2008 Annual Report 1a.Objectives (from AD-416) Objective 1: Develop corn and soybean virus disease control strategies. • Sub-objective 1.A. Characterize the nature of host resistance to virus disease. • Sub-objective 1.B. Identify, map, and clone virus-resistance genes. • Sub-objective 1.C. Determine the effect of combining quantitative virus resistance with insect resistance on virus disease severity. • Sub-objective 1.D. Characterize insect vector/virus relationships. Objective 2: Identify and characterize emerging virus diseases in corn and soybeans. Objective 3: Develop virus-based gene vectors in corn. Soybeans and corn are the two highest value crops grown in the U.S. Although research from this laboratory and others has led to significant improvements in their management, virus diseases continue to be annual threats. Furthermore, history has shown that unanticipated, and often unknown, new virus disease problems can rapidly emerge at any time. In soybeans, the threat of virus diseases has increased even more following the introduction of the soybean aphid into the U.S. in 1999. This is the first soybean-colonizing aphid in the U.S., and the consequences for future virus disease problems in soybeans are unknown. A long-term objective of this program is to reduce corn and soybean losses attributable to virus diseases. Our strategy to do so is to identify corn and soybean viruses when they arise and to characterize their biology and epidemiology (Objective 2), and to then use this information develop practical, effective methods and strategies for minimizing crop losses (Objective 1). Our final objective (Objective. 3)is to use our knowledge and expertise in maize virology to develop new tools for forward and reverse genetic analysis of maize gene function. We will develop gene silencing and expression vectors based on selected maize viruses, and use these vectors immediately to characterize the functions of candidate maize genes thought to be important for virus infection and/or resistance. Because reliable vectors are lacking for monocotyledenous plants such as corn, vectors developed under this objective would be of benefit both to corn geneticists and to those seeking to use corn for the production of non-endogenous materials. 1b.Approach (from AD-416) Our overall approach is to:. 1)identify existing and emerging viruses;. 2)understand their biology; and. 3)develop effective disease control strategies. Known viruses will be identified using existing diagnostics. For previously uncharacterized viruses, we will culture them in healthy plants using mechanical or arthropod transmission, determine their characteristics, and develop diagnostic assays. This knowledge will be used to formulate disease control strategies, although, usually, the most effective and economic control strategy is to use virus-resistant crop varieties and cultivars. Therefore, a primary focus of this project is to identify, characterize, and map virus resistance in maize and soybean germplasm. To develop an understanding of how resistance genes work, they will be isolated and characterized, and their role in the molecular and biochemical changes associated with virus resistance will be examined. Also, factors affecting virus transmission by arthropod vectors will be characterized so that alternative disease control methods can be developed. Maize virus-based gene expression and silencing vectors will be developed to facilitate functional analysis of plant resistance genes using forward and reverse genetics. Such vectors should have broad impact as few are available for cereals. The impact of this research will be to advance our knowledge of virus diseases of corn and soybeans and provide vital information for the development of control strategies to reduce disease losses. 3.Progress Report The majority of our research effort was on the objective to develop corn and soybean virus disease control strategies. Three projects under this objective are nearing completion and publication. We showed that, although the BPMV incidence was lower in semi-dwarf insect-resistant soybeans than in standard insect-susceptible cultivars, virus incidence was also lower in a semi-dwarf, insect-susceptible cultivar. We demonstrated that contributions from ‘modifier genes’ derived from the resistant parent Pa405 are likely to be important for complete resistance of maize to Maize dwarf mosaic virus and Sugarcane mosaic virus, since the introgression of chromosomal regions carrying Mdm1/Scmv1 and Scmv2 into a susceptible background did not by themselves provide complete virus resistance. We showed that Graminella nigrifrons is an inefficient vector of Maize fine streak virus (MFSV). Further, we demonstrated the presence of two populations of MFSV-infected insects: one with high virus titer that includes most of the vectors and one with low virus titer that does not transmit the virus. In ongoing projects under this objective: we are in the third year of trials to evaluate the effects of BPMV on Ohio, North Dakota and Wisconsin soybean cultivars with partial BPMV resistance; results from microarray analysis of the response of resistant and susceptible maize to virus inoculation indicated a small number of genes were differentially regulated by virus four days post inoculation and suggested that a slight modification of our experimental approach was warranted; molecular markers suitable for high throughput identification of cross-over events in the chromosomal regions near two WSMV resistance genes were developed and analysis of several thousand progeny for these cross-overs has begun. Under the objective to identify and characterize emerging virus diseases in corn and soybeans, we have been working with collaborators in Serbia and Ecuador to identify and characterize emerging vector-dependent diseases in maize. With Serbian collaborators we are preparing a manuscript describing the acquistion of stolbur phytoplasma from maize roots by early stage larvae of the vector Reptalus panzeri. The larvae overwinter on wheat roots and emerge as infected adults from wheat fields in late June. Wheat and Johnson grass were shown to be hosts of the stolbur phytoplasma. Preliminary experiments were carried out with an Ecuadoran collaborator to identify viruses and mollicutes assocated with an emerging disease called “cinta roja” (red stripe). For our objective to develop virus-based gene vectors in corn, we are working to make a gene-silencing vector using Maize fine streak virus (MNeSV) and gene expression vectors using Maize fine streak virus (MFSV) and Maize mosaic virus. We showed that all four MNeSV-encoded proteins are required for systemic movement of the virus, and have engineered cloning sites into the virus while maintaining infectivity. We showed that the MFSV nucleoprotein and phosphoprotein can be expressed in Drosophila S2 cells using commercially available plasmids. 4.Accomplishments 1. Bean pod mottle virus (BPMV) movement in insect resistant soybeans. Recently, increased disease and losses caused by BPMV in soybean in the North Central U.S. have been associated with higher populations of its bean leaf beetle vector. No complete genetic resistance to BPMV has been identified in soybean germplasm. We tested whether insect resistance in soybeans could limit BPMV spread in soybeans. Although the BPMV incidence was lower in semi-dwarf insect resistant soybeans than in standard insect susceptible plants, virus incidence was also lower in a semi-dwarf, susceptible cultivar. These data indicate that insect resistance may not be useful for control of BPMV, and that efforts to identify and characterize quantitative resistance to BPMV infection in soybeans are warranted. This project addresses components of the NP-303 Plant Diseases Action Plan including: Biology, ecology, epidemiology, and spread of plant pathogens and their relationships with hosts and vectors; Plant disease resistance; and Biological and cultural strategies for sustainable disease management”. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Active CRADAs 1 Number of New Commercial Licenses Executed 2 Review Publications Hogenhout, S.A., Ammar, E.D., Whitfield, A.E., Redinbaugh, M.G. 2008. Insect vector interactions with persistently transmitted plant viruses. Annual Review of Phytopathology. 46:327-359. De Souza, I., Schuelter, A.R., Guimaries, C., Schuster, I., De Oliveira, E., Redinbaugh, M.G. 2008. Mapping QTL contributing to SCMV resistance in tropical maize. Hereditas. 145(4)167-173.