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ARS Home » Midwest Area » Wooster, Ohio » Corn, Soybean and Wheat Quality Research » Research » Research Project #423065


Location: Corn, Soybean and Wheat Quality Research

2016 Annual Report

1. Monitor and identify emerging insect-transmitted pathogens of maize and soybean, and identify management strategies. 2. Determine whether multiple virus resistance in maize inbred lines is the result of pleiotropic or closely linked genes, and develop and release virus-resistant germplasm to breeders. a) Determine whether resistance to potyviruses is pleiotropic in Pa405. b) Mapping multiple virus resistance in Oh1VI. c) Develop and release virus resistant germplasm. 3. Develop genetic and genomic information on two insect vectors, including the molecular response to feeding on virus-infected plants. 4. Identify virus components important for pathogenesis, insect transmission, and host interactions, and develop virus systems for gene discovery and functional analysis in maize. a) Assess viral protein complements, expression strategies, and functions in maize. b) Develop maize virus-based forward and reverse genetics systems. c) Characterize virus and insect factors needed for virus transmission, and develop methods to study these processes.

1. A sequence-independent approach (SIA) for amplification of viral genome sequences will be used for initial identification of viruses in suspected, symptomatic plants. Mollicutes will be identified using PCR with genus-specific ribosomal DNA (rDNA) primers. The identity of known pathogens will be confirmed with a combination of microscopic, serological and molecular assays. New viruses will be cultured in susceptible plants and characterized. As possible under permit conditions, we will test known vectors of maize and soybean diseases for their ability to transmit pathogens. Mechanical or vector transmission of pathogens will be used to screen maize or soybean germplasm for resistant genotypes. 2. To determine whether the Wsm1 and Wsm2 genes for WSMV resistance confer resistance to multiple potyviruses, to isolate or fine map these two genes in Pa405, and to develop germplasm to fine map or isolate Wsm3. The putative insertional mutants Wsm1µ and Wsm2µ plants identified in the current project will be tested for chromosomal deletions on chr. 6 and 3, respectively, prior to testing for pleiotropic gain of susceptibility to potyviruses. We will clone sequences flanking the insertion sites to identify candidate genes. Genes and cDNAs encoding Wsm1 and Wsm2 will be cloned, and sequences will be used in loss and gain of function assays to confirm gene identity. Because of the risk associated with identifying insertional mutations in Wsm1 and Wsm2, we will continue efforts to develop a fine map Wsm1 and Wsm2, using available recombinant plants and populations. Additional markers will be identified in SNP and microarray analyses. We will develop germplasm to identify mutator insertions and fine map Wsm3. 3. Use second-generation sequence analysis to build and analyze EST libraries for two important vectors of soybean and maize viruses: A. glycines and G. nigrifrons. The vectors will be fed on plants infected with viruses that are transmitted in a non-persistent (SMV), semi-persistent (MCDV), persistent-circulative (SbDV) or persistent-replicative (MFSV) manner. EST libraries will be made with RNA from: 1) A. glycines biotypes 1 and 2 fed on healthy, and SMV or SbDV-infected soybean, and 2) G. nigrifrons fed on healthy, and MCDV- and MFSV-infected maize. Libraries will be sequenced, assembled and annotated. Differential EST expression between different treatments will be verified with quantitative real-time RT-PCR (RT-qPCR), and sequences from A. glycines and G. nigrifrons will be compared with those of other vector genomes. 4. An in vivo protease assay will be used to determine MCDV polyprotein cleavage sites by co-expressing active viral protease with epitope-tagged MCDV polyprotein regions and determining sizes of cleavage products. Antibodies made against predicted small ORF-encoded proteins will be used to test for protein expression in infected plants. MCDV proteins will be tested for subcellular localization and virus protein-protein interactions, and MCDV and MFSV proteins will be tested for their ability to suppress gene silencing in N. benthamiana.

Progress Report
In FY16, we made progress on all objectives. Objective 3 was completed in FY14 and related publications in FY15. Work on Objective 1 included a large effort towards the critical need of characterizing the epidemiology of the devastating Maize lethal necrosis (MLN) outbreak emerging in East Africa, to improve our understanding of the disease, the viruses causing the disease and development of methods to detect them. Another large effort has been focused on disease management efforts, particularly in screening maize germplasm to identify lines that are resistant to one or more of the viruses involved. We completed surveys of Rwanda and Tanzania for MLN-causing viruses and have begun deep sequencing and bioinformatics to characterize virus populations in maize in these samples. We have confirmed genome sequences for one of the previously undescribed viruses identified in samples from Kenya and Uganda, assessed the role of one virus in disease, and determined the incidence for another. Wheat samples were collected across Ohio for assessing virus populations. Field and greenhouse experiments demonstrated differences in cultivar susceptibility and disease potential impact estimates for each of five viruses infecting wheat. For Objective 2, testing of agronomic and resistance responses of selected RIL from the Oh1VI population was completed, as was a study of the responses of U.S. grain and sweet corn hybrids to inoculation with potyviruses. In addition, we selected lines from the Oh1VI population carrying loci associated with potyvirus resistance and tested these for their responses to MLN under artificial and natural inoculation. A project to map MCMV tolerance/resistance in maize was initiated. Results indicate that loci associated with resistance are found on different chromosomes in different maize lines. We have identified sources of host plant resistance to Halyomorpha halys, the brown marmorated stink bug (BMSB) and initiated construction of mapping populations to elucidate QTLs linked to resistance. For Objective 4, for which timelines have been delayed due to reallocation of personnel to Objective 1, we have made progress by identifying a silencing suppressor protein in Maize chlorotic dwarf virus, the first such protein identified in the genus Waikavirus, and mapping the protease cleavage site of the polyprotein precursor of the silencing suppressor. Results have been reported at the American Society for Virology annual meeting, and have been submitted for publication. We have published research on development of a Soilborne wheat mosaic virus infectious clone and adaptation for gene-carrying that is functional for virus-induced gene silencing in the dicot host Nicotiana benthamiana.

1. Characterizing newly identified disease-causing viruses in corn. Next generation sequencing (NGS) is an effective method to rapidly identify emerging viruses and assess existing virus populations, even for previously unknown viruses. ARS researchers at Wooster, Ohio and collaborators surveyed and used NGS to characterize genome sequences for maize and wheat viruses in the United States and East Africa, to develop an in-house plant virus identification pipeline. Partial and complete genome sequence data for maize viruses were obtained, including African isolates of maize chlorotic mottle virus and sugarcane mosaic virus and Ohio isolates maize chlorotic dwarf virus (MCDV) and maize dwarf mosaic virus (MDMV). Sequences for at least three previously undiscovered corn-infecting viruses were also discovered, and have been confirmed in samples. The great diversity found among corn-infecting potyviruses confounds our ability to detect these viruses with conventional diagnostic techniques. Identification of all viruses present in the landscape and understanding the variability in virus population genome sequences is critical to define disease epidemiology, ensure detection of all important virus variants, and limit disease spread.

2. Maize chlorotic dwarf virus encodes a novel silencing suppressor protein that is cleaved by the virus protease from a polyprotein precursor. RNAi (ribonucleac acid interference) or RNA silencing is an important plant defense mechanism that targets and destroys invading viruses in a sequence-dependent manner. In the pathogen-host molecular ‘arms race’, many plant viruses combat this plant defense mechanism by encoding virus suppressors of silencing. ARS scientists at Wooster, Ohio identified a Maize chlorotic dwarf virus (MCDV) protein with silencing suppressor activity. Further, they discovered how this protein is processed from its precursor by a virus-encoded protease. Silencing suppressors have not been previously identified for MCDV or any related virus. This discovery is a significant step in our understanding of gene function and pathogenicity of this major U.S. corn virus.

3. Distribution of Maize Lethal Necrosis (MLN) in Rwanda. ARS researchers from Wooster, Ohio in a collaboration with international partners surveyed maize growing regions of Rwanda for the presence of MLN-causing viruses. Maize chlorotic mottle virus (MCMV), sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) were detected in about 50%, 30% and 50%, respectively, of samples tested using ELISA. Incidence of MCMV and SCMV was somewhat higher in the western vs. the eastern half of Rwanda, and MDMV was not detected in the Eastern or Kigali Provinces. The results indicate the widespread occurrence of the viruses across growing seasons and years. The understanding of the distribution of MLN-causing viruses is critical to defining disease epidemiology, predicting disease outbreaks, anticipating virus spread and for developing and deploying disease control measures.

4. Characterizing resistance and tolerance to maize chlorotic mottle virus in maize. Since 2011, maize lethal necrosis (MLN), caused by infection of maize with two viruses named maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) has caused up to 100% losses for smallholder farmers in sub-Saharan East Africa. ARS researchers in Wooster, Ohio identified maize lines that develop no or few symptoms when inoculated with MCMV, and used these lines to identify genes and quantitative trait loci associated with virus tolerance and resistance. MCMV tolerance/resistance in these lines is quantitative, but has dominant character in some lines and recessive character in others. These lines may form the basis for developing maize hybrids with tolerance or resistance to MLN, providing an affordable and environmentally sound management for this devastating disease.

5. Identifying resistant brown marmorated stink bugs in soybeans. The brown marmorated stink bug (BMSB), Halymorpha halys, is an invasive, polyphagous insect introduced from Asia. As of 2015, BMSB has been detected in 42 states and has established itself as a severe agricultural pest of crops, vegetables and fruits in nine states extending from New York to North Carolina and west into the soybean belt. ARS researchers in Wooster, Ohio have screened over 250 lines from USDA Soybean germplasm collection. Our results indicate that resistance is quantitative and may involve pod wall thickness and seed coat hardness as components to inhibit BMSB stylet penetration into the bean. Resistant germplasm coupled with our new understanding of resistance components will allow for more efficient phenotyping and elucidation of Quantitative Trait Locis. Deploying BMSB resistant varieties will increase the economic value of soybeans through reducing yield loss and dependence on pesticide application and reduce BMSB pressure on other crop systems.

Ongoing research agreement addresses understudied wheat viruses and their impact in Ohio. Activities include semiannual or quarterly meetings with the Ohio Corn & Wheat growers association and the Ohio Small Grains Marketing Program. An ARS scientist at Wooster, OH was invited to be a panel member for a discussion on careers with government and private industry for Ohio State University’s Summer Research Opportunities Program, which is targeted at talented undergraduate students in historically underserved populations.

Review Publications
Todd, J.C., Mian, R.M., Backus, E.A., Finer, J.J., Redinbaugh, M.G. 2015. Feeding behavior of soybean aphid (Hemiptera: Aphididae) biotype 2 on soybean PI 243540, the source of Rag2 resistance. Journal of Economic Entomology. 109:426-433.
Bansal, R., Mittapelly, P., Cassone, B.J., Mamidala, P., Redinbaugh, M.G., Michel, A. 2015. Recommended reference genes for quantitative PCR analysis in soybean have variable stabilities during diverse biotic stresses. PLoS One. doi:10.1371/journal.pone.0134890.
Edwards, M.C., Weiland, J.J., Todd, J., Stewart, L.R., Lu, S. 2016. ORF43 of maize rayado fino virus is dispensable for systemic infection of maize and transmission by leafhoppers. Virus Genes. 52:303-307.
Jarugula, S., Charlesworth, S., Qu, F., Stewart, L.R. 2016. Soil-borne wheat mosaic virus infectious clone and manipulation for gene-carrying capacity. Archives of Virology. 161:2291-2297.
Stewart, L.R. 2015. Sequence diversity of wheat mosaic virus isolates. Virus Research. 213:299-303.
Redinbaugh, M.G. 2016. Mollicutes. In: Munkvold, G., White, D.G., editors. Compendium of Corn Diseases, 4th Edition. Minneapolis, MN:APS Press. p. 16-19.
Stewart, L.R., Louie Jr., R., Redinbaugh, M.G. 2016. Diseases caused by viruses. In: Munkvold, G., White, D.G., editors. Compendium of Corn Diseases. 4th Edition. Minneapolis, MN:APS Press. p. 100-117.