Location: Corn, Soybean and Wheat Quality Research
2018 Annual Report
Objectives
1. Monitor and identify emerging insect-transmitted pathogens of maize and soybean using standard and bioinformatics-based approaches, and develop management strategies. Sub-objective 1.A: Identification and diagnosis of viruses and virus populations in maize. Sub-objective 1.B: Develop tools to characterize emerging maize-infecting viruses. Sub-objective 1.C: Characterize role of E. coryli in damage caused by BMSB.
2. Identify virus factors important for pathogenesis, transmission and host interactions, and develop virus systems for gene discovery and functional analysis in maize and other cereals. Sub-objective 2A: Characterize Maize chlorotic dwarf virus factors important for pathogenesis and interactions with plant hosts. Sub-objective 2B: Develop systems for working with full-length infectious cDNAs of maize viruses. Sub-objective 2C: Define insect vector interactions with plant host and viral pathogens.
3. Identify and characterize mechanisms of action of genetic loci for virus resistance in maize. Sub-objective 3A: Identify and characterize loci providing tolerance/resistance to MCMV in maize and sorghum. Sub-objective 3B: Characterize interactions among potyviruses, MCMV and virus resistance/tolerance in maize. Sub-objective 3C: Characterize and map novel soybean quantitative trait loci (QTL) for host plant resistance to brown marmorated stinkbug (BMSB).
4. Characterize pathogen vectoring relationships of and between emerging insect pests and vectors of maize pathogens using comparative genetic and genomic analyses to identify factors that can be disrupted for disease control.
Approach
Developing control strategies for insect-transmitted diseases requires knowledge about the pathogen, crop host, disease vector, and interactions among them and with the environment. Under Objective 1 we will combine standard serological and molecular approaches for diagnostics with next generation sequencing (NGS) approaches to identify and define population structures for emerging insect-vectored pathogens of maize and soybean. We will use this information to develop targeted molecular and serological diagnostics for emerging diseases, identify virus vectors and identify other factors important for disease development and spread. The identity and populations of yeast of yeast transmitted to soybean by brown marmorated stink bug (BMSB) will be defined using NGS, and traditional plant pathological approaches will be used to determine its role in damage caused by the stink bug. Under Objective 2, molecular biological and biochemical approaches will be used to virual protein structure and function for Maize chlorotic dwarf virus. Molecular biological approaches will be used to develop and improve infectious cloned cDNAs for maize infecting viruses. For Objective 3, methods we previously developed for phenotypic analysis of plant responses to Maize chlorotic mottle virus and BMSB will be used to map resistance in biparental and association mapping populations using molecular and NGS approaches for genotyping. Interactions between known maize potyvirus resistance genes and potyvirus isolates will be assessed in near isogenic lines carrying defined resistance genes and alleles using the development of symptoms and virus titer in inoculated plants. NGS genomic and transcriptomic analyses of leafhoppers feeding on healthy and virus-infected plants under different environmental conditions will be used to develop comparisons of leafhopper species.
Progress Report
The unit has made significant progress on all four major objectives of the project in its first year:
Objective 1, to discover and characterize newly emerging viruses, is ongoing as viruses move, emerge, and are discovered in the U.S. and globally. In the first project year, analyzed samples from Rwanda where maize lethal necrosis (MLN), a major global maize virus disease, is emergent, and collected samples and preliminary data from in-depth surveys of Tanzania, where MLN is also emerging. Conducted surveys of Ohio maize and set up and have a project in progress to survey maize across the U.S. in a variety of regions represented by at least 8 states in this growing season. Set up collaborations with growers and colleagues in Washington, New Mexico, Louisiana, Pennsylvania, Tennessee, Kentucky, Iowa, and Indiana and have already processed and tested survey samples from half of these states (LA, TN, KY, IL, WA) and anticipate completing the collection by the end of the growing season this year. The completed surveys resulted in the discovery of two wheat viruses in Ohio, a new cytorhabdovirus in Peru, and two viruses/strains in MLN-selected samples in East Africa. Developed diagnostic methods and protocols for each of these newly described viruses. These outcomes represent significant progress in the goals to rapidly identify and characterize new pathogens (1.A.1), survey U.S. maize for viruses (1.A.2). Objective 1C: Made significant progress understanding the role of Eremothecium coryli in the damage caused by stink bug pests. Sequences from Halyomorpha halys, Acrosternum hilare, and Euschistus servus were aligned to a core taxonomic repository to identify bacteria, fungi, and plants on and within the three stink bug species. During the analysis sequences from Eremothecium coryli; the causal agent of soybean yeast spot disease were present in all three stink bug hosts but not consistently found across samples. E. coryli hybrid sequences were also identified and validated in additional samples.
In the past year, made significant gains in basic virology research, encompassed by Objective 2, and generated maize dwarf mosaic virus Ohio isolate (MDMV-OH) infectious clones that express a marker gene for green fluorescent protein (GFP). In addition, generated an infectious clone of a resistance-breaking Italian isolate (MDMV-It) and two other variant Ohio MDMV sequences. These infectious clones exceed the planned milestones and provide foundational tools to begin to understand the molecular basis of virulence and resistance breaking in this virus, as well as to track virus movement via a marker gene. As the plant genes underlying resistance and the virus genes or sequence elements resulting in resistance or susceptibility are defined, a complete understanding of interactions that lead to each outcome will be developed. MDMV and related potyviruses are the most ubiquitous viruses of maize in the United States and globally, so this research is of great interest. Also made significant progress researching another U.S. maize virus, maize chlorotic dwarf virus, by developing and testing planned constructs testing the conservation of proteolysis of the N-terminus of the polyprotein across 3 maize chlorotic dwarf virus (MCDV) strains and 2 additional viruses in the genus Waikaivirus. Characterized the virus-encoded protease by creating and testing a series of mutants, identifying residues essential for proteolysis activity. Finally, to better understand how viruses impact the behavior of their insect vectors, conducted available correlation studies with electropenetrography to identify feeding patterns for 2 leafhopper species that transmit maize viruses, Graminella nigrifrons and Dalbulus maidis, providing a baseline for understanding how virus infection of host plant effects insect behavior. The following virus x insect interactions are partially finished, with annotated files prepared for analysis: G. nigrifrons +/- MCDV, Maize fine streak virus, maize rayado fino virus (MRFV).
For Objective 3, to identify and characterize virus resistance loci, an entire maize association mapping population (AMP) was phenotyped for response to potyvirus infection. This work provides a basis for identifying new sources of resistance genes, and potentially identifying new loci for resistance. Resistance QTL were also identified for maize chlorotic mottle virus (MCMV), a major emergent pathogen of maize, and causal agent of the devastating maize lethal necrosis (MLN) epiphytotic in co—infections with potyviruses. These QTL results were published in the past year. In addition to maize phenotyping, a large effort has been focused on identifying QTLs in soybeans for resistance to H. halys; brown marmorated stink bug (BMSB) and identifying new sources of host plant resistance (Objective 3C). Replicated resistance screening in a F5 mapping population and genotyped 184 progenies with 6,000 SNP markers for QTL analysis. To further elucidate this mechanism, initiated a genome wide association study for seed coat hard and begun screening maturity group II, III, and IV plant introductions with exceptional seed coat hardness.
Objective 4 research to characterize vector relationships using genomic analyses made significant progress, as experiments comparing the responses of two leafhoppers that transmit maize rayado fino virus (MRFV), D. maidis and G. nigrifrons, were exposed to uninfected or infected plants under two different temperature regimes (25C and 30C). Four replicates of this experiment were conducted, samples collected, and samples were sequenced to develop a draft transcriptome for D. maidis. Continued analyses will compare transcriptional responses of each leafhopper to temperature and virus exposure.
In the first year of this research project, successfully accomplished the planned milestones, exceeding some, with some adjustments such as changing to a better cross for BMSB resistance mapping in soybean (Sub-objective 3C) and slight lag in qPCR assay development (Sub-objective 1C).
Accomplishments
1. Virus discovery in maize and wheat. In grass crops including corn and wheat, as well as in other crops, viruses impact yield but are often undescribed or undiscovered, in part because they may cause symptoms such as yellowing or stunting that are misattributed to other causes, or because simple diagnostic tests are not available for these undescribed viruses. Using deep sequencing technologies to identify a wide variety of putative viruses not detected by traditional diagnostic methods, ARS researchers at Wooster, Ohio identified previously undescribed viruses and virus strains in maize and wheat. Two viruses identified in Ohio wheat in collaboration with Ohio State University researchers were reported for the first time (Cocksfoot mottle virus and Agropyron mosaic virus). A unique maize-associated cytorhabdovirus was sequenced and identified from samples from Peru, and a polerovirus and strain of Johnsongrass mosaic virus were identified and characterized from East Africa. New sequence-based diagnostics were developed and reported for each of these viruses, which are now available to researchers and diagnosticians. Researchers and diagnosticians now utilize these data and tools, including sequences deposited in NCBI, to identify viruses contributing to disease.
2. Identification of essential residues for virus protease. Viruses utilize a variety of mechanisms to express multiple proteins from a limited genome, including proteolytic cleavage of virus-encoded polyproteins. Maize chlorotic dwarf virus (MCDV) is a maize infecting virus in the U.S. that encodes one large polyprotein that is cleaved into mature individual proteins by a virus-encoded 3C-like protease related to proteases found in a wide range of plant and animal-infecting viruses in the virus families Picornaviridae, Caliciviridae, and Coronoviridae. However, the substrate specificity and catalytic residues of the MCDV protease are not well characterized. To identify essential residues for MCDV protease activity, ARS researchers at Wooster, Ohio created and tested a series of proteases mutagenized at specific residues including putative catalytic sites and identified two residue sites that are required for MCDV activity. This discovery is an important step to understanding the proteolysis of MCDV and related plant viruses, and ultimately understanding the complement of proteins expressed by the virus and their roles in plant pathogenicity.
3. Survey of microbial communities associated with stink bugs. Several stink bug species pose a serious threat to numerous crop, vegetable, and fruit commodities produced in the U.S. Stink bug species can also transmit pathogens that may cause further crop damage. ARS researchers in Wooster, Ohio used next generation sequencing to survey the microbiome of three stink bug species. Each species had distinct microbial communities, but Eremothecium coryli; a fungal pathogen causing soybean yeast spot disease, was intermittently present in all three stink bug hosts. E. coryli like hybrid sequences were observed and validated in all three species. Understanding the microbiomes of stink bug species may help to develop new methods of biocontrol to reduce the threat of stink bug on U.S. agricultural commodities.
Review Publications
Hodge, B.A., Paul, P.A., Stewart, L.R. 2017. First report of Cocksfoot mottle virus infecting wheat (Triticum aestivum) in Ohio. Plant Disease. 102(2):464.
Hodge, B.A., Paul, P.A., Stewart, L.R. 2017. Agropyron mosaic virus detected in Ohio wheat (Triticum aestivum). Plant Disease. 102(2):463.
Massawe, D., Stewart, L.R., Kamatenesi, J., Asiimwe, T., Redinbaugh, M.G. 2018. Complete sequence and diversity of a maize-associated Polerovirus in East Africa. Virus Genes. 54(3):432-437. https://doi.org/10.1007/s11262-018-1560-5.
Jones, M.W., Penning, B., Jamann, T.M., Glaubitz, J.C., Romay, C., Buckler IV, E.S., Redinbaugh, M.G. 2017. Diverse chromosomal locations of quantitative trait loci for tolerance to maize chlorotic mottle in five maize populations. Phytopathology. 108(6):748-758. https://doi.org/10.1094/PHYTO-09-17-0321-R.
Willie, K.J., Stewart, L.R. 2017. Complete genome sequence of a new maize-associated cytorhabdovirus. Genome Announcements. https://doi.org/10.1128/genomeA.00591-17.
Stewart, L.R., Willie, K.J., Wijeratne, S., Redinbaugh, M.G., Massawe, D., Niblett, C.N., Asiimwe, T. 2017. Johnsongrass mosaic virus contributes to maize lethal necrosis in East Africa. Plant Disease. 101(8):1455-1462. https://doi.org/10.1094/PDIS-01-17-0136-RE.
La Mantia, J.M., Mian, R.M., Redinbaugh, M.G. 2018. Identification of soybean host plant resistance to brown marmorated stink bugs in maturity group III plant introductions. Journal of Economic Entomology. 111(1):428-434. https://doi: 10.1093/jee/tox295.
Jarugula, S., Willie, K.J., Stewart, L.R. 2018. Barley stripe mosaic virus (BSMV) as a virus-induced gene silencing vector in maize seedlings. Virus Genes. 54: 616-620. doi.org/10.1007/s11262-018-1569-9.
