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Item No. 1 of 13

ACCESSION NO: 0170088 SUBFILE: CRIS
PROJ NO: IOW03332 AGENCY: CSREES IOW
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 95-37102-2213
START: 01 SEP 1995 TERM: 31 AUG 1999 FY: 1999 GRANT YR: 1995
GRANT AMT: $287,268

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 13

ACCESSION NO: 0175554 SUBFILE: CRIS
PROJ NO: IOW03483 AGENCY: CSREES IOW
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 97-35302-4325
START: 15 AUG 1997 TERM: 31 DEC 2000 FY: 2000 GRANT YR: 1997
GRANT AMT: $90,000

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 13

ACCESSION NO: 0175854 SUBFILE: CRIS
PROJ NO: IOW03490 AGENCY: CSREES IOW
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 97-35301-4405
START: 01 SEP 1997 TERM: 31 AUG 1999 FY: 1999 GRANT YR: 1997
GRANT AMT: $110,000

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 13

ACCESSION NO: 0176951 SUBFILE: CRIS
PROJ NO: IOW03506 AGENCY: CSREES IOW
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 97-35108-5128
START: 15 NOV 1997 TERM: 14 NOV 2002 FY: 2002 GRANT YR: 1997
GRANT AMT: $211,097

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. 5 of 13

ACCESSION NO: 0174285 SUBFILE: CRIS
PROJ NO: IOWR-9600735 AGENCY: CSREES IOWR
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 96-35300-3740
START: 01 NOV 1996 TERM: 31 OCT 1999 FY: 1998 GRANT YR: 1996
GRANT AMT: $204,602

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.


Item No. 6 of 13

ACCESSION NO: 0189458 SUBFILE: CRIS
PROJ NO: MO-BCCG0080 AGENCY: CSREES MO.
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2001-35318-11221 PROPOSAL NO: 2001-03423
START: 15 SEP 2001 TERM: 30 SEP 2004 GRANT YR: 2001
GRANT AMT: $175,000

INVESTIGATOR: emerich, D.; mawhinney, T.

PERFORMING INSTITUTION:
BIOCHEMISTRY
UNIVERSITY OF MISSOURI
COLUMBIA, MISSOURI 65211

ALANINE EXCRETION BY SOYBEAN NODULE BACTERIODS: METABOLIC FATE OF ALANINE

OBJECTIVES: The overall objective of this proposal is to determine the role of alanine in the soybean-B. japonicum symbiosis. Two specific objectives are proposed to initiate investigation toward the overall goal. Specific Objective 1.: Determine the symbiotic phenotype of the alanine dehydrogenase mutant. The symbiotic phenotype will indicate the degree to which alanine can serve as the nitrogen source for the plant. In peas, alanine excretion increased the efficiency or capacity of nitrogen transport to the plant, but was not essential. Our preliminary results suggest there will be a greater contribution of alanine to the nitrogen economy in soybeans. Specific Objective 2.: Characterize the alanine transport mechanisms of B. japonicum bacteroids. The uptake and the excretion of alanine appear to be separate processes and infer a futile cycle. Net transport of fixed nitrogen to the plant would require preference of excretion over uptake. Thus, characterization of these two alanine transport mechanisms would facilitate our understanding of nitrogen metabolism and flux within nodules.

APPROACH: For Specific Objective 1 we will inoculate plants with the mutant and observe the symbiotic phenotype of the soybean plant. Some of the parameters to be monitored are acetylene reduction activity, plant dry weight, and nodule morphology. Bacteroids will be isolated and measured for alanine excretion and ammonia diffusion. For Specific Objective 2 we will measure the mutant bacteroids for their ability to excrete and take up radiolabeled alanine. The use of inhibitors will further characterize the nature of these two transfer processes.

NON-TECHNICAL SUMMARY: Symbiotic nitrogen fixation by leguminous crop plants is an economical and environmentally friendly process relative to the application of commercial fertilizers. The chemical identity of the principal nitrogen-containing molecules transported to the plant remains in question. This research will attempt to determine the chemical nature of the principal forms of nitrogen that are transported between the symbiotic bacteria and the plant cells.

PROGRESS: 2001/01 TO 2001/12
The overall objective of this proposal is to determine the role of alanine in the soybean-B. japonicum symbiosis. The initial experimental procedure was to generate a mutant of B. japonicum deficient in alanine dehydrogenase and to determine its symbiotic phenotype. A second alanine dehydrogenase was found in the genome of B. japonicum. The two enzymes are highly similar in their N-terminal ends but are quite different in the remainder of their sequence. Each enzyme may have a unique physiological role in this dimorphic organism. The second alanine dehydrogenase is being isolated via the polymerase chain reaction and will be genetically inactivated by insertion of a kanamycin resistance cassette.

IMPACT: 2001/01 TO 2001/12
The presence of two alanine dehydrogenases suggests that each of the two enzymes performs a different metabolic role. Genetic inactivation will determine the role of each. If alanine is a primary transport compound from the bacteroid to the plant, then the process can be genetically modified to improve the nitrogen fixation capacity of the symbiosis and enhance plant productivity.

PUBLICATIONS: 2001/01 TO 2001/12
No publications reported this period

PROJECT CONTACT:

Name: Emerich, D.
Phone: 573-882-4252
Fax: 573-882-5635
Email: emerichd@missouri.edu2001

Item No. 7 of 13

ACCESSION NO: 0166107 SUBFILE: CRIS
PROJ NO: MO-PSCG0047 AGENCY: CSREES MO.
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 94-37305-0800
START: 15 SEP 1994 TERM: 14 SEP 1999 FY: 1999 GRANT YR: 1996
GRANT AMT: $130,942

INVESTIGATOR: Krishnan, H. B.

PERFORMING INSTITUTION:
PLANT SCIENCES
UNIVERSITY OF MISSOURI
COLUMBIA, MISSOURI 65211

CULTIVAR SPECIFICITY GENES AND NODULATION OF SOYBEAN BY RHIZOBIUM FREDII

OBJECTIVES: PROJ. #9403617. The first objective is to determine how two nodulation genes, nolW and nolX are regulated in Rhizobium fredii USDA257. The second objective is to identify the function of the nodulation genes in the nolBTUVWX locus.

APPROACH: We will run primer extension assays to identify transcript start sites for the genes and use gel retardation to determine if upstream regions bind regulatory proteins. We will also examine a poorly conserved nod-box region that overlaps the initiation codon for nolX. These experiments will emphasize nolX and nolW, but we will examine other genes as time permits. In addition, use will determine the effects of nolBTUVWX on Nod factors and surface polysaccharides of USDA257.

PROGRESS: 1994/09 TO 1999/09
Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on unimproved soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. We are trying to understand why some combination of soybean cultivars and S.fredii strains yield nitrogen fixing root nodules, while others do not. We have shown that incompatibility of USDA257 with McCall is controlled by negatively acting genes in the microsymbiont. Sequence analysis of the negative cultivar specificity locus, nolXWBTUV, indicated that these genes has no significant homology to conventional nod genes, and that none is associated with a consensus nod box promoter. However, computer searches have identified sequence homologies between the nolXBTUV locus and components of TypeIII protein secretion system of Gram-negative pathogens of plants and animals. Primer extension experiments had identified transcriptional start sites for nolX, nolW, and the nolBTUV transcriptional unit, and we have discovered that expression of nolX and nolBTUV is flavonoid-inducible and thus independent of conventionally positioned nod boxes. Loci homologous to nolXWBTUV are also present in cultivar nonspecific strain USDA191 and Rhizobium sp. NGR234, which does not form nodules on soybean. Inactivation of nolXWBTUV inhibits the flavonoid-dependent extracellular protein production by S. fredii USDA257.

IMPACT: 1994/09 TO 1999/09
S. fredii USDA257 form nitrogen-fixing nodules on soybean roots. The root-nodules capture nitrogen from the atmosphere and provides it to the plant without cost in an environmentally sustainable manner. Our research will enable others to rationally manage and enhance the process of biological nitrogen fixation there by benefiting the soybean farmers.

PUBLICATIONS: 1994/09 TO 1999/09
1. Vinardell, J.M., Ollero, F.L., Krishnan, H.B., Espuny, M.R., Villalobo, E., Pueppke, S.G. and Ruiz-Sainz, J.E. 1997. Isolation and characterization of ISRf1, an insertion sequence from the nitrogen-fixing bacterial symbiont, Rhizobium fredii. Gene 204: 63-69.
2. Krishnan, H.B., and Pueppke, S.G. 1998. Ineffective nodulation of Sesbania macrocarpa by Sinorhizobium meliloti strain RCR2011. FEMS Microbiol. Letters 165: 207-214.


Item No. 8 of 13

ACCESSION NO: 0179832 SUBFILE: CRIS
PROJ NO: MO-PSCG0090 AGENCY: CSREES MO.
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 98-35303-6741 PROPOSAL NO: 9802040
START: 15 DEC 1998 TERM: 31 MAY 2000 FY: 1999 GRANT YR: 1998
GRANT AMT: $120,000

INVESTIGATOR: Johal, G. S.

PERFORMING INSTITUTION:
PLANT SCIENCES
UNIVERSITY OF MISSOURI
COLUMBIA, MISSOURI 65211

MOLECULAR ANALYSIS OF ADULT PLANT RESISTANCE TO LEAF SPOT AND EAR ROT IN MAIZE

OBJECTIVES: Our goals will be evaluated by the following specific aims; to clone two maize genes that confer adult plant resistance to Cochliobolus carbonum race 1, the causal organism of a severe leaf spot and ear rot pathogen of maize; to compare the structure of these genes with each other as well as with the standard Hm1 disease resistance gene of this pathosystem; to compare the expression patterns of these genes with each both in the presence and the absence of the pathogen; to generate transgenic maize in which the expression of a reporter gene is tied with the promoter of the Hm2 adult plant resistance gene.

APPROACH: This study is aimed at understanding the molecular basis of adult plant resistance (ADR) in plants. To accomplish this aim we have selected the well characterized maize-Chochliobolus carbonum race 1 disease system as a model. Two maize genes (Hm1A and Hm2) that confer ADR against this disease have been identified. These genes will be cloned, characterized and compared with each other as well as with the standard Hm1 disease resistance gene to hopefully discern the factor(s) that specify the ADR behavior of these genes. We will also determine how the expression of both of these genes varies in relation to developmental aging and pathogen induced stresses. In addition, we will genetically engineer plants in which the expression of the Hm2 gene could be visually monitored to elucidate exactly how much and where in various plant parts this gene is normally active. Although these studies are of a basic nature, they do have the potential to lead to the conception, deployment or enhancement of strategies that may be used to increase the effectiveness of resistance in plant disease.

PROGRESS: 1998/12 TO 2000/12
The focus of this grant proposal (of two-year duration) was to understand the molecular basis of adult plant resistance (APR) in maize against race 1 of Cochliobolus carbonum, the causal agent of a severe leaf spot and ear-mold disease. The plan was to first clone two partially dominant, maize disease resistance genes Hm1A and Hm2, both of which become fully effective only around anthesis. Once cloned, the structures of these genes (especially in the promoter region) were to be compared and contrasted with each other as well as with the promoter of the standard Hm1 gene, which provides completely dominant, absolute resistance in all maize tissues at all stages of development. An additional component was to determine (using both conventional and transgenic approaches) how the expression of these genes (especially Hm2) varies in relation to developmental aging and pathogen induced stresses. However, the project had to be terminated about a year after its commencement because of the move of the Johal lab to Pioneer Hi-Bred Int. Inc., in Iowa. The progress accomplished before the move is as follows: Both Hm1A and Hm2, which are structurally related to Hm1, were cloned and confirmed. The sequence of the coding region was fully determined for both of these genes. While the coding region of Hm1A is identical to Hm1, the sequence of Hm2 is only about 80% homologous to Hm1. The subclones from the genomic region for both Hm1A and Hm2 were isolated but were not sequenced. For expression analysis, tissue samples were collected from a number of developmental stages of both Hm1A and Hm2 plants and total RNA was extracted from these samples.

IMPACT: 1998/12 TO 2000/12
The major impact of this study would have been an insight into the basis of adult plant resistance, which is a grossly under-investigated form of plant resistance of very great practical importance.

PUBLICATIONS: 1998/12 TO 2000/12
No publications reported this period


Item No. 9 of 13

ACCESSION NO: 0188441 SUBFILE: CRIS
PROJ NO: MOR-2000-02756 AGENCY: CSREES MO.R
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2001-35316-10000 PROPOSAL NO: 2000-02756
START: 15 NOV 2000 TERM: 14 NOV 2002 FY: 2001 GRANT YR: 2000
GRANT AMT: $100,000

INVESTIGATOR: Hibbard, B. E.

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
COLUMBIA, MISSOURI 65211

ESTABLISHMENT AND MOVEMENT OF WESTERN CORN ROOTWORM LARVAE

OBJECTIVES: Specific objectives are to: 1) Determine the effects of competition and resource availability on establishment and subsequent movement of WCR larvae on susceptible corn, 2) Evaluate alternate hosts for their potential importance in the WCR life cycle, 3) Determine establishment success of WCR larvae on host roots of differing maturities, and 4) Determine the behavioral response of WCR larvae to transgenic sources of resistance.

APPROACH: The first field experiment involves infesting individual plants with 100, 200, 400, 800, or 1,600 viable western corn rootworm (WCR) eggs. Sampling for larvae will begin immediately after the first larva is detected by hanging whole root balls in onion bags and allowing them to dry over water pans. In addition, the host status of grasses common to corn fields and adjacent grassy areas will be reexamined in the greenhouse and the field. Giant foxtail, yellow foxtail, green foxtail, baryardgrass, wooly cupgrass, fall panicum, crabgrass, and Johnson grass are among the more common grassy weed species. Each will be evaluated for their ability to support WCR growth and development along with those which were among the better alternate hosts for WCR larvae in previous studies and others common in or near corn. Host establishment will be evaluated by infesting individual plants every 7 days for 10 weeks starting at planting and recovering larvae or adults from plants infested at different growth stages of corn. Lastly, a variety of field and laboratory techniques will be used to evaluate the behavioral response of WCR larvae to transgenic sources of resistance.

NON-TECHNICAL SUMMARY: The introduction of transgenic crops for managing insect pests is one of the most important events in the last 50 years of agriculture. Since more crop land is treated with insecticide for corn rootworms than any other U.S. insect pest, the recent development of this technology for corn rootworm control by several seed companies may offer an environmentally friendly alternative to one of largest markets for insecticides. Maintaining susceptibility to transgenic crops (resistance management) is in the interest of growers, the EPA, and industry. Efforts to develop resistance management plans for prevention of corn rootworm adaptation to transgenic corn cultivars requires knowledge of rootworm larval movement and alternate host utilization that are not currently available. We propose to develop a thorough understanding of corn rootworm larval movement patterns in susceptible corn, transgenic corn, and alternative larval host micro-habitats, which are crucial in deploying effective resistance management strategies. Specifically we propose to: 1) Determine the effects of competition and resource availability on establishment and subsequent movement of WCR larvae on susceptible corn, 2) Evaluate alternate hosts for their potential importance in the WCR life cycle, 3) Determine establishment success of WCR larvae on host roots of differing maturities, and 4) Determine the behavioral response of WCR larvae to transgenic sources of resistance. As a result of this research, timely basic biological information will be generated within a two-year time frame that will assist the EPA in

PROGRESS: 2001/10 TO 2002/09
If registered for use by the Environmental Protection Agency (EPA), the introduction of transgenic corn will offer a viable alternative to insecticides for managing the western corn rootworm, one of the most economically important pests of corn. Maintaining susceptibility to transgenic crops (resistance management) is in the interest of growers, the EPA, and industry, but little is known about many aspects of corn rootworm biology. We have conducted a series of experiments to evaluate larval movement by the western corn rootworm. Previously, two row spacings and two plant spacings were evaluated with a single infestation level to determine if movement occurred. In the summers of 2001 and 2002, infestation levels of 100, 200, 400, 800, 1,600, and 3,200 viable eggs on a central plant were evaluated to determine the effect of egg density and subsequent plant damage on movement after establishment. Movement up to three plants down the row and across a 0.46 m row have been clearly documented after initial establishment. No significant post-establishment larval movement has occurred at lower infestation levels. Larvae apparently move when plants are highly damaged and/or when competition for food exists, but the extent that this movement occurs under normal field infestations (without a single point source) remains unclear. Another aspect of rootworm biology related to resistance management for which little information is available is the level of importance that alternate hosts play in the life cycle of the western corn rootworm. Previously, we evaluated 28 species of major grassy weeds for their host status with western corn rootworm larvae in the greenhouse. Twenty-one of the species evaluated supported western corn rootworm larval growth at least to the third instar and most of these grasses can be found in or near corn fields. In field experiments in 2001 and 2002, we evaluated grassy weed species by themselves, with isoline corn and with transgenic corn. We demonstrated that adults were produced from five of nine grassy weed species evaluated, and more adults were produced from a combination of grass and transgenic corn than either the transgenic corn or the grass alone.

IMPACT: 2001/10 TO 2002/09
Grasses in, near, and perhaps even distant from corn may play more of a role in the rootworm life cycle than most rootworm entomologists would have guessed, implying that resistance management models for corn rootworm adaptation to transgenic corn (computer simulations at this point) may need to have some of their assumptions adjusted.

PUBLICATIONS: 2001/10 TO 2002/09
Hibbard, B.E., D.P. Duran, M.R. Ellersieck, and M.M. Ellsbury. 2003. Post-establishment movement of western corn rootworm larvae (Coleoptera: Chrysomelidae) in Central Missouri corn. J. Econ. Entomol. (Accepted, In Press).

PROJECT CONTACT:

Name: HIBBARD, B. E.
Phone: 573-882-6281
Fax: 573-882-7850
Email: hibbard@missouri.edu

Item No. 10 of 13

ACCESSION NO: 0186823 SUBFILE: CRIS
PROJ NO: MOR-2000-03364 AGENCY: CSREES MO.R
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2001-35318-10097 PROPOSAL NO: 2000-03364
START: 01 DEC 2000 TERM: 30 NOV 2002 FY: 2001 GRANT YR: 2001
GRANT AMT: $225,000

INVESTIGATOR: Tipton, P. A.

PERFORMING INSTITUTION:
BIOCHEMISTRY
UNIVERSITY OF MISSOURI
COLUMBIA, MISSOURI 65211

SOYBEAN URATE OXIDASE AND THE UREIDE PATHWAY

OBJECTIVES: To trap and identify intermediates that form during the catalytic cycle of the enzyme urate oxidase, and to clone the gene encoding hydroxyisourate hydrolase from soybean root nodules.

APPROACH: To identify intermediates in the urate oxidase reaction, rapid-mixing chemical quench techniques are being applied. Components in the quenched reaction mixtures are purified by HPLC and identified using mass spectrometry. Cloning of the gene encoding hydroxyisourate hydrolase is being accomplished with PCR-based techniques using primers that have been designed based on the amino acid sequence of tryptic fragments of purified hydroxyisourate hydrolase.

NON-TECHNICAL SUMMARY: Nitrogen is a limiting nutrient for most crops; legumes such as soybeans occupy a special place in agriculture because they possess the ability to convert atmospheric nitrogen into ammonia. The metabolic pathway by which the ammonia is converted into organic compounds that can be used by the plant to support its growth is called the ureide pathway. Nitrogen fixation is an energetically costly process for the plant, and a worthwhile goal for agricultural research is to enhance the efficiency of the process by which nitrogen becomes available to the plant in usable form. A prerequisite for doing so, however, is gaining a detailed understanding of the ureide pathway and its component enzymes. One of the enzymes in the ureide pathway is urate oxidase, which catalyzes the conversion of urate to 5-hydroxyisourate. It is now known how 5-hydroxyisourate is converted to allantoin, which is the metabolite that the plants transport from the roots, where nitrogen fixation and the urate oxidase reaction occur, to the stem and leaves, where the nitrogen is used in amino acid biosynthesis. One current goal is to characterize 5-hydroxyisourate hydrolase, which may be a hitherto unrecognized constituent of the ureide pathway. A second goal is to continue mechanistic studies of urate oxidase by isolating and identifying intermediates formed during the catalytic cycle. These studies are leading to an understanding of how urate oxidase can operate in the absence of any of the cofactors that typically mediate dioxygen-dependent enzymatic reactions.

PROGRESS: 2001/10 TO 2002/09
Experiments conducted in the past year demonstrated that allantoin produced by the action of the ureide pathway in soybean root nodule slices is optically active, which indicates that every step in the pathway must be enzyme-catalyzed. Therefore, there remains at least one more enzyme in the pathway to be discovered and characterized. We determined the organellar and subcellular localization of hydroxyisourate hydrolase, and defined the temporal expression of its gene in relation to onset and cessation of nitrogen fixation. All of the data are consistent with hydroxyisourate hydrolase participating in the ureide pathway. Experiments were conducted to characterize the chemical mechanism of hydroxyisourate hydrolase. Sequence alignments suggested that it shares considerable homology with well-characterized retaining glycosidases, which utilize glutamate residues at the active site to mediate substrate hydrolysis. We identified analogous glutamate residues in hydroxyisourate hydrolase and showed by site-directed mutagenesis that they play critical roles in the catalytic reaction. The addition of anionic nucleophiles to the reaction medium accelerated the rate of the enzymatic reaction, which suggests that it proceeds via formation of an enzyme-substrate covalent intermediate. We conducted mechanistic studies of urate oxidase; specifically, rapid-mixing chemical quench experiments were performed to characterize the timecourse for urate dianion formation during the catalytic cycle. Site-directed mutagenesis was used to identify the amino acid residues required for substrate deprotonation.

IMPACT: 2001/10 TO 2002/09
Our recent data strongly suggest that hydroxyisourate hydrolase is a legitimate constituent of the ureide pathway, and clearly demonstrate that another, as yet unidentified enzyme, participates in the pathway as well. Thus, it is clear that this important metabolic pathway remains poorly characterized. It is anticipted that a better understanding of the ureide pathway is required for efforts to improve the efficiency of nitrogen utilization by soybeans. The mechanistic studies of urate oxidase continue to define the parameters and characteristics of cofactor independent oxygen-mediated enzymatic reactions.

PUBLICATIONS: 2001/10 TO 2002/09
No publications reported this period

PROJECT CONTACT:

Name: TIPTON, P. A.
Phone: 573-882-7968
Fax: 573-884-4812
Email: tiptonp@missouri.edu

Item No. 11 of 13

ACCESSION NO: 0176198 SUBFILE: CRIS
PROJ NO: MOR-9701357 AGENCY: CSREES MO.R
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 97-35300-4391
START: 01 AUG 1997 TERM: 31 JUL 2001 GRANT YR: 1997
GRANT AMT: $364,000

INVESTIGATOR: Mcmullen, M. D.; Lee, E. A.; Byrne, P. F.; Snook, M. E.; Wiseman, B. R.; Widstrom, N. N.

PERFORMING INSTITUTION:
PLANT GENETICS
AGRICULTURAL RESEARCH SERVICE
COLUMBIA, MISSOURI 65211

GENETIC CONTROL OF CORN EARWORM RESISTANCE FACTORS IN MAIZE

OBJECTIVES: 9701357. Determine the role of known genes in the flavonoid pathway in controlling maysin synthesis. Determine the relationship of expression of flavonoid genes with QTL controlling maysin synthesis and corn earworm resistance. Relate information gained to enhance corn earworm resistance in maize lines.

APPROACH: Use QTL analysis to determine the role of known genes in the flavonoid pathway in controlling maysin synthesis. Use insect bioassays to correlate maysin levels with antibiosis. Use gene expression studies to determine the role of transcription activation in determining QTLs for maysin synthesis.

PROGRESS: 1997/10 TO 1998/09
With this project we will continue studies to determine the relationship of specific genes in the flavonoid pathway with QTLs for flavone synthesis and corn earworm (CEW) resistance. This summer we initiated experiments with five new populations with the following specific objectives. The population (T218 x GT119)F2 to determine the genetic basis of the salmon silk phenotype and to study epistatic interaction of the p1 and sm1 loci. The population (W23al x GT119)F2 to determine the effect of a block in the 3-deoxyanthocyanin pathway on flavone synthesis and CEW resistance. The population (Mo1W & Mo20W x A619)F2s to study flavone synthesis and CEW resistance directed by pl-wwb alleles. Two populations are being studied to determine the relationship of QTLs for flavone synthesis and levels of gene expression of genes of the flavonoid pathway, the population (GT114 x GT119)F2 to study activation of the flavone pathway by variation at p1, and the population (GE37 x Mp464)F2 to study the relative expression of the two chalcone synthase genes, c2 and whp1. During the summer of 1997 silks were collected from all these populations and genetic and chemical analyses are in progress.

PUBLICATIONS: 1997/10 TO 1998/09
1. MCMULLEN, M.D., BYRNE, P.F., SNOOK, M.E., WISEMAN, B.R, LEE, E.A., WIDSTROM, N.W., and COE, E.H. (1998) Quantitative trait loci and metabolic pathways. Proc. Natl. Acad. Sci. USA 95:1996-2000.
2. BYRNE, P. F., MCMULLEN, M. D., WISEMAN, B. R., SNOOK, M. E., MUSKET, T. A., THEURI, J, M., WIDSTROM, N. W., and COE, E. H. (1998) Maize silk maysin concentration and corn earworm antibiosis: QTLs and genetic mechanisms. Crop Sci. 38:461-.
3. LEE, E.A., BYRNE, P.F., MCMULLEN, M.D., SNOOK, M.E., WISEMAN, B.R., WIDSTROM, N.W., and COE, E.H. (1998) Genetic mechanism underlying apimaysin and maysin synthesis and corn earworm antibiosis in maize (Zea mays L.). Genetics 149:1997-2006.


Item No. 12 of 13

ACCESSION NO: 0180498 SUBFILE: CRIS
PROJ NO: MOR-9803871 AGENCY: CSREES MO.R
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 98-35311-6688
START: 15 SEP 1998 TERM: 30 SEP 2001 FY: 2001 GRANT YR: 1998
GRANT AMT: $50,000

INVESTIGATOR: Stuart, M. K.

PERFORMING INSTITUTION:
MICROBIOLOGY & IMMUNOLOGY
KIRKSVILLE COLL OF OSTEO MED
KIRKSVILLE, MISSOURI 63501

ELONGATION FACTOR 1-ALPHA AS A POTENTIAL TARGET FOR DISRUPTION OF INSECT METABOLISM

OBJECTIVES: 9803871. The primary goal of this project is the production of monoclonal antibodies that can inactivate eucaryotic translation elongation factor-1a(EF-1a) from the fall armyworm, Spodoptera frugiperda. This is the first step toward the long-range goal of generating biopesticides capable of inhibiting protein synthesis in target pests, a goal that will be achieved by engineering baculoviruses to express EF-1a-specific single-chain antibodies within the cytoplasm of host cells.

APPROACH: Monoclonal antibodies specific for EF-1a will be generated from mice immunized with 1) synthetic peptides that are based on amino acid sequences unique to S. frugiperda EF-1a, and 2) whole-molecule EF-1a isolated from the S. frugiperda cell line, Sf21. An antibody shown to recognize native EF-1a by its ability to immunoprecipitate the nondenatured enzyme from cellular homogenates will be further examined for its effects on protein synthesis in cell-free in vitro translation systems established from Sf21 and BTI-TN-5BI-4 cells, the latter derived from the cabbage looper, Trichoplusia ni. The ability of antibodies to inhibit protein synthesis will be determined by adding luciferase mRNA and purified antibody to translation-competent insect cell lysates pre-treated with micrococcal nuclease to remove endogenous mRNA, and then examining new translation products for luciferase protein. Incorporation of biotinylated lysines into newly synthesized luciferase will be detected by a non-radioactive colorimetric method after the translation products have been subjected to SDS-PAGE and transferred to nitrocellulose.

PROGRESS: 1998/09 TO 2001/09
Our LONG-RANGE GOAL is to enhance the baculovirus as a microbial insecticide by engineering it to express single-chain antibodies capable of inactivating essential enzymes in insect cells. Our first target for this technology is elongation factor-1 alpha (EF-1a), an enzyme necessary for protein synthesis. The SPECIFIC AIMS of the current project were to: 1) PRODUCE MURINE MONOCLONAL ANTIBODIES (MABS) TO EF-1a FROM THE FALL ARMYWORM, SPODOPTERA FRUGIPERDA. This goal was met by immunizing mice with EF-1a passively eluted from SDS-PAGE gels loaded with homogenates of Sf21 cells, and fusing the spleen cells to myelomas. Seventy-five hybridoma lines secreting MABs specific for EF-1a were obtained. The identity of the antigen recognized by the MABs was confirmed by internal amino acid sequence analysis, and by the antigen's cross-reactivity with a monospecific rabbit antiserum generated to EF-1a from Dictyostelium discoideum. Four MABs were chosen for further analysis. All four antibodies reacted with EF-1a in eggs and first through fifth instars of the fall armyworm, but did not recognize EF-1a from a human cell line. 2) IDENTIFY THOSE MABS THAT RECOGNIZE EF-1a IN ITS NATIVE CONFORMATION. This goal was met, as evidenced by the ability of the MABs to immunoprecipitate EF-1a from homogenates of Sf21 cells prepared in a non-denaturing buffer. 3) ESTABLISH A CELL-FREE IN VITRO TRANSLATION SYSTEM DERIVED FROM SF21 CELLS. This goal was met, as evidenced by autoradiographic analysis of acid-precipitable proteins that incorporated [35S]-methionine during synthesis by Sf21 cell lysates. 4) IDENTIFY THOSE MABS WITH THE ABILITY TO INHIBIT PROTEIN SYNTHESIS IN AN INSECT-DERIVED CELL-FREE TRANSLATION SYSTEM. This goal was met by adding purified MABS to the Sf21-derived in vitro translation system and measuring changes in protein synthesis by scintillation. Of the four EF-1a-specific MABs tested, three significantly inhibited incorporation of [35S]-methionine into acid-precipitable proteins (and thus protein synthesis) compared to a negative control antibody matched for isotype (P<0.001, one-way ANOVA; followed by Dunnett's test, P<0.05). In future studies, hybridomas that secrete inhibitory MABs will be used as the source of DNA used to create recombinant baculoviruses secreting single-chain antibodies specific for EF-1a. Of the three hybridoma lines secreting inhibitory MABs identified during the current study, two are particularly well suited for the next phase of the project involving production of recombinant viruses. MABs 7D6 and 7G3 demonstrated no cross-reactivity for High Five (BTI-TN-5B1-4) cells derived from the cabbage looper, Trichoplusia ni. Such species specificity is fortuitous because it will allow the High Five cell line to be used to propagate recombinant baculoviruses to control fall armyworm infestations. Since the antibodies do not bind to EF-1a from High Five cells, there should be no toxic effects to the culture system that would prevent the maturation of infectious virions containing the antibody genes.

IMPACT: 1998/09 TO 2001/09
The baculovirus is a safe alternative to chemical insecticides for controlling pest insects because it does not infect fish, birds, or mammals. However, the baculovirus often requires a week or more to kill insects under field conditions. Through recombinant DNA technology, we intend to equip the baculovirus with genes encoding antibodies that can rapidly shut down insect cell metabolism, leading to more rapid insect death or cessation of feeding, thus enhancing the value of the baculovirus as a microbial pesticide. In the current study, we have produced antibodies that are capable of inhibiting protein synthesis in insect cells. The genes encoding these antibodies are good candidates for use in producing the enhanced baculoviruses.

PUBLICATIONS: 1998/09 TO 2001/09
Stuart MK and Chamberlain NR (2001): Monoclonal antibodies to elongation factor-1a inhibit in vitro translation in lysates of Sf21 cells. Submitted to Archives of Insect Biochemistry and Physiology (MS #01-061).


Item No. 13 of 13

ACCESSION NO: 0183283 SUBFILE: CRIS
PROJ NO: MOR-9902652 AGENCY: CSREES MO.R
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 99-35302-8164 PROPOSAL NO: 1999-02652
START: 01 OCT 1999 TERM: 30 SEP 2001 GRANT YR: 1999
GRANT AMT: $100,000

INVESTIGATOR: Bultman, T. L.

PERFORMING INSTITUTION:
DIVISION OF SCIENCE
TRUMAN STATE UNIVERSITY
100 E. NORMAL
KIRKSVILLE, MISSOURI 63501

EFFECT OF NOVEL FUNGAL ENDOPHYTES ON PEST AND BENEFICIAL INSECTS

OBJECTIVES: I propose to address two important gaps in the understanding of how endophytic fungi protect grasses from insect herbivores. First, while substantial evidence for detrimental impacts of endophytes on insect herbivores has accumulated over the past 15 years, considerable variation in the strength of these impacts is apparent. The source of this variation is not well understood. Second, our understanding of endophyte-insect herbivore interactions is hindered by a lack of knowledge about what impact endophytes have on the third trophic level. The proposed research will explore these gaps by addressing the following questions. 1) Do grass endophytes mediate wound-induced resistance to insect herbivores. Using two cultivars of tall fescue, I will manipulate infection by fungal endophyte and simulate herbivory by livestock using clipping. The performance of fall armyworm and bird cherry-oat aphid on experimental plants will be monitored to assess the presence of and magnitude of constitutive and induced resistance. 2) Do endophytes influence the performance of natural enemies of fall armyworm and bird cherry-oat aphid. Using two cultivars of tall fescues, I will manipulate infection by fungal endophyte and allow herbivores (bird cherry-oat aphid) to feed on plants. Aphids will be allowed to be parasitized by Aphelinus asychis. Survival and development of parasitoids will be compared when reared from hosts fed endophyte-infected plants versus uninfected plants. 3) What are the individual and combined effects of fungal and plant genotype on resistance (constitutive and induced) to pest insects and on beneficial insects. I will assess resistance to fall armyworm and bird cherry-out aphids as well as effects on aphid parasitoids using two artificially introduced (novel endophytes) fungal genotypes and wild type endophyte within each of two plant cultivars.

APPROACH: Biomass gain, consumption and survival will be monitored in caterpillars, while reproduction of aphids will be assessed. Nutritional chemicals (protein nitrogen) and potential allelochemicals (ergots, lolines and peramine) in plants will also be assessed. In an additional experiment, potential indirect effects of endophytes on parasites of aphids will be assessed. Survival and growth of parasites will be assessed under host diets that vary with respect to plant and fungal genotype and the presence of fungal endophyte.

NON-TECHNICAL SUMMARY: Fungal endophytes of forage grasses have detrimental effects on livestock. Ergot alkaloids produced by the fungi appear to be the primary cause for these effects. In addition, fungus-produced loline and peramine alkaloids provide infected plants with enhanced resistance to insect herbivores. The fungi, and the toxins they produce, result in annual revenue losses of over $600 million in the US. A current strategy by plant breeders is to develop grasses with fungal strains that reduce the negative effects on livestock but retain resistance to insects. The proposed research utilizes newly developed experimental endophyte strains that were artificially introduced (i.e., novel) into two tall fescue cultivars and tests their effects on pest and beneficial insects. I will test for resistance to caterpillars and aphids in two separate laboratory experiments in which prior plant damage and the presence and genetic strain of fungi will be manipulated. Biomass gain, consumption and survival will be monitored in caterpillars, while reproduction of aphids will be assessed. Nutritional chemicals (protein nitrogen) and potential allelochemicals (ergots, loline and peramine) in plants will also be assessed. In an additional experiment, potential indirect effects of endophytes on parasites of aphids will be assessed. Survival and growth of parasites will be assessed under host diets that vary with respect to plant and fungal genotype and the presence of fungal endophyte. Results will 1) assess effects of experimental combinations of plant and fungal genotypes on pest and beneficial insects.

PROGRESS: 1999/10 TO 2000/09
Experimentation - Over the past 12 months we have conducted five experiments aimed at testing the central thesis of the grant proposal - that novel endophytes of tall fescue influence the performance of herbivores and their enemies. Four of those experiments used herbivores (fall armyworm and bird cherry-oat aphid). Each herbivore has been tested with Jesup and Georgia tall fescue cultivars and fungal genotype has been manipulated within each cultivar. Our results have, for the most part, mirrored results of previous work on similar grass endophyte systems in my laboratory. The most exciting result to date is that we found a strong mediation of wound-induced resistance by endophytes in the Jesup cultivar, particularly plants infected with endophyte strain 542. In an experiment using Georgia tall fescue, Wild and 502 endophyte strains, and E- (uninfected) fescue, aphids showed reduced performance (F2,144=22.5; p (F3,178=19.2; p (2000). Nonetheless, through the efforts of my students, we did make progress. However, yet another disruption in the project will soon occur. I have accepted an academic position at another institution and will be moving at the end of this semester. For this reason I will be unable to complete the entire project within the time frame of the original proposal and will be contacting my program director shortly about a 1 year no-cost extension.

IMPACT: 1999/10 TO 2000/09
The proposed research utilizes new experimental endophyte strains and begins the process of determining their effects on pest and beneficial insects. The information gathered in the proposed work will be crucial to an evaluation of the experimental grass/endophyte varieties for commercial use.

PUBLICATIONS: 1999/10 TO 2000/09
1. Pawlitz, R.J. and T.L. Bultman. 2000. Host selection by a mycophagous fly and its impact on fly survival. Ecography 23:41-49.
2. Bultman, T.L., A.M. Welch, R.Boning, and T.I. Bowdish. 2000. The cost of mutualism in a fly-fungus interaction. Oecologia 124:85-90.
3. Faeth, S.H. and T.L. Bultman. 2001. Endophytes and multitrophic plant-insect interactions. In: T. Tscharntke and B.A. Hawkins (eds.) Multitrophic level interactions. Cambridge University Press.

PROJECT CONTACT:

Name: BULTMAN, T. L.
Phone: 660-785-7252
Fax: 660-785-4045
Email: tbultman@truman.edu