrate attenuation in re-establied riparian buffers, 2) to assess a chronosequence of re-established riparian buffer vegetation of 0 to 9 years of age for nitrate attenuation and compare these with riparian zones under crops, cool-season grasses, and mixed timber, and 3) to develop a decision-aid for assessing the efficacy of re-established riparian buffers to attenuate nitrate.

APPROACH: This research is part of an ongoing project to develop a riparian management system model for agricultural watersheds in the Midwestern US. The focus of the riparian restoration is the Bear Creek Watershed where we have already re-established over 6.5 km of streamlength to riparian buffer since 1990. This research will utilize existing groundwater and vadose zone monitoring networks to assess nitrate flux in these buffers and will determine the relative importance of dilution, denitrification, or plant and microbial uptake in subsurface nitrate attenuation. Nitrate flux will also be assessed within a chronosequence of re-established buffers and riparian zones in crops, cool-season grasses and mixed timber using multi-level piezometers and direct-push groundwater monitoring. N transformations will be related to surrogate variables such as soil type, vegetation cover, depth to water table, soil organic C and N content, pH, and topography to develop a decision-aid tool to a priori assess the efficacy of established buffers in removing nitrate.

PROGRESS: 1998/12 TO 2002/12
This research is part of an ongoing project to develop a riparian management system model for agricultural watersheds in the Midwestern US. The focus of the riparian restoration is the Bear Creek Watershed in Central Iowa, where riparian buffer has been re-established on over 6.5 km of stream length since 1990. The sum of this research suggests that riparian buffers re-established on previously cropped or pastured land have tremendous potential to remediate nonpoint source pollution in agricultural ecosystems. One component of this research was to determine the effectiveness of an established multi-species buffer in trapping sediment, nitrogen, and phosphorus from cropland runoff. Results during natural rainfall events demonstrate that a 16.3 m wide switchgrass/woody buffer removed 97% of the sediment, 94% of the nitrate-nitrogen, 91% of the total phosphorus, and 80% of the phosphate-phosphorus. A second component of the research has been to assess the cascading impacts of the composition and age of the re-established riparian vegetation on soil quality and nitrogen biogeochemistry within riparian soils. When using soil-water infiltration as an index, the established multi-species buffer vegetation improved soil quality after six years, with average 60-min cumulative infiltration five times greater under the buffers than under cultivated fields and pasture. Soil bulk densities under the multi-species buffer vegetation were also significantly smaller than in the crop fields and pasture. Soil respiration, microbial biomass, aggregate stability and potential denitrification rates were significantly greater within re-established riparian buffers than in cropped fields, indicating greater soil biological activity within the buffers. Riparian buffers have been shown to remove nitrate from groundwater, but the processes controlling removal are not well documented. Previous research in the Bear Creek Watershed suggests that geology influences groundwater velocity, residence time, denitrification rate, and ultimately how well the buffer functions. A multi-electrode electrical resistivity imaging system was used to characterize the extent and distribution of alluvial materials beneath buffers. Seven locations were selected for further groundwater investigations based on resistivity data and buffer maturity. Hydraulic gradient and hydraulic conductivity data from multilevel piezometers were used to assess controlling factors on nitrate removal in buffers. Several buffers consistently removed over 95 percent of nitrate. However, a few sites often had little effect on nitrate removal. Nitrate removal was favored in locations with available dissolved organic carbon and low groundwater velocities (long residence time). Lack of dissolved oxygen in these locations suggests denitrification as the removal mechanism. Based on data from the six sites, the water quality benefits of buffers are most dependent on geology, groundwater residence time and geochemical environment. Five graduate students were partially or wholly supported on this project.

IMPACT: 1998/12 TO 2002/12
This research demonstrates that riparian buffers re-established on previously cropped or pastured land have tremendous potential to remediate nonpoint source pollution in agricultural ecosystems. The research also provides insight into the geomorphic, hydrologic, and biologic factors controlling the effectiveness of streamside buffers. This information is being used to improve the performance of buffers in the field and help realize the promise of conservation buffer technology.

PUBLICATIONS: 1998/12 TO 2002/12
1. Lee K, TM Isenhart and RC Schultz. 2003. Sediment and nutrient removal in an established multi-species riparian buffer. Journal of Soil and Water Conservation 58(1):1-8.
2. Bharati L, K-H Lee, TM Isenhart and RC Schultz. 2002. Riparian zone soil-water infiltration under crops, pasture and established buffers. Agroforestry Systems 56:249-257.
3. Dornbush ME, TM Isenhart and JW Raich. 2002. Assessing the importance of fine roots: and improved alternative to litterbags. Ecology 83(11):2985-2990.
4. Simpkins WW, TR Wineland, RJ Andress, DA Johnston, GC Caron, TM Isenhart and RC Schultz. 2002. Hydrogeological constraints on riparian buffers for reduction of diffuse pollution: examples from the Bear Creek Watershed in Iowa, USA. Water Science and Technology 45(9):61-68.
5. Wineland TR. 2002. Assessing the role of geology for nitrate fate and transport in groundwater beneath riparian buffers. M.S. Thesis. Iowa State University. 122 pp.
6. Dornbush ME. 2001. Fine root decay: a comparison among three species. M.S. Thesis. Iowa State University. 109 pp.
7. Marquez CO. 2001. Soil aggregate dynamics and aggregate-associated carbon under different vegetation types in riparian soils. Ph.D. Dissertation. Iowa State University. 214 pp.
8. Tufekcioglu A. 2000. Biomass, carbon, nitrogen, and soil respiration within riparian buffers and adjacent crop fields. Ph.D. Dissertation. Iowa State University. 104 pp.
9. Webber D. 2000. Comparing estimated surface flowpaths and sub-basins derived from digital elevation models of Bear Creek watershed in central Iowa. M.S. Thesis. Iowa State University. 82 pp.
10. Andress RJ. 1999. Fate and transport of nitrate in groundwater within a riparian buffer in the Bear Creek Watershed. M.S. Thesis. Iowa State University. 93 pp.
11. Hameed S. 1999. Spatio-temporal modeling in an agricultural watershed. Ph.D. Dissertation. Iowa State University. 237 pp.
12. Lee KH. 1999. Effectiveness of a multi-species riparian buffer system for sediment and nutrient removal. Ph.D. Dissertation. Iowa State University. 139 pp.
13. Pickle JE. 1999. Microbial biomass and nitrate immobilization in a multi-species riparian buffer. M.S. Thesis. Iowa State University. 86 pp.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 7 of 21

INVESTIGATOR: Beattie, G. A.

PERFORMING INSTITUTION:
MICROBIOLOGY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

ROLE OF PLANT-BACTERIAL SURFACE INTERACTIONS IN EPIPHYTIC COLONIZATION

OBJECTIVES: I) To evaluate the influence of various features of plant cuticles on the growth, survival, retention, and entry into leaves of two bacterial phytopathogens and one saprophyte, and II) to evaluate how modulation of a major bacterial surface trait, the presence of extracellular polysaccharides, affects the relations between cuticular features and bacterial colonization of leaves.

APPROACH: A collection of maize mutants that were altered in wax biosynthetic loci were previously evaluated for their leaf surface properties, including leaf surface hydrophobicity and wax quantity, structure and distribution. Mutants that represent a range of surface properties will be tested for differences in their ability to retain bacteria following a waterborne immigration event, to foster bacterial growth and survival under various environmental conditions, and to influence bacterial localization on and in the leaf. These studies will be performed with several bacterial species to identify species-dependent effects. Mutants of Pseudomonas syringae pv. syringae that are deficient in the production of the extracellular polysaccharides alginate and levan will be constructed by a targeted mutagenesis. In planta production, and loss of in planta production, will be evaluated using EPS-specific antibodies and fluorescence microscopy. The influence of bacterial and plant surface interactions on bacterial leaf colonization will be evaluated by performing colonization studies with both the maize mutants and the bacterial EPS-deficient mutants.

NON-TECHNICAL SUMMARY: We have limited knowledge of the factors that influence bacterial colonization of leaves. Knowledge of these factors should contribute to effective strategies for reducing disease losses caused by bacterial phytopathogens, and for controlling phytopathogens using bacterial biocontrol agents. The goal of this proposal is to identify how specific surface properties of plants and bacteria influence the growth and survival of bacteria on leaves. Because the major point of contact between leaf surface bacterial populations and the plant host is at the waxy layer called the cuticle, the cuticle is likely to be a dominant plant trait influencing the leaf surface as a habitat for bacteria. This proposal will examine how this seemingly inhospitable waxy surface and bacterial cell surfaces interact to influence distinct colonization processes. The results of these studies will provide targets in plants and bacteria for hindering colonization by plant pathogens or for fostering colonization by biocontrol agents. They will also improve our ability to predict foliar diseases and biocontrol effectiveness by indicating how bacterial populations are influenced by changes in plant cuticles. Such cuticle changes may result from air pollution, water stress, pesticides, or genetic engineering of plants for enhanced drought resistance.

PROGRESS: 2002/01 TO 2002/12
Extracellular polysaccharides (EPS) are likely to have a dominant role in bacterial leaf surface ecology because their presence defines the environment immediately surrounding the bacteria. EPS-deficient mutants of Pantoea agglomerans were constructed by marker-exchange mutagenesis directed toward homologs of several Pantoea stewartii cps genes, which are involved in stewartan synthesis, and toward two Pseuodmonas syringae lsc genes, which are involved in levan synthesis. Mutants exhibited decreased production of EPS based on colony size and appearance on EPS production-promoting media. In two separate field trials, a levan-deficient mutant and a levan- and "stewartan-like EPS"-deficient double mutant maintained significantly smaller population sizes on leaves than did the wild-type strain and the "stewartan-like EPS"-deficient mutant. Although these results suggest that distinct types of EPS in P. agglomerans contribute differentially to the fitness of the organism on leaves, the impact of the antibiotic resistance cassettes on fitness have not yet been taken into account.

IMPACT: 2002/01 TO 2002/12
Knowledge of the bacterial traits that influence their fitness on leaves is important to strategies for successfully deploying bacteria onto leaves to promote plant health, such as by controlling foliar microbial pathogens. These studies are expected to demonstrate a role for one bacterial trait in fitness.

PUBLICATIONS: 2002/01 TO 2002/12
1. Marcell LM and GA Beattie. 2002. The effect of leaf surface waxes on leaf colonization by Pantoea agglomerans and Clavibacter michiganensis. Molecular Plant-Microbe Interactions 15:1236-1244.
2. Beattie GA and LM Marcell. 2002. Comparative dynamics of adherent and non-adherent bacterial populations on maize leaves. Phytopathology 92:1015-1023.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 8 of 21

INVESTIGATOR: Baum, T. J.; Rodermel, S. R.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

MUTANTS AND SIGNAL TRANSDUCTION: TWO KEYS TO UNDERSTANDING HETERODERA PARASITISM

OBJECTIVES: This project aims to explore the interactions of the sugar beet cyst nematode (Heterodera schachtii) with the host plant Arabidopsis thaliana at the molecular level. Plant genes that are involved in these interactions will be identified and characterized with the goal to understand the signal transduction events that take place in the host plant during nematode infection.

APPROACH: Two approaches will be used to identify genes involved in the nematode-plant interactions. In the first approach, previously identified A. thaliana genes that are differentially expressed during the early stages of nematode parasitism will be further characterized. For this goal, these genes are subjected to sequence analyses, hybridization experiments, and expression analyses using reporter genes. In the second approach, ethylmethane sulfonate mutagenized A. thaliana plants that exhibit altered susceptibility to the sugar beet cyst nematode, will be characterized phenotypically, and the mutant genes responsible for the aberrant phenotypes then will be mapped in preparation for positional cloning.

NON-TECHNICAL SUMMARY: Cyst nematodes (Heterodera spp.) are major pests of agricultural crops worldwide. The long-term goal of this project is to genetically engineer plants that are resistant to cyst nematodes. We are characterizing mutant A. thaliana plants that exhibit altered susceptibility to H. schachtii, the sugarbeet cyst nematode. Furthermore, we are exploring the functions of A. thaliana genes that change activity in response to the parasitizing nematode.

PROGRESS: 1999/11 TO 2002/10
In this project we targeted the understanding of the molecular interactions between the sugar beet cyst nematode and the plant Arabidopsis thaliana as a model for economically important cyst nematode pathosystems. We approached this task by designing a genetic mutant screen for Arabidopsis mutants with altered susceptibility to the cyst nematode. The idea behind this approach is that once altered susceptibility mutants are found, the responsible mutant gene can be identified using standard genetic approaches. We established a functional mutant screen and were able to identify promising mutants. One mutant in particular was further examined. This mutant, rhd1, is approximately twice as susceptible to the cyst nematode as the wild-type. We established that the nature of this mutation is due, in part, to an altered plant response to the plant hormone ethylene. Based on this discovery, we tested other Arabidopsis ethylene mutants and found a pronounced positive correlation between ethylene signal transduction in Arabidopsis and susceptibility to the cyst nematode. Genetic analyses using double mutants of rhd1 with known ethylene mutants further confirmed a role of ethylene in the rhd1 phenotypes. We cloned the rhd1 gene and determined it to be a glucose epimerase gene. The exact reason why rhd1 is hypersusceptible remains to be determined. The second approach to understanding the interactions between Arabidopsis and the cyst nematode relied on the identification of plant genes that change expression following nematode infection. We used differential display as well as microarray analyses to identify a large panel of such genes. To further characterize these genes functionally, we assayed their expression patterns using transgenic reporter gene plants. We focused on four genes that we determined to change expression in close vicinity to the invading cyst nematode and, hence, stand a high chance of being functionally involved in the interactions between plant and parasite. These genes code for metabolic enzymes as well as for components in signal transduction mechanisms. Their exact roles remain to be elucidated in future research projects.

IMPACT: 1999/11 TO 2002/10
The impact of this project is that it established Arabidopsis as a genetic model for nematode research in addition to the already known nematological applications. Second, we established the effects of ethylene in plant susceptibility and identified a panel of genes likely involved in the plant - nematode interaction. These facts generated valuable resources for the further study of the economically important cyst nematodes, which will ultimately lead to novel control measures against these important plant pathogens.

PUBLICATIONS: 1999/11 TO 2002/10
Puthoff DP, D Nettleton, SR Rodermel and TJ Baum. 2003. Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles. Plant Journal:in press

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 9 of 21

INVESTIGATOR: Bronson, C. R.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

ROLE OF FUNGAL EXTRACELLULAR MATRIX IN LESION DEVELOPMENT

OBJECTIVES: To test whether the outer layer of extracellular matrix surrounding hyphae of the fungus Cochliobolus heterostrophus is important in its ability to cause disease on maize, and, in particular, whether it is important after hyphae have penetrated maize leaves.

APPROACH: To test the importance of the outer layer of extracellular matrix, we will clone from the fungus a gene (Ecm1) specifying the production of the outer layer and use it to disrupt the resident gene in a wild-type strain. If the disrupted strain has a reduced ability to cause disease, then the layer is important. We will use microscopy to confirm the presence/absence within maize leaves of the layer around the hyphae of wild-type/mutant strains, respectively.

NON-TECHNICAL SUMMARY: The overall goal of this research is to understand how fungi cause plant disease. In particular, we want to test the importance of the slime layers that surround many fungi. These slime layers form the closest point of contact between such fungi and their plant hosts; thus, they have the potential to perform a variety of functions important in disease, especially once the fungus is inside the plant. To test whether fungal slimes are important inside plants, we will clone a gene from the fungus Cochliobolus heterostrophus, causal agent of southern corn leaf blight. This gene encodes a slimy, mucilaginous layer that surrounds the fungus and is dispensable when the fungus is on the surface of corn leaves, but appears to be essential when it is inside. We will then use the cloned gene to disrupt the resident gene in a wild-type strain. If the disrupted strain loses the ability to cause disease, then the slime layer was needed inside the plant. We will use microscopy to confirm the presence/absence within the plant of the slime layer around the wild-type/mutant strains, respectively. If this research demonstrates that fungi need slimes when they are within plant tissues, then it may be possible to control at least some plant diseases by engineering plants to degrade the slime material.

PROGRESS: 1999/09 TO 2002/09
We examined the structure of the extracellular matrix of Cochliobolus heterostrophus in plants and on surfaces. Wild-type strains have two layers: an inner layer involved in adhesion and an outer "slime" layer. The outer layer of the matrix consists of a fibrillar scaffolding that holds in place protein-containing islands. In the Ecm1 mutant, which lacks the outer layer, the fibrillar material is present, but is not interconnected. This results in a collapse of the scaffolding (and thus the apparent absence of the outer layer) and a release of the protein-containing islands. The release of these protein-containing islands is associated with an increased resistance response and decreased disease in maize plants inoculated with the mutant versus the wild-type strain. We hypothesize that the outer layer of extracellular matrix in C. heterostrophus functions to hold extracellular fungal materials in place, providing them a favorable environment for their activity or concealing them from the host, thus preventing or slowing plant defense responses. We further hypothesize that it is the release of the protein-containing islands or other materials from the collapsed scaffolding that causes the reduced pathogenicity of the Ecm1 mutant. Our attempts to clone the Ecm1 locus have been unsuccessful. However, we were able to identify a number of YAC clones containing an AFLP linked to Ecm1. We also placed RFLP linked to Ecm1 on a sequence contig of chromosome 4, better defining the possible location of Ecm1.

IMPACT: 1999/09 TO 2002/09
These results suggest that plants modified to degrade fungal slimes may show increased resistance to certain diseases. The results therefore may prove useful in the development disease resistant crop varieties.

PUBLICATIONS: 1999/09 TO 2002/09
Tsuchiya D, A Matsumoto, SF Covert, CR Bronson and M Taga. 2002. Physical mapping of plasmid and cosmid clones in filamentous fungi by fiber-FISH. Fungal Genetics and Biology 37:22-28.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 10 of 21

INVESTIGATOR: Liebman, M.; Buhler, D. D.

PERFORMING INSTITUTION:
AGRONOMY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

IMPACTS OF COMPOSTED SWINE MANURE ON WEED SEED SURVIVAL, SEEDLING EMERGENCE, GROWTH, AND COMPETITIVE ABILITY

OBJECTIVES: This project will (1) identify how application of an organic amendment to soil (composted swine manure) alters weed population dynamics and weed-crop interactions; and (2) determine the influence of weed seed size on weed responses to soil conditions. Information generated by this project will provide insight into the possibility of developing "weed suppressive soils" through the use of manure management practices that foster efficient nutrient recycling, diminish odor problems, and reduce opportunities for water contamination.

APPROACH: Effects of composted swine manure on weed seeds and seedlings will be studied by creating artificial weed seed banks in field plots amended or not amended with composted manure. Seeds of three common Midwestern weed species (Abutilon theophrasti, Amaranthus rudis, and Setaria faberi) will be added to soil contained in PVC rings and then recovered at various times after placement. Seed survival and germination will be determined. Soil and weed seeds collected from field plots will processed in a microbiology laboratory and analyzed for the presence and impacts of seed-inhabiting bacteria and fungi. To determine whether composted swine manure alters weed-crop competition, seeds of A. theophrasti, A. rudis, and S. faberi will be sown with corn in field plots that have or have not received composted manure. Weed seedlings will be thinned shortly after emergence to fixed densities. A weed-free, corn-only control treatment will also be established. Weed and corn growth, nutrient uptake, final biomass, and seed production will be measured. Laboratory bioassays will be used to determine the effects of composted swine manure on 10 weed species (Ipomoea hederacea, Abutilon theophrasti, Brassica kaber, Solanum ptycanthum, Amaranthus rudis, Sorghum bicolor, Eriochloa villosa, Setaria faberi, Echinochloa crus-galli, and Panicum dichotomiflorum) that differ in seed weight. Pre-germinated seeds will be placed on soil drawn from field plots amended or not amended with composted manure, and seedling length and disease incidence will be measured after incubation. A glasshouse experiment will be conducted to determine the effects of composted swine manure on seedling growth of the same 10 weed species over a five-week period.

NON-TECHNICAL SUMMARY: Midwestern agriculture is currently characterized by heavy reliance on herbicides and concentrated livestock production, both of which can threaten environmental quality. This project examines the impacts of composted swine manure on weeds in corn and soybean fields. Because of their seed bank and fixed root habits, annual weeds are highly responsive to soil conditions. Thus application of composted swine manure to soil is likely to alter weed dynamics. The compost used in this study will be produced in hoop structures that are increasingly popular because of low capital investment costs, lower risks of water contamination, and reduced odor emissions. Two field experiments, a laboratory bioassay experiment, and a glasshouse experiment will be conducted to examine compost effects on seed and seedling survival, establishment, growth, and competitive ability of ten weed species commonly infesting Midwestern U.S. corn and soybean fields. We will use species spanning a range of seed weights and seedling emergence times because we believe those are key factors affecting the structure and function of weed communities. The project will test whether the use of an organic matter amendment to soil, such as composted swine manure, can foster the development of weed-suppressive soil conditions, through changes in physical, chemical, and biological characteristics.

PROGRESS: 2002/01 TO 2002/12
Field plot, growth chamber, and glasshouse experiments were conducted from 1999 to 2002 to determine soil, weed, and crop responses to compost made from swine manure and corn stalks. Application of compost (at 8 Mg C per hectare) to Clarion (Typic Hapludolls) and Nicollett (Aquic Hapludolls) loam soils in Boone, IA, increased soil moisture content, and soil nitrate-N, P, K, and organic matter levels. In plots in which different weed species grew with corn, compost increased common waterhemp (Amaranthus rudis) height and biomass in each of three years, and increased velvetleaf (Abutilon theophrasti) height and biomass in one year. Compost had no effect on the height or biomass of giant foxtail (Setaria faberi) grown with corn. Seed production by common waterhemp, velvetleaf, and giant foxtail was proportional to biomass production. Compost consistently increased corn height, leaf K concentration, and stalk nitrate-N concentration, but typically did not increase corn grain yield under weed-infested conditions. In some years, yield of weed-infested corn was lower with compost than without compost, indicating that compost could increase weed competition against corn. Results from plots in which soybean grew with common waterhemp sown at different times indicated that the impact of compost depended on the timing of weed emergence. When common waterhemp emerged before soybean reached the second-node stage, compost increased the weed's height, biomass, competitive ability, and seed production. In contrast, when common waterhemp emerged after the soybean second-node stage, weed growth and competitive impact on soybean were negligible, regardless of compost application. To evaluate the effect of composted swine manure on crop and weed germination in a growth chamber, seeds of corn, soybean, wheat, common waterhemp, velvetleaf, and giant foxtail were placed in moistened germination paper with 100 g of soil amended with compost at rates equivalent to 0, 3.5, 7.0, or 10.7 g of C per kg of soil. Compost inhibited germination in a manner that was concentration dependent and inversely proportional to seed size: larger seeded species (the three crops) were less inhibited than the smaller seeded species (the three weeds). To evaluate the impact of compost on emergence and growth of the three crops and three weeds in a glasshouse, seeds were sown in pots containing compost at the concentrations described above, and seedling density and biomass were determined periodically. Compost did not affect crop emergence, but reduced weed emergence. Without compost, relative growth rates of the crop and weed species were similar, whereas with compost, relative growth rates of the weeds were higher than those of the crops. Overall, results indicate that compost can reduce weed emergence, but can also increase weed growth, seed production, and competitive ability against crops. Seed size appears to be an important variable determining differential responses to compost among crop and weed species.

IMPACT: 2002/01 TO 2002/12
Deep-bedded hoop structures are becoming increasingly popular for swine production in Iowa and other Midwestern states because they can reduce capital investment costs, odor emissions, and water contamination risks associated with conventional swine production systems. Hoop buildings used for swine production are typically bedded with corn stalks or cereal straw, and mixtures of the bedding and manure can be composted readily and spread on farm fields as soil amendments. Results of this project indicate that composted swine manure has beneficial effects on soil characteristics and crop nutrition, and that it can reduce weed emergence. However, once weeds emerge successfully, this type of compost can promote weed growth, competitive ability, and seed production. Consequently, effective weed management strategies should be in place when composted swine manure is used as a soil amendment.

PUBLICATIONS: 2002/01 TO 2002/12
1. Liebman M, T Richard, DN Sundberg, DD Buhler and FD Menalled. 2002. Impacts of composted swine manure on maize and three annual weed species. Proceedings of the 5th Workshop of the European Weed Research Society Working Group on Physical and Cultural Weed Control (11-13 March 2002, Pisa, Italy), p. 173. Institut de Malherbologie, Ste. Anne de Bellevue, Quebec, Canada.
2. Menalled FD, M Liebman and DD Buhler. 2002. Differential response of crop and weed seeds to composted swine manure applications. Ecological Society of America 2002 Meeting Abstracts: http://199.245.200.45/pweb/?SOCIETY=esa&YEAR=2002&ID=5483.
3. Menalled FD, M Liebman and DD Buhler. 2002. Impact of composted swine manure on crop and weed establishment and growth. Proceedings of the 5th Workshop of the European Weed Research Society Working Group on Physical and Cultural Weed Control (11-13 March 2002, Pisa, Italy), p. 183. Institut de Malherbologie, Ste. Anne de Bellevue, Quebec, Canada.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 11 of 21

INVESTIGATOR: Miller, W. A.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

REGULATION OF BARLEY YELLOW DWARF VIRUS GENE EXPRESSION

OBJECTIVES: (1) Identify structures on BYDV RNA that facilitate synthesis of viral subgenomic RNAs, and determine the mechanism(s) of this process. (2) Test a model of trans-regulation of translation of the viral mRNAs in which subgenomic RNA2 (sgRNA2) specifically shuts off translation of genomic RNA while allowing translation of subgenomic RNA1. This would facilitate a switch from early to late gene expression. We will also investigate whether sgRNA2 shuts off host translation.

APPROACH: For objective 1 we will place the putative subgenomic RNA promoter in a nonessential portion of the BYDV genome in our full-length, infectious clone, PAV6. The effects of deletion and site-directed mutations on accumulation of sgRNAs 2 and 3 in oat protoplasts inoculated with the mutant viral RNA will be examined, as we have done previously with the sgRNA1 promoter. Transcripts comprising sgRNA promoters will be probed with structure-sensitive nucleases and chemicals to determine their secondary structures. Transcripts corresponding full-length sgRNAs will be co-inoculated with PAV6 RNA to determine if sgRNAs can be replicated in trans, without dependence on full-length genomic RNA template. In objective 2, immunoblots and northern blots will be used to measure the accumulation of viral proteins and RNAs, respectively, over time, in synchronous infections of oat protoplasts. The effect of sgRNA2 on protein expression from the other viral mRNAs will be observed by making mutations in its translation enhancer sequence, and by expressing it from the unrelated vector, brome mosaic virus, and in transgenic oat plants. The effect of virus infection, and sgRNA2 in particular, on host translation will be investigated. Cells will be inoculated with a GFP-containing BYDV genome. Infected (GFP-expressing) cells will be sorted from uninfected cells by fluorescence-activated cell sorting. Infected cells will be tested for host protein synthesis by labeling proteins with radioactive amino acids.

NON-TECHNICAL SUMMARY: Like most viruses, barley yellow dwarf virus (BYDV) consists of an RNA molecule surrounded by a protein coat. Like DNA, RNA codes for the viral genes. The viral genes are much more densely packed than typical genes on the DNA of the host organism. The ways in which the information in these genes is converted to the proteins (the process of gene expression) needed for virus propagation differs in many remarkable ways from gene expression mechanisms of normal host genes. The aim of this research is to understand how BYDV genes are expressed, and how this controls replication (copying) of the viral RNA. To achieve these aims, we will genetically engineer infectious clones of this virus and study gene expression and replication in plant cell culture. This research will improve our understanding of replication and gene expression relevant to many plant and animal RNA viruses and of how plants themselves express genes. It may also provide ways to use viruses as tools to express useful proteins in plants. Finally, this research may also lead to new means of controlling and preventing diseases caused by RNA viruses, including BYDV, the most widespread and economically important virus of wheat, barley and oats.

PROGRESS: 2002/01 TO 2002/12
Control of BYDV gene expression by its 3' untranslated region (UTR) was investigated. The 3' cap-independent translation (protein synthesis) element (TE) is contained within the subgenomic (sg) RNA2 promoter. To determine whether the same RNA substructures that control cap-independent translation also control sgRNA2 synthesis, mutations in the TE with known effects on translation were introduced into a viral replicon and their effects on sgRNA2 synthesis were observed. Some substructures necessary for TE function were not necessary for sgRNA2 synthesis. Thus different factors probably interact with this sequence for the two functions. A sequence adjacent to the sgRNA3 promoter basepairs to a sequence 4 kb upstream to facilitate the ribosomal frameshifting necessary for translation of the viral polymerase. We propose that the long-distance base pairing needed for frameshifting and for translation initiation (last year's report) serves as a 'molecular traffic signal' which allows the replicase proceeding from the 3' end to prevent translation far upstream by disrupting the base pairing. This would clear the viral RNA of ribosomes which would otherwise block the replicase and prevent replication. In the second part of the project, expression of TE-containing sgRNA2 was predicted to inhibit host translation as observed in vitro. None of the 24 transgenic lines (engineered with collaborator D. Somers, U. Minn.) showed significant expression of sgRNA2. This is expected if high levels of TE RNA are lethal due to translation inhibition. Translation from the abundant sgRNA2 in virus-infected, non-transgenic cells was not detectable using serological methods or by replacement of ORF 6 with a GFP gene. We predict that sgRNA2 lacks sequences needed for circularization of mRNA that is needed for translation in vivo. Mutations in and around the start codon of ORF6 are lethal to virus replication but did not affect cap-independent translation of genomic RNA. We deduce that these bases, but not the encoded protein are necessary for virus replication. We conclude that sgRNA2 is a regulatory RNA rather than an mRNA. To further analyze the role of the TE, Tobacco necrosis virus (TNV) was used because its genome is 2 kb smaller than that of BYDV and unlike BYDV, it is mechanically transmissible and accumulates to high titers in plants. Deletion analysis of a full-length infectious TNV clone (from R. Coutts, Imperial College) revealed that a predicted BYDV-like 3' TE indeed confers cap-independent translation. TNV at least transiently infects Arabidopsis ecotype C24 plants, and the 3' TE's of both TNV and BYDV provide cap-independent translation of a reporter gene in Arabidopsis protoplasts. To test the effect on host translation, transgenic Arabidopsis plants were constructed that express sgRNA2 behind the estradiol-inducible UVX promoter (from N.-H. Chua, Rockefeller Univ.). No conclusions can be drawn at this early stage. The translation experiments revealed that BYDV is more closely related to TNV (in a different virus family) than it is to other viruses in BYDV's family, suggesting that these viruses should be reclassified.

IMPACT: 2002/01 TO 2002/12
This basic research may lead to (1) revision of taxonomic relationships in plant viruses which is important for epidemiology and possibly quarantine regulations, (2) more efficient engineering of plants for production of useful proteins, (3) understanding of control of replication by positive strand RNA viruses and thus better ways to prevent their occurrence. Some of this research has influenced researchers working with flaviviruses (e.g. West Nile, dengue, yellow fever), and possibly hepatitis C virus, which have similar RNA interactions to those of BYDV.

PUBLICATIONS: 2002/01 TO 2002/12
1. Koev G, Liu S, Beckett R, Miller WA (2002) The 3'-terminal structure required for replication of barley yellow dwarf virus RNA contains an embedded 3' end. Virology 292:114-126.
2. Atkins JF, Baranov PV, Fayet O, Herr AJ, Ivanov IP, Matsufuji S, Miller WA, Moore B, Prere MF, Wills NM, Jiadong Zhu J, Gesteland RF (2001)(published in 2002) Over-riding standard decoding enriches gene expression: Implications of recoding for ribosome function. In: Cold Spring Harbor Symposium on Quantitative Biology, vol. 66: The Ribosome. Cold Spring Harbor Laboratory Press, New York. pp. 217-232.
3. Barry JK, Miller WA (2002) A -1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA. Proc Natl Acad Sci USA 99:11133-11138.
4. Miller WA, Liu S, Beckett R (2002) Barley yellow dwarf virus: Luteoviridae or Tombusviridae? Molecular Plant Pathology 3:177-183.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 12 of 21

INVESTIGATOR: Horton, R.; Jaynes, D. J.

PERFORMING INSTITUTION:
AGRONOMY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

WATER FLOW AND PREFERENTIAL SOLUTE TRANSPORT PROPERTIES OF FIELD SOILS

OBJECTIVES: Develop a new method that can rapidly and simultaneously measure both hydraulic properties: saturated hydraulic conductivity and the macroscopic capillary length, and chemical transport properties: especially mobile water content, mass transfer coefficient, and dispersion coefficient in fields. Use the new method to measure the spatial distributions of hydraulic and solute transport properties of agricultural fields. Evaluate the new method using both observed data and numerical simulations.

APPROACH: The new method combines a point source method with time domain reflectometry (TDR). Irrigation drippers and TDR probes installed beneath the irrigation drippers will be used to simultaneously determine both hydraulic and chemical transport properties at multiple field locations. The method is suited to characterize spatial distributions of surface hydraulic and chemical transport properties of field soils. Once soil hydraulic and chemical transport properties are measured, the properties will be used in existing solute transport models to predict water and chemical leaching through soil profiles. The predictions of water and solute transport through soil profiles will be compared with observed tracer leaching through soil profiles. Comparison of predicted and measured tracer distributions will determine how well the surface hydraulic and transport properties obtained from the new method can be used to predict chemical leaching.

NON-TECHNICAL SUMMARY: There are no practical field methods to measure both soil water and chemical transport properties. Therefore, we are not able to screen for how quickly chemicals leach through soil. The purpose of this study is to develop and evaluate a new method that can rapidly measure both water and chemical transport properties of field soil.

PROGRESS: 2002/01 TO 2002/12
We performed a field experiment to evaluate the effect of surface solute transport properties on subsurface leaching. The field plot was 12 m by 12 m with a tile drain crossing its center at a depth of 1.2 m. Water with relatively low electrical conductivity (EC) was applied to the plot by a portable sprinkler system until a steady state water condition was attained. After reaching the steady state condition, about 14 cm of water with calcium chloride of 0.1 M concentration and 8 cm of water with low EC were applied consecutively by the same sprinkler system. A time domain reflectometry (TDR) set up was utilized to determine the near-surface chemical transport properties by measuring the bulk electrical conductivity in the top 2 cm of soil. In order to ascertain the distribution of surface solute transport properties, a total of 45 TDR probes were installed at different locations covering the various field operational management practices such as crop row, traffic and non-traffic furrows. The electrical conductivity (EC) of the tile flow was measured continuously. The breakthrough curves measured by the TDR probes will be used to estimate the surface solute transport properties that will subsequently be used to predict the tile flow EC.

IMPACT: 2002/01 TO 2002/12
New data have been obtained for describing soil chemical transport properties. These new measurements will enable better characterization of soil properties. They also enable quantitative evaluation of soil management system effects on chemical leaching.

PUBLICATIONS: 2002/01 TO 2002/12
1. Al-Jabri SA, R Horton and DB Jaynes. 2002. A point-source method for rapid simultaneous estimation of soil hydraulic and chemical transport properties. Soil Sci. Soc. Am. J. 66:12-18.
2. Al-Jabri SA, R Horton, DB Jaynes and A Gaur. 2002. Field determination of soil hydraulic and chemical transport properties. Soil Sci. 167:353-368.
3. Lee J, R Horton and DB Jaynes. 2002. The feasibility of shallow time domain reflectometry probes to describe solute transport through undisturbed soil cores. Soil Sci. Soc. Am. J. 66:53-57.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 13 of 21

INVESTIGATOR: Bhattacharyya, M. K.

PERFORMING INSTITUTION:
AGRONOMY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

SIGNAL TRANSDUCTION IN THE EXPRESSION OF DISEASE RESISTANCE IN SOYBEAN

OBJECTIVES: Resistance of soybean against the root pathogen Phytophthora sojae is conferred by a series of Rps genes. A signal pathway involved in the expression of host defenses is activated following the recognition event determined by Rps genes. Our long-term goal is to understand these recognition and signal transduction processes and apply the knowledge and resources gathered during these studies to develop soybean lines with broad-spectrum resistance. The central hypothesis of the proposed research is that a common signal pathway is involved in mediating resistance of soybean against a range of pathogens. Here we intend to investigate the involvement of a common signal pathway in the expression of resistance against four diverse pathogens P. sojae, Pseudomonas syringae pv. glycinea, soybean mosaic virus and soybean cyst nematode. We are in the process of cloning Rps1-k, the most stable Phytophthora resistance gene for the last two decades. We will overexpress this gene to induce the signal pathway(s) for the Rps-specific resistance, and then test if that pathway alters the level of resistance against these selected pathogens. We will also investigate the involvement of Rps1-k in global gene transcription through a signal pathway(s) in mediating broad-spectrum resistance against selected pathogens. The proposed research will significantly advance our fundamental knowledge of signal transduction in the expression of disease resistance in soybean, and also of the general field of plant-pathogen interactions. Such knowledge is expected to assist us in developing new soybean germplasm with broad-spectrum resistance and, therefore, will contribute significantly to the sustainability of the U.S. agriculture. We plan to test our central hypothesis and accomplish the overall goal of this application by carrying out the following specific research objectives. 1. Identify the Rps1-k gene through complementation analysis 2. Investigate the influence of Rps1-k in the global gene transcription through a signal pathway(s) in conferring broad-spectrum resistance.

APPROACH: Objective 1: Identification of Rps1-k through the complementation analysis. We have initiated complementation analysis for three non-TIR NBS-LRR like genes identified from BAC18, 99 and 160, respectively. Currently non-TIR NBS-LRR like sequences being sub-cloned in the binary vector pTF101.1. The recombinant molecules will be transferred to Agrobacterium tumefaciens strain EH101 and used in transforming cotyledonary explants. Objective 2. Investigate the influence of Rps1-k in the global gene transcription through a signal pathway(s) in conferring broad-spectrum resistance. Transgenic soybean plants will be analyzed by Southern blot analysis to estimate the number of integrated transgene copies. Northern blot analysis will be carried out to determine the Rps1-k-transcript levels among the transgenic lines. Transgenic lines showing elevated levels of the Rps1-k transcript will be evaluated for the extent of induced resistance against virulent races or strains of P. sojae, Pseudomonas syringae pv. glycinea, soybean cyst nematodes, soybean mosaic virus. The level of resistance in these plants will be compared to that of the vector transformed control plants. We will characterize the transgenic lines with high Rps1-k transcript levels and broad- spectrum resistance for changes in i) the pattern of global gene transcription, ii) phenotypes at the cellular or tissues levels, and iii) metabolites that are considered as signal transducing effectors. Global gene regulation will be studied for the transgenic lines with the highest level of Rps1-k transcripts and/or broad-spectrum resistance. Microarray analysis will be carried out in collaboration with Dr. L. Vodkin. Microarrays carrying all soybean unigenes (~30,000) including those identified from the incompatible soybean-P. sojae-interaction will be analyzed as follows. The poly(A)+ samples prepared from the etiolated hypocotyls for the following treatments will be used in studying the transcript profiles for different genes. They are: i) water control for the vector-transformed Williams (rps1-k); ii) water control for the transgenic Williams (Rps1-k) showing broad-spectrum resistance; iii) P. sojae race 1 infected vector-transformed Williams (rps1-k); iv) P. sojae race 1 infected transgenic Williams (Rps1-k) with the broad-spectrum resistance; and v) P. sojae race 1 infected Williams 82 (Rps1-k). Hybridization probes will be prepared using high quality poly(A)+ isolated from the above treatments. Verification of specific genes identified in microarray analysis will be accomplished by northern blot analyses or quantitative PCR. Changes in phenotypes at the cellular or tissue level will be determined if the over-expression causes any cell death at the macroscopic or microscopic level among the transgenic plants. Any changes in autofluorescent materials will be determined according to method of Dietrich and his co-workers (1994) and cell death by the method of Shigaki and Bhattacharyya (1999).

NON-TECHNICAL SUMMARY: Soybean suffers yield loss valued 0.12 billion dollars annually from the root and stem rot disease caused by Phytophthora sojae. The number of races of P. sojae is increasing rapidly. This is a highly variable pathogen that exists in the soil in a wide variety of virulence phenotypes to which most soybean resistance genes are ineffective. Therefore, severe crop losses from the infestation by this pathogen could be inevitable. Genetic engineering of soybean with novel and stable broad-spectrum resistance is, therefore, crucial for protecting this important crop from this and also other pathogens. The purpose of this project is to investigate the extent to which overexpression of a Phytophthora resistance gene alters global gene transcription in enhancing resistance against P. sojae and other soybean pathogens.

PROGRESS: 2002/01 TO 2002/12
A positional cloning strategy was applied in cloning five genes with high identity to the coiled-coil nucleotide-binding site leucine-rich repeat-type disease resistance gene from the soybean Rps1-k locus. The genes were classified into two classes. Members of each of the two classes contain identical open reading frames. Three representative genes were expressed in transgenic soybean plants. Each gene was shown to confer Phytophthora resistance in stable transgenic soybean plants. These results indicate that recent duplications of two similar progenitor genes resulted in the evolution of a family of active genes that together confer stable resistance against a large number of Phytophthora sojae races. Southern blot analysis indicates that there are 33 additional paralogous sequences of the Rps1-k gene family in the soybean genome. High-resolution mapping showed that most members of the Rps1-k paralogous sequences are located in six loci in a 3 cM region including the Rps1-k locus at one end. The rate of recombination is highly suppressed in the Rps1-k region. Of the two recombinants identified for the Rps1-k region one carries both rps1-k- and Rps1-k-haplotype-specific allelomorphs of two physically linked molecular markers. Presumably, an unequal crossing over event resulted in sequence duplication in this recombinant. These results strongly suggest the existence of a local gene duplication mechanism in the Rps1-k region. Most likely unequal crossing over leads to gene duplication in the Rps1-k region for evolution of new Phytophthora resistance genes. To understand the mechanism of Phytophthora resistance 4737 clones were sequenced from a cDNAs library. The cDNA library (Gm-c1084) was constructed by using polyA(+) RNAs prepared from infected etiolated hypocotyls 2 and 4 h following inoculation with P. sojae zoospores. cDNA clones were then compared to ~152,000 soybean expressed sequence tags (ESTs) that were isolated from cDNA libraries generated from unstressed organs. Two hundreds and twenty-five ESTs were found to be specific to the Gm-c1084 library.

IMPACT: 2002/01 TO 2002/12
The proposed research will significantly advance our fundamental knowledge of signal transduction in the expression of disease resistance in soybean, and also of the general field of plant-pathogen interactions. Such knowledge is expected to assist us in developing new soybean germplasm with broad-spectrum resistance and, therefore, will contribute significantly to the sustainability of the U.S. agriculture.

PUBLICATIONS: 2002/01 TO 2002/12
No publications reported this period

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 14 of 21

INVESTIGATOR: Miller, W. A.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

SYMPOSIUM ON POST-TRANSCRIPTIONAL CONTROL OF GENE EXPRESION IN PLANTS

OBJECTIVES: This project is a symposium: "Post-transcriptional Control of Gene Regulation in Plants" to be held on May 10 - 13, 2001 at Iowa State University. The symposium will focus on regulation of and by mRNA, from the moment of transcription until translation terminates. Exciting new discoveries have revealed a plethora of post-transcriptional events ranging from subtle alterations of transcribed genes to entirely new processes with profound effects. These events play diverse roles in plant development, and responses to pathogens and environmental variation. Topics include: post-transcriptional gene silencing, suppression of silencing, mRNA turnover, pre-mRNA splicing, viral translation mechanisms, translational control of plant gene expression, responses to biotic and abiotic stresses, and bioinformatics and genomics approaches. The major goal is to maximize opportunities for interactions between a variety of established scientists, students, and post-doctoral fellows, who might not encounter each other in less focused meetings, so they will learn from each other, develop new ideas and concepts together, initiate new collaborations and strengthen on-going collaborations.

APPROACH: Fifteen speakers who are world-wide leaders in their fields have accepted invitations to give plenary talks on the topics listed in Field 21 (Objectives). These well-known scientists should attract many other active researchers and interested participants. The symposium will be announced well in advance by mass mailings of a glossy brochure, mass emailings, and an informative web site. The symposium will mix plenary talks with poster sessions and short talks to be selected by the Organizing Committee from the submitted abstracts. Posters will remain up throughout the symposium to allow ample time for participants to view and discuss posters. Interactions will be facilitated by the small size of the meeting: we anticipate that the number of participants will be less than 250. Interaction will also be encouraged by frequent breaks, and by a dessert party after the first full day. This last event has proved to be a real facilitator of informal interactions in previous meetings and has provided conference goers with a unique and convivial experience.

NON-TECHNICAL SUMMARY: Every cell in a plant contains the complete set of genes (genome) of that plant. Yet cells within a plant can be very different, and the growth, development and composition of cells change in response to stimuli, such as light, drought, and pathogen attack. These differences are due to expression of different sets of genes that encode the proteins that specify cell function. Much of gene expression is controlled by transcription: copying of DNA sequence into messenger RNA (mRNA). However, the mRNA can undergo splicing, degradation, localization in the cell, and selective interaction with ribosomes (the machines that translate mRNA sequence into protein), prior to protein synthesis. These post-transcriptional processes are studied by scientists in diverse fields that often don't interact. The Symposium will bring together these scientists from around the world in a format that will maximize interaction between world-class scientists, lesser known scientists, postdocs and graduate students. There will be a limited number of participants, informal atmosphere, and ample time for discussion. Participants will leave with an awareness of the amazing diversity of gene expression mechanisms, possible new collaborations, and a better idea of the future research necessary for to understand plant gene expression. Knowledge gained from this research has direct application to improvement of crop yield, disease resistance, tolerance of harsh growing conditions, and creation of novel high-value traits, to give just a few examples.

PROGRESS: 2001/07 TO 2002/06
This project provided partial funding for the symposium: "Post-transcriptional Control of Gene Regulation in Plants" held at Iowa State University. Exciting new discoveries have revealed a plethora of post-transcriptional events ranging from subtle alterations of transcribed genes to entirely new processes with profound effects. These events play diverse roles in plant development, and responses to pathogens and environmental variation. Topics included post-transcriptional gene silencing and suppression of silencing, mRNA turnover, pre-mRNA splicing, viral translation mechanisms, translational control of plant gene expression, and bioinformatics and genomics approaches. The meeting succeeded in its aims of bringing together plant scientists in a variety of disciplines that rarely interact, exposing Midwestern American graduate students and postdocs to a large number of European scientists, and fostering new research collaborations. Participants left with an awareness of the amazing diversity of gene expression mechanisms, and with new ideas on how to carry out the research necessary for us to have even a rudimentary understanding of the forthcoming complete sequence of a plant genome. It generated intense, productive discussions among many participants from around the world late into the night. This was especially evident in the fast-moving field of post-transcriptional gene silencing and suppression of silencing. Collaborations among labs studying translation mechanisms were also facilitated.

IMPACT: 2001/07 TO 2002/06
This meeting had direct relevance to US agriculture, from understanding the mechanisms of transgenic crops, to preventing crop damage by pathogens or weather-induced stresses. Research presented at the meeting was published in prominent journals such as Cell, Nature and Plant Cell. The meeting led to at least one fruitful collaboration that would not have happened otherwise (between my own lab and a French lab), that resulted in a significant discovery on virus gene function. Other collaborations among other labs also ensued. The meeting so successful that two participants agreed immediately afterward to host a fourth meeting on this topic in Scotland in 2003.

PUBLICATIONS: 2001/07 TO 2002/06
Miller WA, Browning KS, Brown JWS, and Waterhouse PM. 2001. The RNA World in Plants: Post-transcriptional control III. (Meeting Review) Plant Cell 13:1710-1717.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 15 of 21

INVESTIGATOR: Owen, M. D.; Liebman, M. Z.; Jurik, T. W.

PERFORMING INSTITUTION:
AGRONOMY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

LIGHT, SEED WEIGHT, AND EMERGENCE TIMING EFFECTS ON WEED RESPONSE TO NITROGEN

OBJECTIVES: We hypothesize that light interception, weed seed weight, and weed emergence date partially explain the variable weed response to soil fertility in previous studies. We propose the investigation of these on three factors, with specific objectives to: 1) determine the effect of nitrogen application date and crop density on the competitiveness of selected weeds in field corn; 2) determine the effect of light availability and seed weight on interspecific weed response to nitrogen availability; and 3) determine the effect of weed seedling emergence time relative to corn seedling emergence on weed response to nitrogen availability.

APPROACH: Understanding how light availability, seed weight and emergence time influence weed response to soil fertility will allow producers to optimize fertilization and planting configuration strategies to reduce weed competitiveness for nutrients and light. The objectives will be met by a three-year field study of the effect of two nitrogen application dates and two corn population densities on the competitiveness of three weed species. The experiment will describe the treatment effects on early and mid-season vegetative growth, as well as the seed production of both crop and weed species. Furthermore, we will grow six weeds in sand culture. The weeds will be grown in environments with high and low nitrogen availability and light availability. Last, corn and Setaria faberi will be grown in competition and varying the weed emergence timing relative to the corn. These plants will be grown in sand culture environments with high or low nitrogen. Functional growth analysis will determine the rapidity and severity of each species' response to the treatment combinations.

NON-TECHNICAL SUMMARY: The role of soil fertility management in integrated weed management systems is not well understood. The research will investigate the response of six weeds to varying nitrogen and light availability to determine the impact of different corn populations and nitrogen application timing on weed growth.

PROGRESS: 2002/01 TO 2002/12
We studied the responses of eight crop and weed species, in light and shade, to daily fertilization with either 7.5 or 0.2 mM NH4NO3 in a growth chamber. Dry weights and leaf areas of all species at 18 days after emergence (DAE) were greater with high N than with low N. These favorable responses to high N were also greater in light than in shade in all species. Dry weights with high N were up to 100% greater in shade, and up to 700% greater in light, than with low N. These responses suggested that shade reduced the benefit of N to the plants. Dry weight and leaf area responses to N were positively correlated with their respective relative growth rates under high N conditions. The regression slopes of relative growth rates with high N on relative growth rates with low N were less than unity. These slopes demonstrated an ecological trade-off in which the fastest-growing species with high N suffered greater proportional decreases in growth with low N. Relative growth rates were also negatively correlated with mean seed weights among species. We compared the effect of pre-emergence (PRE N) and post-emergence (POST N) ammonium nitrate applications on the competitive abilities of giant foxtail, velvetleaf, and waterhemp in 'Pioneer 33V08' corn. The N timing effects were studied in corn densities of 5.4 and 7.9 plants per meter in order to understand the influence of other resources, including shading, on N response in weeds. By late June of each year, PRE N had increased shoot dry weight by over 50% for corn, 100% for velvetleaf, and at over 20% for giant foxtail, compared to POST N. Common waterhemp was unaffected by N timing. Corn density increased corn shoot dry weight by 21 to 32%, but had little effect on weeds. Corn density did not generally affect weed shoot dry weights when evaluated in late June. Corn grain and weed seed yields were significantly affected by interactions between N timing and weed species. Corn yield was decreased 13 to 20% by velvetleaf for PRE N compared to POST N. Conversely, corn yield was decreased 12 to 15% by POST N compared to PRE N. Similarly, PRE N increased velvetleaf seed weight by 13 to 195% compared with PRE N, but decreased giant foxtail seed weight by 55%. Velvetleaf and giant foxtail seed weights with the greater corn density were decreased by 23 to 56% and 30 to 62%, respectively. Neither N timing or corn density affected corn yield loss or weed seed production in treatments that included common waterhemp.

IMPACT: 2002/01 TO 2002/12
Relative growth rate and seed size may predict weed community responses to ecological weed management strategies that rely on shade and nitrogen manipulation. Economically important weeds are affected by nitrogen application timing, which may be incorporated into integrated weed management (IWM) strategies that reduce environmental impacts of crop production and crop yield losses. Nitrogen application timing also has an important effect on corn growth that may be used to increase the competitiveness of that crop thus improving the efficiency of herbicide use and further reduce crop yield losses.

PUBLICATIONS: 2002/01 TO 2002/12
Harbur MM and MDK Owen. 2002. Effect of Nitrogen application timing and corn density on the competitiveness of three Iowa weed species. Proc. North Cent. Weed Sci. Soc. 57:54

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 16 of 21

INVESTIGATOR: Liebman, M.; Hartzler, R. G.; Dixon, P. M.

PERFORMING INSTITUTION:
AGRONOMY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

UNDERSTANDING WEED DYNAMICS IN CONTRASTING CROP ROTATION SYSTEMS: COMBINING A PULSE/CHASE FIELD EXPERIMENT WITH MATRIX MODELS

OBJECTIVES: Our project will use a large-scale cropping systems experiment and matrix population models to meet three objectives: (1) By adding a pulse of Setaria faberi (giant foxtail) and Abutilon theophrasti (velvetleaf) seeds to plots and following their fates in subsequent years, we will determine mortality and fecundity rates of these species in contrasting rotation systems. Special attention will directed toward measuring weed seed mortality resulting from the activities of vertebrate and invertebrate seed predators. (2) By creating areas in which weed seed additions to the soil are prevented after an initial pulse of seeds is applied, we will estimate rates of seed bank decline in contrasting rotation systems. (3) We will use data from these manipulative experiments to determine how variations in mortality and fecundity affect weed population growth rates (lambda). As a result of these analyses, we will identify critical "choke points" in weed life cycles where control tactics can be targeted for greatest impact.

APPROACH: Weed population dynamics will be compared in two-, three-, and four-year rotations. The two-year rotation sequence (corn-soybean) dominates cash grain farming systems of north-central Iowa and much of the Midwestern USA. The three-year rotation is suitable for farming systems without ruminant livestock and includes a fall-sown small grain (triticale) and a legume green manure (red clover), in addition to two spring-sown crops (corn and soybean). The four-year rotation is suitable for farming systems with ruminants and includes a hay crop (alfalfa), in addition to corn, soybean, and triticale. Treatment plots will be arranged in a randomized complete block design with four replicates at a site maintained by Iowa State University in Boone County, IA. Each 0.153-ha plot will be18.3 m wide (i.e., 24 rows of corn or soybean planted at 76-cm row spacing) by 83.8 m long. Every crop entry point in each rotation system will be present in each year of the experiment. In September 2002, each main plot will be subdivided into areas where S. faberi and A. theophrasti seeds will be broadcast together on the soil surface at rates of 2,000 and 500 seeds per m2, respectively. In half of the subplots, weed populations will be allowed to grow and add new seeds to the soil seed bank within the constraints of the farming practices used (the "Add/Allow" treatment). In the other set of manipulated subplots, after seeds of S. faberi and A. theophrasti are added in September 2002, additional seed deposition will be prevented. Weed seedlings in these subplots will be pulled and destroyed after emergence. This set of subplots will comprise an "Add/Prevent" treatment. Weed seed and seedling densities, weed seed production, rates of seed bank decline, and post-dispersal loss of weed seeds to vertebrate and invertebrate granivores will be determined from 2002 to 2005. Using data generated in our field experiments, we will construct matrix population models to identify "choke points" within weed life cycles where control tactics can be targeted for greatest impact.

NON-TECHNICAL SUMMARY: In most of the Midwestern USA, cropping systems are characterized by short rotation sequences and heavy reliance on herbicides. This project will determine whether longer rotations can provide weed suppression with greater reliance on ecological processes and less dependence on chemical inputs. It will also enhance the knowledge base necessary for bringing an additional feed grain (triticale) and forages (red clover and alfalfa) into areas that are currently dominated by corn and soybean. We will use field experiments and matrix population models to understand how contrasting rotation systems affect the population dynamics of Setaria faberi (giant foxtail) and Abutilon theophrasti (velvetleaf) in two-, three-, and four-year rotation systems.

PROGRESS: 2002/01 TO 2002/12
A field plot experiment was established in Boone, IA, in 2001 and 2002, to determine the impacts of 2-, 3-, and 4-year crop rotation systems on population dynamics of velvetleaf (Abutilon theophrasti) and giant foxtail (Setaria faberi). Soil samples were collected in November 2001 to assess initial seed bank densities of these species. Velvetleaf was not detected and giant foxtail was found only at low densities (mean = 44 viable seeds per square m to a depth of 20 cm; range: 0-632). In November 2002, locally produced velvetleaf and giant foxtail seeds were added at rates of 500 and 2000 seeds per square m, respectively, to sub-plot areas of each rotation system x entry point main plot. Fates of these seeds will be followed until fall 2005. To study sources of weed mortality, preliminary seed predation trials were conducted in corn and alfalfa plots in September 2002. Results indicated that 40-100% of velvetleaf seeds presented on cards or rough pads were removed within six days. Population dynamics of common waterhemp (Amaranthus rudis), which already infests the experimental site, also will be studied in the different rotation systems. In soil samples collected in November 2001, common waterhemp dominated the soil seed bank (mean = 2080 viable waterhemp seeds per square m to a depth of 20 cm; range: 0-10739). Common waterhemp also produced 90% of the total weed biomass measured in September 2002. Large differences in weed biomass were evident among rotation systems and entry points. Common waterhemp produced <1 g biomass per square m in corn in all rotation systems, as well as in triticale and alfalfa in the 4-year rotation. It produced a substantial amount of biomass (48 g per square m) in triticale in the 3-year rotation. Common waterhemp biomass in soybean in the 2-year rotation (3.6 g per square m) was lower than in the 3-year (175 g per square m) and 4-year (177 g per square m) rotation systems. Differences in weed infestation in soybean were attributed to differences in herbicide use and to infection by soybean cyst nematode (SCN). In the 3- and 4-year rotations, where soybean received less herbicide than in the 2-year rotation, SCN-damaged soybean plants failed to competitively suppress common waterhemp. Prospective and retrospective perturbation analyses of periodic matrix models were used to examine cropping system effects on giant foxtail population growth rate. Comparisons were made between corn-soybean-wheat/red clover (+RC) and corn-soybean-wheat (-RC) rotations, using data collected in field experiments conducted in Boone, IA, in 1999-2001. Without herbicides or cultivation, the projected population growth rate of giant foxtail was more than twice as great in the +RC treatment compared to -RC treatment. With herbicides or cultivation, giant foxtail density declined more quickly with red clover than without red clover. Much of the difference between cropping systems in the weed's population trajectory was due to seed predation, which was greater in the +RC treatment. Observations and pitfall trap samples indicated that field crickets, carabid beetles, and rodents attacked giant foxtail seeds under field conditions.

IMPACT: 2002/01 TO 2002/12
In most of the north central USA, cropping systems are characterized by short rotation sequences and heavy reliance on herbicides. This project will determine whether longer rotations can provide weed suppression with greater reliance on ecological processes and less dependence on chemical inputs. It will focus particular attention on weed seed bank dynamics and seed predation by resident vertebrates and invertebrates. Modeling work completed to date indicates that prospective and retrospective perturbation analysis of periodic matrices can provide valuable insight into the impacts of crop rotation systems on weeds, and that these modeling approaches can help identify points within weed life cycles that are especially vulnerable to changes in management practices.

PUBLICATIONS: 2002/01 TO 2002/12
1. Davis AS. 2002. Cropping system effects on giant foxtail demography. Ph.D. dissertation. Iowa State University, Ames, IA.
2. Davis AS, PM Dixon and M Liebman. 2002. Retrospective perturbation analysis of cropping system effects on weed population growth rate. Ecological Society of America 2002 Meeting Abstracts: http://199.245.200.45/pweb/?SOCIETY=esa&YEAR=2002&ID=4716.
3. Davis AS and M Liebman. 2002. Response of giant foxtail (Setaria faberi Herrm.) demographic parameters in maize (Zea mays L.) to varied tillage and soil amendment practices: empirical and modeling studies. D.C. Cloutier (ed.), Proceedings of the 5th Workshop of the European Weed Research Society Working Group on Physical and Cultural Weed Control (11-13 March 2002, Pisa, Italy), p. 5. Institut de Malherbologie, Ste. Anne de Bellevue, Quebec, Canada.

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 17 of 21

INVESTIGATOR: Isenhart, T. M.; Schultz, R. C.; Simpkins, W. W.

PERFORMING INSTITUTION:
NATURAL RESOURCE ECOLOGY & MANAGEMENT
IOWA STATE UNIVERSITY
AMES, IOWA 50011

ASSESSMENT AND PREDICTION OF THE FATE OF NITRATE IN RE-ESTABLISHED RIPARIAN BUFFERS

OBJECTIVES: The overall goal of this work is to determine the effectiveness of riparian buffers re-established on previously cropped land in regulating nitrate flux to surface and groundwaters. The central hypothesis is that the riparian buffers will reduce nitrate movement to surface and groundwaters, but that such benefit is dependent upon the hydrogeologic setting (i.e. surface topography, soils, stratigraphy, and hydraulic and geochemical properties of geological units) and the composition of the vegetation and age of the buffer ecosystems. Specific research objectives are to 1) Quantify nitrate flux within a chronosequence of re-established riparian buffers, 2) Evaluate geomorphic and hydrogeologic controls of nitrate transport within riparian zones, and 3) Evaluate the mechanistic linkages between the composition and age of riparian vegetation, soil quality, and nitrate loss processes.

APPROACH: The long-range goal of the Bear Creek Watershed Project is to develop locally-acceptable watershed management systems that increase the sustainability of agriculture in the Midwestern United States with respect to surface and ground water quality, while improving the integrity of the aquatic and terrestrial ecosystems. These systems include re-establishing a suite of perennial plant-based conservation buffers, constructing or restoring strategically placed wetlands, and implementing management practices that are meant to complement in-field best management practices. As part of the project, we have already established over 11 km of riparian buffer along an agricultural stream in North-Central Iowa, providing a chronosequence ranging from 0 to 11 years since establishment. We have also defined and collected data on comparison sites of riparian zones under crops, cool-season grasses, and forest. Many sites are already implemented with the networks of groundwater and vadose zone monitoring equipment crucial to assessing shallow groundwater chemistry along groundwater flow paths. Objectives of this research will be achieved through intensive sampling and experimental approaches focused on these existing re-established riparian buffers and comparison sites. We will take advantage of the intensity of monitoring at these locations to define the mechanisms of nitrate attenuation and to assess the spatial and temporal heterogeneity of nitrate removal within established riparian buffers. Alluvium depth, texture, and extent beneath re-established riparian buffers and other vegetation will be assessed using geophysical techniques and core sampling. An analytic element model will be used to simulate the groundwater flow system at the watershed scale. The quantity and quality of carbon by depth and other soil quality parameters will be assessed beneath the re-established riparian buffers of different ages and compared with riparian zones under other vegetation. These soil quality parameters will be mechanistically linked to the ability of riparian soils to denitrify or immobilize nitrate. This information will be used to improve the ecological performance of buffers and help realize the stewardship goals of the National Conservation Buffer Initiative.

NON-TECHNICAL SUMMARY: Surface waters within intensively agricultural watersheds of the Midwestern USA contain some of the highest nitrate concentrations in the nation. Such nitrate loads have potentially widespread impacts on both public health and ecosystem function. This research examines the effectiveness of re-established riparian buffers in regulating nitrate flux to surface and groundwaters.

PROGRESS: 2002/01 TO 2002/12
The central hypothesis of this research is that riparian buffers will reduce nitrate movement to surface and groundwaters, but that such benefit is dependent upon the hydrogeologic setting (i.e. surface topography, soils, stratigraphy, and hydraulic and geochemical properties of geological units) and the composition of the vegetation and age of buffer ecosystems. The research focus area consists of nearly 11 km of re-established riparian buffer ranging in age from 1 to 12 years since establishment within the Bear Creek Watershed in Central Iowa. Ongoing monitoring includes monthly sampling of water-table monitoring wells for nitrate-nitrogen, dissolved oxygen, temperature, specific conductance, and chloride and quarterly sampling for dissolved organic carbon, pH, and alkalinity. Nitrate concentrations in groundwater show well-defined decreases at two of the three intensively monitored sites. In order to assess the effectiveness of riparian buffers in regulating nutrient flux at the watershed scale, a regional, two-dimensional analytic element model (GFLOW 2000), which simulates groundwater and surface water conjunctively, was developed to represent the region. The constructed model represents approximately 2200 km2 of central Iowa (with a near-field of about 600 km2) and is centered on Bear Creek. Unconfined conditions were assumed for the model simulations. Model input parameters include hydraulic conductivity (K=1.7 x 10-5 m/s), aquifer thickness (137 m), areal recharge (81 mm/yr), porosity (0.2), pumping from municipal wells, and discharge from drainage tiles into Bear Creek. Inhomogeneities along the creeks and rivers, which consist of alluvial and outwash deposits, were assigned K values of 3.5 x 10-4 m/s. Model calibration utilized up to six years of hydraulic head data from 41 piezometers along Bear Creek and stream discharge data from 3 gaging stations. Preliminary simulations show a deviation of 5 to 11 ft between actual and modeled heads (mean squared difference=8.5), which suggests that K and recharge need to be further optimized in the model to more closely match observed heads. Once an optimized solution is obtained, the model results can assist in calculating potential nitrate flux and removal through buffered areas of the watershed. Ongoing process-based studies are investigating denitrification potential and dissolved organic carbon (DOC) at five depths in warm season and cool season grass buffers. Denitrification potential was found to be much greater in surface soils than in subsurface soils. Cool season grass surface soils had higher denitrification potential than warm season grass. DOC is generally higher in surface soils and in cool season grass plots. DOC is not a strong predictor of denitrification potential in these sites. Carbon additions increase DEA more than nitrate additions compared to the control group. This suggests that carbon is limiting to denitrification in these soils. Two graduate students are supported on this project.

IMPACT: 2002/01 TO 2002/12
This research demonstrates that riparian buffers re-established on previously cropped or pastured land have tremendous potential to remediate nonpoint source pollution in agricultural ecosystems. The research also provides insight into the geomorphic, hydrologic, and biologic factors controlling the effectiveness of streamside buffers. This information is being used to improve the performance of buffers in the field and help realize the promise of conservation buffer technology.

PUBLICATIONS: 2002/01 TO 2002/12
No publications reported this period

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

Item No. 18 of 21

INVESTIGATOR: Dhanwada, K. R.

PERFORMING INSTITUTION:
BIOLOGY
UNIVERSITY OF NORTHERN IOWA
CEDAR FALLS, IOWA 50514

IDENTIFICATION OF REGULATORY ELEMENTS IN HORMONE-RELATED LARVAL SPECIFIC GENES FROM TOBACCO

OBJECTIVES: The objectives of this project are to identify tissue- and larva-specific factors that regulate larval epidermal cuticle and pigment gene expressions. The information obtained from this study should be useful for designing new generation of insecticides that target specific tissue and growth stage of pest insects. The insecticyanin A (INS-a) and larval-specific endocuticular protein LCP14 genes are selected for this study, Ecdysteroid and juvenile hormone (JH) control expression of both LCP14 and INS-a genes. Genomic DNA sequences of the 5' flanking region and introns of both INS-a and LCP14 genes have shown remarkable similarity in the 5' flanking regions. There is an 80% sequence similarity (from -856 to -1135 bp in INS-a and from -1004 to -1280 bp in LCP14) upstream of transcription starts site in INS-a and LCP14 genes. This highly conserved 270 bp sequence in the 5' flanking region in two larval specific genes may be indicative of a specific regulatory sequence necessary for their regulation in the epidermis. In this proposal, I will concentrate on finding whether tissue-specific factors interact with this 270 bp conserved DNA sequences, which may regulate the temporal and developmental expression of both LCP14 and INS-a genes in larval epidermis. Those regulatory factors will be the targets for future study, which can provide information for designing a new generation of insecticides.

APPROACH: In vitro study of regulatory elements in insecticyanin and LCP14 genes. In vitro transcription assays will be conducted to determine tissue specific transcriptional regulation of the LCP14 and INS-a genes. The nuclear extract containing specific transcription factors will be tested for its ability to transcribe from the promoters of the genes. If all needed factors are in the nuclear extract, transcription of the gene will be detected by assaying for the transcribed messenger using labeled probes for the gene. If a specific promoter sequence is deleted from the intact promoter, and the transcriptional activity is affected, then the importance of the promoter sequence can be deduced. If a tissue-specific transcription factor enhances the transcription from some of the truncated promoter but not others, then the regulatory elements can be deduced. Insecticyanin A and LCP14 gene regulatory elements and their nuclear binding protein. A gel mobility shift assay is a useful tool for analysis of interactions between regulatory elements in a gene and nuclear proteins that are involved in that regulation. The upstream sequences from INS-a and LCP14 genes will be used in gel mobility shift assays to determine the element's capacity for nuclear protein binding. The length of DNA sequences of the regulatory elements indicated by transcription analysis will be guiding information to determine which DNA probes should be used in gel mobility shift assays. To identify nuclear proteins interacting with various regulatory elements in the INS-a and LCP14 genes, nuclear protein extracts from Manduca sexta epidermal cells of larval, pre-pupal and pupal stages will be used. To define the precise sequences that interact with nuclear proteins, DNA footprinting experiments will be conducted. Once the precise DNA sequences bound by different transcription factors are revealed by DNA footprinting analysis, the information can be used for isolating larval and tissue specific transcription factors that may be the target genes for designing new insecticides.

NON-TECHNICAL SUMMARY: This research proposal aims at a deeper understanding of developmental-specific gene regulation by insect hormones. Insect's growth and metamorphosis are under the regulation of two major hormones, ecdysteroid and juvenile hormone (JH). Two of the developmental-specific genes have been isolated from Manduca sexta, the insecticyanin A (INS-a) and LCP14 genes. Ecdysteroid indirectly switches on and off these genes during larval molts and metamorphosis, and juvenile hormone determines whether ecdysteroid-induced gene expressing changes are transient or permanent. However, the regulatory mechanisms whereby this occurs are unknown. The goal of the experiments proposed here is to identify the relevant regulatory sequences in these genes and the hormone-induced nuclear factor(s) that interact with those sequences. The regulatory DNA sequences in both genes will be studied using epidermal tissue and cultured cells. Nuclear proteins that interact with those DNA regulatory sequences will be identified in nuclear protein extracts from specific development stages of Manduca sexta using assays that detect specific protein/DNA interaction. My long-term research goal is to reveal the mechanisms that regulate insect growth and development by insect hormones at the molecular and cellular levels. This study may provide new information on JH-directed cellular development and coordination of hormones in regulating gene expression. Understanding the molecular mechanisms underlying the action of insect growth regulators, especially the insect hormones, is essential for the development of new

PROGRESS: 1999/09 TO 2002/09
The aims of this study were to identify the relevant regulatory sequences in the Manduca sexta larval epidemeral pigment insecticyanin A (INS-a) and cuticle protein LCP14 genes and the hormone-induced nuclear factors that interact with those sequences. INS-a and LCP14 genes are larval specific and expressed in epidermal tissues during the intermolt period. Insect molting hormone 20-hydroxyecydysone (20E) suppresses the expression of both INS-1 and LCP14 genes directly. Deletion constructs of INS-a promoter were made and transfected into GV1 cells. Half of the cells were treated with 20-hydroxyecdysone (20E) for 12 hours. Cells were collected and assayed for beta-galactosidase activity and CAT protein. We studied the effect of the INS-a upstream region because there is 80% sequence similarity present between the transcription start sites of both genes. We wanted to see if this sequence was involved in tissue/developmental specific regulation of these genes. The removal of this sequence would result in higher constitutive activation of the INS-a promoter. A deletion from the promoter did increase the activity of INS-a promoter. Two constructs were made. One contained the entire 80% homologous intact and a very large portion of the 5' flanking region intact and the other did not have the 80% homology region and a majority of the 5' regulatory region. The large fragment had no change in its transcriptional activity to any significant level in the presence or absence of 20E. But the very short fragment responded to 20E-treatment positively. But no ecdysone responsive element has yet been shown within the region. We do not know if this 20E response was a direct or indirect response since we have not yet tested a short-term 20E-treatment. Work has also been done to show DNA-protein interactions between the INS-a promoter and nuclear extracts from Manduca sexta larvae. We isolated the promoter sequence from INS-a promoter and conducted GMSA using nuclear extracts from dorsal epidermis of different larval stages. Results show that all 3 oligonucleotides formed protein/DNA complexes with nuclear extracts from 4th instar larva Day 2, Day 3, Wandering Day 1 (5th instar). We focused on 2 oligos of the 5' and 3' sequences of the homologous region since these 2 fragments seemed to have higher specific binding with proteins in competition assays. There was no detectable stage-specific DNA/protein complex when either of these 2 oligos was used in GMSAs. One complex was common to all nuclear extracts from treated stages. The high molecular weight of the complex suggests that multiple proteins may be binding to the oligonucleotide sequences. Work was also done with the LCP-14 gene. The expression of LCP14 overlaps with expression of the MHR3 gene, a transcription factor in the nuclear hormone receptor family induced by 20-E. Gel mobility shift analysis was performed using MHR3 with the LCP14 intron. It was done to determine if MHR3 was interacting with the LCP14 gene since a MHR3 responsive element was found within the LCP14 fragment. MHR3 did shift the fragment indicating in preliminary experiments that this transcription factor may be interacting with LCP14.

IMPACT: 1999/09 TO 2002/09
By studying the molecular and cellular regulation of insect genes that are tissue and developmental stage specific, we can get a better understanding of the actions of the many regulatory proteins found in insects. Understanding the interactions between insect proteins and other growth regulators such as insect hormones will give us the necessary information to aid in the development of new insecticides

PUBLICATIONS: 1999/09 TO 2002/09
No publications reported this period

PROJECT CONTACT:

Name: LAN, Q.
Phone: 319-273-2327
Fax: 319-273-7125
Email: que.lan@uni.edu

Item No. 19 of 21

INVESTIGATOR: Jaynes, D. B.; Saleh, A.; Arnold, J. G.

PERFORMING INSTITUTION:
USDA/ARS
NATIONAL SOIL TILTH LABORATORY
2150 PAMMEL DR
AMES, IOWA 50014

MODIFICATION AND EVALUATION OF SWAT MODEL FOR ARTIFICIALLY DRAINED WATERSHED

OBJECTIVES: There are two main objectives to this proposed work. The first is to modify the computer code for the watershed-scale SWAT model and develop the model protocols for simulating the unique effects that distributed subsurface tile drainage systems have on hydrology and nutrient fate and transport within surface streams. The second objective is to evaluate the accuracy and robustness of the modified model against the extensive and lengthy data set collected for the tile-drained Walnut Creek watershed in central Iowa.

APPROACH: In much of the Midwest, subsurface drains were installed piecemeal over an extended period of time. Tile maps are either nonexistent, inaccurate, or not up-to-date. Many of these tile drainage systems are distributed throughout the watershed, connecting areas of poor drainage to surface streams or ditches. Thus, precise knowledge of number and location of tile drains is not possible and instead an effective drainage network must be used to simulate the impact of artificial subsurface drainage on hydrology and nutrient contamination of surface waters. We will generate a subbasin map for the Walnut Creek watershed from the a digital elevation map using the r.watershed command in the GRASS (Geographic Resources Analysis Support System) GIS program. The Walnut Creek watershed will be divided into 7 subbasins based on the elevation map and the major sampling sites within this watershed. A virtual basin approach will be used to divide the Walnut Creek watershed into multiple units (virtual subbasins) for SWAT simulation. Each virtual subbasin will be assumed to have homogeneous land use, management, tile drain system, and soil characteristics. This approach provides the opportunity to better describe the different conditions including tile-drain systems, management practices, and soil characteristics within the Walnut Creek watershed. Once simulation of each virtual subbasin is completed, the SWAT program, revised to explicitly account for tile drains, will route the computed water flow, sediment, and nutrient loading from each virtual subbasin to the outlet of the sub-watershed. Each of the 7 subwatersheds in Walnut Creek watershed will be divided into 10-30 virtual subbasins. This will divide the Walnut Creek watershed into a total 70 to 210 virtual basins. The area of each virtual basin will range from 24 to 73 ha. Predicted flow, sediment, and nutrient loading will be verified against the measured values at each sub-watershed. The first phase of calibration and testing of the modified SWAT model will use Walnut Creek stream and county drain data from 1992. Evaluation of model performance will include comparison at daily and monthly time scales of water discharge and nitrate concentration and loads. Multiple comparison criteria including maximum error, root mean square error, coefficient of determination, modeling efficiency and coefficient of residual mass will be used to evaluate SWAT model performance. After initial calibration and testing of the model with 1992 data, the model will be used to predict watershed response for the years 1991 and 1993-1998 to test the robustness of the model - calibrated parameter set to accurately predict watershed for a range of weather patterns. If satisfactory, the model will be used to test the impact of various N management practices on water quality within Walnut Creek. Initially, the model will be used to investigate the effect of switching from a fall to spring N-fertilizer application scenario and predictions compared to the results of a watershed project currently underway within Walnut Creek using this strategy.

NON-TECHNICAL SUMMARY: States will be required to identify surface waters impaired by excess nutrients and develop total maximum daily loads (TMDLs) for nutrients in these impaired watersheds. Proper TMDL calculations will require accounting for nutrients derived from all sources, including agriculture, and may lead to mandated reductions in fertilizer use. Because stream databases are poorly developed for most watersheds, computer models will be relied upon for the determination of both TMDLs and the identification of best management practices for pollutant reduction efforts. To be affective, these models must accurately predict water flow and nutrient loads within complex watersheds, which will require the accurate description of all major hydrologic processes operating within a watershed. Currently, there are no watershed-scale models that include the effect of subsurface drainage tiles. This is especially important for much of the Midwest corn belt where 50% of the crop land in some states is drained by tiles. In this research, we will modify the extensively used computer model SWAT, to specifically include the efects that tiles have on surface hydrology and nutrient fate and transport. Once modified, we will evaluate the model for its accuracy and reliability in predicting stream flows and nitrate concentrations. This testing will use the multi-year data set being collected on Walnut Creek in central Iowa - a stream contamination by nitrate due to agricultural activity in a watershed dominated by tile drains. This data set includes stream flow and nitrate concentration measurements covering

PROGRESS: 2001/10 TO 2002/09
A Specific Cooperative Agreement has been executed between ARS and Tarleton State University to transfer grant funds to Dr. Saleh as specified by the proposal. Data from Walnut Creek has been verified by Dr. Jaynes and collected into data sets that can be directly imported into SWAT. This data has been shared with all co-investigators on the project. A post-doctoral investigator has been hired at Tarleton State University to provide the majority of the programming and model simulation effort. Initial modification and testing of the SWAT model has been completed. The model is faithfully matching the monthly hydrographs measured within Walnut Creek over the 1992 - 2000 seasons. The nitrogen component is currently being modified and evaluated against measured data. Early results showed that the model underestimated nitrate concentrations during the summer and fall months. Model simulations are being overseen by Dr. Saleh and model modifications are being made by Dr Arnold.

IMPACT: 2001/10 TO 2002/09
This research will develop and test a computer model that will allow States and others to quantify the impact of specific farming practices on water quantity and quality at the watrshed scale. The model will be specifically modified to simulate watersheds in the Midwest corn belt where extensive artificial subsurface or "tile" drainage networks have been installed and serve as the primary source of nitrate contamination of surface waters.

PUBLICATIONS: 2001/10 TO 2002/09
Arnold, F.G., R. Srinivasan, M. DiLuzio, S.L. Neitsch, K.W. King. 2002. SWAT2000 - Capabilities and improvements in watershed modeling. Proc. ASAE Annual Meeting. Paper No. 02-022137, Chicago, Il. July 2002.

PROJECT CONTACT:

Name: JAYNES, D. B.
Phone: 515-294-8243
Fax: 515-294-8125
Email: jaynes@nsti.gov

Item No. 20 of 21

INVESTIGATOR: Wise, R. P.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
IOWA STATE UNIVERSITY
351 BESSEY HALL
AMES, IOWA 50010

STRUCTURAL AND FUNCTIONAL GENOMICS OF MLA-MEDIATED RESISTANCE SIGNALING IN BARLEY

OBJECTIVES: 1) Define the complete primary sequence of the Mla complex. 2) Determine the identity of the Mla1, Mla6, and Mla13 resistance specificities. 3) Identify and characterize downstream signaling mutants of the Mla6 and Mla13 specificities.

APPROACH: An absolute minimum tiling path consists of 160-kb BAC. We will sequence the overlapping 120-kb BAC to define the entire primary structure of the Mla cluster. We will also identify which candidate sequence corresponds to each specificity via mutational studies, cDNA expression analyses, and, finally, via complementation via a transient transformation assay. We will genetically characterize these mutants and determine if they define new genes in the Mla signaling pathway.

PROGRESS: 2001/10 TO 2002/09
Powdery mildew of barley is caused by the fungal pathogen, Blumeria graminis f. sp. hordei and is an ideal system for investigating fungal diseases in cereal crops. At least 30 resistance variants have been identified at a major determinant for resistance to this disease, designated Mla, however, the mechanisms of regulation of plant disease resistance genes and resistance gene products are largely unknown. A novel variant of the Mla locus, Mla13, was isolated by the Wise group (Corn Insects and Crop Genetics Research Unit, Ames, IA). We demonstrated for the first time, that expression of Mla6 and Mla13, as well as two additional genes required for Mla resistance, Rar1 and Sgt1, are coordinately induced during the same time frame that powdery mildew feeding structures make contact with the host cell plasma membrane. Three small genes upstream of Mla13 control the regulation of downstream protein synthesis and appear to be required for proper activity of the Mla resistance gene. Our results indicate that regulation of Mla transcription is not constitutive and that induction is coordinately controlled by recognition-specific plant factors. The sudden increase in transcription could account for the rapid defense response phenotype conferred by Mla13, while simultaneous control of the amount of protein synthesis would prevent uncontrolled cell death.

IMPACT: 2001/10 TO 2002/09
Powdery mildew disease causes reduced grain yield, kernel weight and grain protein. The Mla (powdery mildew resistance) locus is of particular interest because of its highly variable, multi-component nature. Host-plant resistance is the most desirable control strategy because it can be highly effective, is environmentally benign, and requires little or no additional expense to producers. This research enables breeders to utilize resistance more effectively.

PUBLICATIONS: 2001/10 TO 2002/09
Wei, F., Wing, R., Wise, R.P., 2002. Evolution and genome dynamics of the Mla (powdery mildew) resistance locus in barley. The Plant Cell 14:1903-1917.

PROJECT CONTACT:

Name: Wise, R. P.
Phone: 515-294-9756

Item No. 21 of 21

INVESTIGATOR: Wise, R. P.; Bush, A. L.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
IOWA STATE UNIVERSITY
AMES, IOWA 50011

GENETIC AND PHYSICAL ANALYSIS OF RESISTANCE TO CROWN RUST INAVENA

OBJECTIVES: 9600735. Develop recombinant inbred populations segregating for rust resistance.Position additional molecular markers tightly linked to R345 in the D526 rust resistance population via high resolution genetic analysis. Establish the degree of microsynteny of rice Bacterial Artificial Chromosomes (BAC) with the R345 rust resistance region in the D526 population and attempt to establish physical linkage of R345 with tightly-linked molecular markers.

APPROACH: We have developed a number of diploid and hexaploid mapping populations and derived recombination inbreds. These mapping populations will be used to map additional crown rust resistance loci. We will utilize high-resolution genetic analysis, microcolinearity among cerreal crops, and PCR-based amplification of resistance gene homologues to initiate positional-based cloning of genes for resistance to rust. Knowledge gained from in-depth analysis will enable us to design compound resistance gene clusters to control rust diseases. Furthering our understanding of these complex genetic interactions will be an asset in breeding programs, alleviating the problems associated with genetic uniformity and susceptibility to new races of pathogens.

PROGRESS: 1996/11 TO 1999/10
Avena (oat) plants with the appropriate genotype frequently respond to P. coronata by eliciting hypersensitive (HR) cell death at the sites of fungal infection. To investigate the genetic and cellular basis for this response, the Wise group (Corn Insects and Crop Genetics Research Unit, Ames, IA) analyzed the segregation of HR cell death on the A. strigosa (C.I. 3815) x A. wiestii (C.I. 1994) recombinant inbred (RI) population. Two newly described loci mediate HR cell death; rdh (resistance dependent hypersensitive cell death) is recessive and mediates response in resistant plants infected with P. coronata isolate 276, and, Rih (Resistance independent hypersensitive cell death) is dominant and mediates response in both resistant and susceptible plants infected with P. coronata isolate 290. These results indicate that HR cell death is not essential for resistance to crown rust and that multiple independent pathways play a role in defense to this disease. To effectively utilize current and future maps for positional cloning of agronomically important genes in Avena, a significant increase in marker density is necessary. The Wise group (Corn Insects and Crop Genetics Research Unit, Ames, IA) used two PCR (polymerase chain reaction) based methods, AFLP (amplified fragment length polymorphisms) and S-SAP (sequence-specific amplification polymorphisms) to establish a saturated map for the A. strigosa x A. wiestii RI population. Five hundred and thirteen markers were mapped to seven linkage groups, creating a framework for public utilization. This is the first saturated AFLP map created for the diploid oat genome, and provides a foundation upon which further gene-isolation efforts will be based.

IMPACT: 1996/11 TO 1999/10
Worldwide, rust diseases pose a substantial challenge to the production of small grains. The primary method of control is through genetic resistance. Host-plant resistance is the most desirable control strategy because it can be highly effective, is environmentally benign, and requires little or no additional expense to producers. This research enables breeders to utilize resistance more effectively.

PUBLICATIONS: 1996/11 TO 1999/10
1. Bush, A. L. and Wise, R. P. 1998. High-resolution mapping adjacent to the Pc71 crown-rust resistance locus in hexaploid oat. Molecular Breeding 4:13-21.
2. Kianian, S. F., Fox, S. L., Groh, S., Tinker, N., O'Donoughue, L. S., PJ Rayapati, P. J., Wise R. P., Lee, M., Sorrells, M. E., Tanksley, S. D., Fedak, G., Molnar, S. J., Rines, H. W., and Phillips, R. L. Molecular marker linkage maps in diploid and hexaploid oat (Avena sp.). DNA-based markers in plants', July 2000. 2nd edition (in press).
3. Yu, G. X. and Wise, R. P. 2000. An anchored AFLP and retrotransposon-based map of diploid Avena. Genome 43:736-749.