Progress 05/22/17 to 05/09/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This project has reached its termination date and has been replaced by project 5082-22000-002-00D, ⿿Detection and Characterization of Genetic Resistance to Corn and Soybean Viruses. Objective 1, to discover and characterize emerging viruses. Samples were tested for viruses from the U.S., S. America, and Africa. Two wheat viruses in Ohio, a cytorhabdovirus in Peru, and two viruses/strains in E. Africa were discovered and diagnostic tests were developed. Brome mosaic virus, a maize and wheat virus, was found to reduce wheat yields by up to 60%. An emerging polerovirus, maize yellow mosaic virus, is of growing interest given its close relationship to viruses in the U.S. and extensive presence in E. Africa, Asia, and S. America. Diagnostic assays were developed, and tolerant maize lines were identified. The polerovirus was found to be transmitted by at least two aphid species: R. maidis and R. padi. Synergism among maize yellow mosaic virus, maize chlorotic mottle virus (MCMV), and sugarcane mosaic virus (SCMV) was found to amplify disease in co-infected plants. Virus surveys were conducted in nine states and maize dwarf mosaic virus, maize chlorotic dwarf virus, foxtail mosaic virus, and high plains wheat mosaic virus were detected by next generation sequencing or immunoassay. At least one virus was detected in all states. These data provide insight concerning the distribution and importance of maize viruses within the U.S. Maize lethal necrosis (MLN) infected samples from E. Africa were collected and diagnostics were completed. Assays for MCMV and SCMV were developed. Deep sequencing detected known, emerging, and/or novel viruses including MCMV, SCMV, maize yellow mosaic virus, and barley yellow dwarf virus. SCMV isolates were obtained from Rwanda and isolated by passaging through sorghum. Antisera from Agdia, Bioreba and NanoDiagnostics and generic potyvirus strips had sufficient sensitivity and specificity to detect all SCMV isolates, indicating existing serological diagnostic tests are suitable for use in E. Africa. The diagnostic tools and improved understanding of the epidemiology and etiology of MLN are valuable for developing control and breeding strategies. Microbiome sequences from three stinkbug species were aligned to core taxonomic repositories to identify bacteria, fungi, and plants present within the gut of each species. Eremothecium coryli, the causal agent of soybean yeast spot disease, was present in all three species. E. coryli inoculations of seed from different soybean lines were conducted to evaluate variation in seed infection between lines. However, results were inconclusive due to variable seed coat permeabilities. Objective 2, identify virus factors important for pathogenesis and transmission and develop virus systems for gene discovery and functional analysis. Maize chlorotic dwarf virus constructs were generated, and polyprotein cleavage, pathogenesis, and vector interactions were characterized. The first infectious clone of this manipulation- recalcitrant virus was generated and launched in maize, the first infectious clone for any waikavirus. Virus constructs were created to test the conservation of protein cleavage sites across MCDV strains and other waikavirus species. Residues essential for proteolysis activity were identified by testing this series of mutants. Infectious potyvirus clones of differential virulence were generated to test resistance breaking. Potyviruses are ubiquitous pathogens of maize, instigating up to 30% yield loss individually and are involved in synergistic viral diseases including MLN. A maize dwarf mosaic virus clone was engineered for multi-gene virus induced gene silencing. Infectious maize yellow mosaic virus and maize rayado fino virus clones were developed. The latter clone is a valuable tool for gene silencing. Significant progress was made towards a full-length infectious clone for the challenging rhabdovirus, maize fine streak. Minireplicons with evidence of replication in a model host were generated. These tools will help elucidate the molecular basis of virulence and resistance breaking, track virus movement by green fluorescent protein, and conduct gene silencing for functional genomics research. Electropenetrography was conducted on leafhoppers: Graminella nigrifrons and Dalbulus maidis, to evaluate how viruses affect insect feeding behavior on maize chlorotic dwarf virus, maize rayado fino virus, or maize fine streak virus infected maize. This technique detects insect feeding patterns by measuring waveforms generated by creating a circuit between the insect and host. A conductive wire is attached to the insect and low levels of electrical current are run through the host plant, thus creating a circuit when feeding occurs. Despite major plant physiological changes induced by these viruses, only subtle differences in feeding behavior were found. All three viruses require prolonged phloem feeding for transmission, thus they may not have evolved to change vector feeding behavior. Electropenetrography was also used to characterize soybean aphid feeding patterns on soybean mosaic virus and bean pod mottle virus infected plants. Few effects of soybean mosaic virus on the aphid were found, despite it being a soybean mosaic virus vector. However, the beetle-transmitted bean pod mottle virus caused aphid feeding difficulties. The results explain previous findings of reduced aphid fitness on bean pod mottle virus-infected plants. These studies improve our understanding of virus-vector-host interactions. Objective 3, identify and characterize virus resistance mechanisms in maize. A genome wide association study for MCMV resistance in the maize 282 diversity panel was conducted, revealing the presence of seven MCMV resistance loci. A recombinant inbred line population derived from one of the most MCMV resistant lines in the 282 panel was used to map a major MCMV resistance quantitative trait locus on chromosome 10. Recombinant families derived from MCMV resistant maize lines were genotyped and evaluated for resistance. The lines were backcrossed multiple times to the susceptible parent to fine map MCMV tolerance loci present in the MLN resistant maize line N211. A MCMV resistance gene was identified in the MLN resistant maize line KS23-6 in collaboration with Corteva/Pioneer Hi- Bred as part of a CRADA. These results facilitate identification of new MCMV resistance genes. A sorghum association mapping population was also evaluated for MCMV resistance. However, results were inconsistent between replicates suggesting resistance breakdown in sorghum may be highly influenced by environmental variance. A maize synthetic population, OhMCMV-1, derived from five MCMV resistant parents was developed and released. This population contains lines with better resistance to MCMV, potyviruses, and MLN than any of the individual parents. MCMV resistance loci derived from N211, KS23-5, and KS23-6 were introgressed into 15 elite inbreds for use in E. African breeding programs. These same three lines were also converted to white endosperm color to broaden the diversity of MCMV resistant lines and appeal to varied consumer preferences. The unit supported ARS⿿s Germplasm Enhancement of Maize project, by evaluating 233 lines for resistance to three maize viruses. This project is a cooperative effort to broaden the genetic base of maize in the U.S. Seventeen SCMV, 23 MCMV, and 34 maize dwarf mosaic virus tolerant lines were identified. These efforts support rapid development and release of virus tolerant corn cultivars worldwide. Near isogenic lines with potyvirus resistance loci introgressions from three resistance inbred lines were developed. The SCMV isolates from E. Africa described previously were tested on these near isogenic lines and donor lines to assess virulence. African isolates were more virulent than those from Ohio and Germany and one overcame resistance even among the most potyvirus resistant control lines. Greater virulence among E. African potyvirus strains may contribute to the MLN epidemic in the region. Viral titers of MCMV and SCMV were measured in all combinations in resistant and susceptible lines. Resistant lines were found to have as much as 100,000-fold reduced MCMV titer compared to susceptible controls. Both potyvirus and MCMV resistance are important for reducing MLN virus titer and severity. A soybean mapping population was assessed for resistance to brown marmorated stinkbug and genotyped. However, resistance loci were not consistently detected in replicated experiments, revealing the complexity of this trait. Researchers believed stinkbug resistance is associated with seed coat hardness. A genome wide association study detected several seed coat hardness genomic locus that co-localized with genes and loci reported elsewhere. However, seed coat hardness is problematic for soybean and thus selecting for this trait is undesirable. Objective 4, characterize relationships between maize rayado fino virus and two leafhopper vectors D. maidis and G. nigrifrons, was completed. Transcriptomic analysis was used to assess the effects of virus infection on vector gene expression. The leafhoppers expressed a full repertoire of immunity genes and several of these were responsive to virus infection, though the response of each species was unique. This is the first transcriptome for the corn leafhopper and provides a basis to identify genes limiting or promoting virus infection and transmission, serving as targets for future advanced management strategies like RNAi. ACCOMPLISHMENTS 01 Maize germplasm with elite resistance to maize lethal necrosis disease. Maize lethal necrosis (MLN) is a synergistic virus disease of maize that was first detected in the United States in the 1970s. The disease has since spread globally, recently causing devastating yield losses in East Africa, Southeast Asia, and South America. ARS researchers at Wooster, Ohio developed a maize population with elite resistance to MLN and its causal viruses, maize chlorotic mottle virus (MCMV) and potyviruses. The population, named OhMCMV-1, was made from five parental lines with elite MLN resistance. Breeding populations were developed, from which the most virus resistant lines were selected. These lines were used to develop the OhMCMV-1 population. Lines in this population are significantly more resistant to MLN, MCMV, and potyviruses than the parental lines. The strong virus resistance in this population will be used by breeders to develop new, elite breeding lines, hybrids, and cultivars to mitigate the impact of MLN worldwide. OhMCMV-1 was publicly released and deposited in the National Plant Germplasm System, where it is available for research and development. 02 Novel maize chlorotic mottle virus resistance loci detected in a diverse maize population. Maize chlorotic mottle virus (MCMV) is the most important virus causing maize lethal necrosis (MLN), a devastating virus disease of corn. ARS researchers at Wooster, Ohio detected the presence of new genomic loci that are significantly associated with MCMV resistance. The maize Goodman 282 diversity panel, a population of lines representing the global diversity of corn, was evaluated for resistance to MCMV. Several lines with strong MCMV resistance were identified. Using more than 10 million genetic markers, seven genomic loci were found to be associated with MCMV resistance in this population. Subsequently, a major MCMV resistance locus on chromosome 10 was identified in one of the most resistant lines from this population. These studies identified 12 lines with strong MCMV resistance and several candidate genes that can be used by breeders and seed companies to improve MCMV and MLN resistance in maize by traditional breeding or gene editing approaches. New sources of genetic resistance to MLN are of major interest to seed companies and the most desirable tool for solving MLN epidemics, most notably ongoing in East Africa.
Impacts
(N/A)
Publications
- Jones, M.W., Ohlson, E.W. 2022. Registration of the Maize Synthetic Population OhMCMV-1. Journal of Plant Registrations. 16(2):394-399. https:/ /doi.org/10.1002/plr2.20195.
- Gentzel, I.N., Ohlson, E.W., Redinbaugh, M.G., Wang, G. 2022. VIGE: virus- induced genome editing for improving abiotic and biotic stress traits in plants. Stress Biology. 2, Article 2. https://doi.org/10.1007/s44154-021- 00026-x.
- Ohlson, E.W., Wilson, J.R. 2022. Maize lethal necrosis: Impact and disease management. Outlooks on Pest Management. 33(2):45-51(7). https://doi.org/ 10.1564/v33_apr_02.
- Ohlson, E.W., Redinbaugh, M.G., Jones, M.W. 2022. Mapping maize chlorotic mottle virus tolerance loci in the maize 282 association panel. Crop Science. 62(4):1497-1510. https://doi.org/10.1002/csc2.20762.
- Todd, J.C., Stewart, L.R., Redinbaugh, M.G., Wilson, J.R. 2022. Soybean aphid (Hemiptera: Aphididae) feeding behavior is largely unchanged by soybean mosaic virus but significantly altered by the beetle-transmitted bean pod mottle virus. Journal of Economic Entomology. https://doi.org/10. 1093/jee/toac060.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. Scientists in Wooster, Ohio made significant progress on all four objectives of the project in its fourth year: Objective 1, to monitor, characterize, and develop management strategies for emerging viruses, is ongoing as maize and soybean viruses spread, emerge, and are detected across the U.S. and worldwide. Bioinformatic analysis of a 2018 maize virus survey was completed, and at least one potentially novel virus was identified in silico. Validation and characterization of novel viruses and virus variants are needed and ongoing. Synergistic interactions between maize yellow mosaic virus (MaYMV), sugarcane mosaic virus (SCMV), and/or maize chlorotic mottle virus (MCMV) were characterized and indicated that MaYMV has measurable and significant interactions with each virus. MaYMV caused significant stunting and leaf reddening symptoms under growth chamber conditions. Evaluation of more than 30 maize inbreds for MaYMV resistance was completed and ten asymptomatic lines were identified. Potyvirus samples, that were previously collected from East Africa, were successfully purified to eliminate contamination by other viruses and will facilitate downstream virulence testing. These accomplishments represent significant progress towards the completion of goals to rapidly identify and characterize pathogens of maize and soybean and to identify genetic resistance (1.A.1), survey U.S. maize for virus sequences (1.A.2), and develop tools for understanding epidemiology of maize lethal necrosis (MLN) in East Africa (1.B). Under Objective 2, substantial progress has been made in basic virology research. Maize chlorotic dwarf virus (MCDV) constructs were built for in planta assays to evaluate putative polyprotein proteolysis sites. Eleven MCDV-S protease mutants were created and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that lose catalytic activity. MCDV-M1 and MCDV-Severe infectious clones were created, P51 protein was swapped to evaluate their effects on disease severity in maize, and P27 protein was deleted and replaced with GFP to evaluate P27 as the helper component in MCDV vector transmission. An MDMV infectious clone was developed to modify gene expression in maize, allowing overexpression or knocking down of multiple genes simultaneously. These results support the characterization of MCDV virus factors involved in pathogenesis and host- pathogen interactions (2.A) and development of systems for working with full-length infectious clones of maize viruses (2.B). Significant progress has been made towards the identification and characterization of loci of genetic resistance in maize as part of Objective 3. A genome wide association study (GWAS) analysis of MCMV tolerance in the maize Goodman 282 population led to the identification of several significant tolerance loci. Quantitative resistance loci were mapped in a recombinant inbred line population derived from one of the most MCMV tolerant lines identified from the Goodman population. This work led to the identification of a major MCMV resistance locus. Several maize recombinants in the MCMV tolerance regions derived from N211 were identified on chromosomes 3 and 5 and will be used to facilitate development of fine mapping populations. Evaluation of MCMV and potyvirus levels in MCMV tolerant line, N211, and susceptible OH28 were performed. Results from this experiment indicated that MCMV titer in the tolerant line was reduced by approximately 100 thousand-fold two weeks post infection. Multiplexed RT-qPCR assays for MCMV and SCMV were refined to improve accuracy and reproducibility. Development of a maize synthetic population, OhMCMV-1, was completed and evaluated for MCMV and MLN resistance. The population was developed by intermating five MCMV tolerant maize inbred lines and conducting one complete cycle of recurrent selection for MCMV resistance. The OhMCMV-1 population has significantly higher levels of MCMV and MLN resistance than found among the five parents. This population is planned for public release and will serve as a valuable breeding tool for MLN resistance. Continued progress was made towards the conversion of N211, KS23-5, and KS23-6 lines from yellow to white endosperm through marker assisted backcrossing. KS23-5 and KS23-6 have been successfully converted to white endosperm, while N211 is partially converted. Endosperm conversion will allow for more rapid deployment and development of MLN tolerant varieties in East Africa. Screenings of a population of 233 lines from the germplasm enhancement of maize (GEM) project for resistance to MDMV and SCMV were completed and submitted to the GEM database. These efforts support the identification, characterization (3.A.1, 3.B.1, 3.B.2), and fine mapping (3.A.2) of virus resistance loci in maize. Objective 4, genetic and genomic characterization of pathogen vectoring relationships between insect vectors of maize pathogens, is nearly complete moving into the final year of the project. Leaf hopper data sets were analyzed and virus-responsive and temperature responsive differentially expressed transcripts were detected. Annotation of these datasets is ongoing. Strand specific RT-PCR assays were developed and used to demonstrate MFSV replication in non-vector leaf-hopper species Dalbulus maidis and Macrosteles quadrilineatus. Research performed as part of this objective has helped to inform pathogen vectoring relationships between important leaf-hopper pests and viruses of corn and led to identification of several potential factors that could be disrupted for disease control. Record of Any Impact of Maximized Teleworking Requirement: Maximized teleworking had a major impact on our ability to conduct research since the start of the pandemic. Several experiments had to be discarded or reduced in scope. Growth chamber breakdowns and delayed repairs caused the loss and postponement of experiments. Personnel limitations in the lab greatly limited our ability to generate new research data and conduct experiments. Benchwork was severely limited. Bioinformatic analyses that are dependent on computing resources and pipelines that are unavailable under telework, could not be accessed for more than six months and analyses had to be conducted using inefficient and alternative approaches. ACCOMPLISHMENTS 01 A recently detected corn virus causes disease and interacts with co- infecting viruses that cause maize lethal necrosis (MLN). A new corn- associated polerovirus, often termed maize yellow mosaic virus (MaYMV) was recently discovered and found to be highly prevalent globally (Asia, Africa, South America, but not yet described in the U.S.) along with other corn viruses that cause a devastating disease called maize lethal necrosis (MLN) in co-infected corn plants. Whether the newly discovered polerovirus caused disease or contributed to MLN was unknown. ARS scientists at Wooster, Ohio, elucidated that MaYMV causes significant stunting, but other symptoms such as leaf reddening are often very mild. This indicated that the virus is likely to impact yield but evade facile visual detection, and that identification and breeding of genetic resistance in corn to this virus is advisable due to its high potential for yield reduction. Disease instigated by MaYMV was evaluated in single and mixed infections by the virus alone and in all possible combinations with two other corn viruses. MaYMV had measurable interactions with each of the two other viruses in mixed infections, causing significantly increased disease severity. These discoveries were published in Plant Disease and have spurred further research to identify sources of genetic resistance to MaYMV and improved information on management of MaYMV and MLN in corn, which is of great benefit to growers, breeders, and seed companies. 02 Maize dwarf mosaic virus-based tool for simultaneous gene silencing and expression in corn. Ever since the first genome sequence of corn was developed, there has been a need for research tools to examine and understand the function of genes, gene families, and genetic pathways in the plant. Historically, such tools have been very labor-intensive (generating transgenic plants or random mutagenesis), but viruses provide a shorter timescale method to target individual genes to study their function. ARS scientists at Wooster, Ohio, in collaboration with researchers at The Ohio State University (OSU), developed a new virus- based tool from maize dwarf mosaic virus (MDMV) with unique and unprecedented capability for corn. The MDMV-based tool can simultaneously overexpress and knock down expression of multiple corn genes, allowing study of either individual genes or entire genetic pathways in maize without the generation of transgenic or mutagenized plants. MDMV-based clones have already been shared under Material Transfer Agreements with two North Carolina State University and one Ohio State University laboratory, the Army Corps of Engineers via Defense Advanced Research Projects Agency, and have been requested from two international laboratories shortly after publication to enable studies of corn gene function and biology. An invention disclosure was made for this work.
Impacts
(N/A)
Publications
- Stewart, L.R. 2021. Sequiviruses and Waikaviruses (Secoviridae). Encyclopedia of Virology. vol. 3, pp. 703-711. Oxford: Academic Press.
- Mlotshwa, S., Xu, J., Willie, K.J., Khatri, N., Marty, D., Stewart, L.R. 2020. Engineering maize rayado fino virus for virus-induced gene silencing. Plant Direct. 4(8). Article e00224. https://doi.org/10.1002/pld3.224.
- Stewart, L.R., Willie, K.J. 2021. Maize yellow mosaic virus interacts with maize chlorotic mottle virus and sugarcane mosaic virus in mixed infections, but does not cause maize lethal necrosis. Plant Disease. Article 33736468. https://doi.org/10.1094/PDIS-09-20-2088-RE.
- Xie, W., Marty, D., Xu, J., Khatri, N., Willie, K.J., Buckner Moraes, W., Stewart, L.R. 2021. Simultaneous gene expression and multi-gene silencing in Zea mays using maize dwarf mosaic virus. BMC Biology. 21. Article 208. https://doi.org/10.1186/s12870-021-02971-1.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. ARS scientists at Wooster, Ohio, made significant progress on all four major objectives of the project in its third year: Objective 1: This objective, to monitor and identify emerging insect- transmitted pathogens of maize and soybean, saw major advances in the past year. Ongoing work on Sub-objective 1.A., focusing on virus discovery and diagnosis, resulted in a published survey of maize viruses in Rwanda, where Maize lethal necrosis (MLN) is emergent and highly destructive. In addition, a comprehensive survey catalog of, and incidence data on, wheat viruses in Ohio, including several that also infect maize, was also completed and published. A survey using current sequencing technology has not previously been done in the region for wheat and virus data were lacking. A new ryegrass mosaic virus (RGMV) sequence variant or strain in Ohio wheat was identified, with the complete genome sequence report soon to be published. Biological characterization and vector identification were completed for a recently discovered and globally prevalent maize polerovirus often referred to as maize yellow mosaic virus (MaYMV), and results were published. This work is the first to isolate this virus from other co- infecting viruses found in samples, and is seminal in definitively identifying disease symptoms caused by and biological attributes of this virus, also found in South America and with as yet unknown presence and distribution in North America. Sub-objective 1.B., developing tools to further characterize viruses discovered, also had major progress as antisera against virus proteins were developed for maize fine streak virus (MFSV), maize dwarf mosaic virus (MDMV), maize chlorotic dwarf virus (MCDV); and for detection of MaYMV. Antisera against multiple proteins from MFSV, MDMV, and MCDV were generated. Preliminary tests on MDMV and MaYMV antisera were conducted, but further testing to optimize detection efficacy is needed. Sub-objective 1.C., work to characterize the role of yeast, associated with brown marmorated stinkbug (BMSB), in soybean seed pod damage caused by insect feeding resulted in new information on seed imbibition. BMSB causes damage to soybean seeds by interfering with seed development, and in doing so, often cause yeast spot disease induced by Eremothecium coryli. An E. coryli inoculation experiment was conducted to determine the differences in seed infection between soybean lines, but the results were inconclusive due to presumed variation in seed coat permeability among the lines. However, the experiment led to tests of seed imbibition, that successfully identified the confounding effects of seed coat permeability on seed imbibition and E. coryli infection. Molecular genetic analyses (a genome-wide association study (GWAS) and quantitative trait loci (QTL) analyses) verified single nucleotide polymorphisms (SNPs) in a major seed coat composition gene involved in the domestication of Glycine soja (wild soybean) to Glycine max (domesticated soybean). Objective 2: Major breakthroughs were achieved in Sub-objective 2.A. on virus gene and functional analyses to characterize molecular mechanisms of MCDV pathogenesis and plant interactions. Infectious clones were developed, which were a key tool in this process. Infectivity of full- length clones of MCDV was demonstrated by their ability to launch infection in maize. MCDV has been highly recalcitrant to cloning, with high bacterial toxicity of sequences. Thus many years of effort by other researchers were unable to produce infectious clones. Optimization of MCDV clone-based infection of maize is still required as infection rates are very low, but this tool represents a major breakthrough for mutational analyses of virus functions leading to vector transmission and pathogenicity in maize. Progress on Sub-objective 2.B., to develop full-length clones and optimize working with them, overlaps with the key progress on MCDV molecular biology. In addition, infectious clones of MaYMV were developed and we are the first to successfully launch infectious clones of this virus in their natural host, maize. Major improvements in infection rates and clone stability were also achieved for MDMV infectious clones, first developed in our unit in 2012. Progress towards a full-length infectious clone of the rhabdovirus MFSV was also substantial in the past year. MFSV minireplicons were constructed which showed evidence of replication in a model host with full-length virus clones being constructed. Rhabdoviruses are well known among virologists to be extremely challenging for developing infectious clones, due to recalcitrant cloning and segmented negative-sense genomes that require simultaneous expression of three replicase proteins with the genomic RNA to launch infection in a single cell. Sub-objective 2.C., progress to characterize virus-vector interactions included completion of electropenetrography (EPG) experiments, an established technique for electronically monitoring feeding by hemipterans, comparing leafhopper feeding patterns on healthy and virus- infected maize. We hypothesized that there would be measurable differences between the leafhopper⿿s activities when feeding on uninfected and MCDV, MFSV, or maize rayado fino virus (MRFV)-infected maize plants. Each of the tested viruses has a different biological relationship with the leafhopper, which we expected to influence the leafhoppers in a manner consistent with their distribution in plants and mode of transmission. We used the leafhopper vectors Graminella nigrifrons and Dalbulus maidis. Analyzed results showed much subtler vector behavioral differences than expected despite major changes in infected host plant physiology, and analyzed results have been compiled into a manuscript draft for scientific publication. The project⿿s completion overlapped with a similar experiment using the soybean aphid, Aphis glycines, feeding on virus-infected soybeans. The aphid project was also completed, and analyzed results compiled into a manuscript draft. Objective 3: Progress on identifying and characterizing mechanisms of action of genetic loci for virus resistance in maize was substantial in the past year. For Sub-objective 3.A., to identify and characterize loci providing tolerance/resistance to MCMV in maize and sorghum, all work progressed. A synthetic population Ohio maize chlorotic mosaic virus-1 (OHMCMV-1) completed one full cycle of first generation self-pollination (S1) recurrent selection and is ready to release. The release is pending an evaluation of progress in MCMV tolerance. This population combines five unique resistance sources with the potential to generate inbred lines with superior resistance MCMV than currently available. It also has potential to combine resistance to sugarcane mosaic virus (SCMV) with MCMV resistance. The highly MCMV tolerant inbreds N211, KS23-5, and KS23- 6 have undergone six generations of backcrossing to convert them to white endosperm and will be self-pollinated to fix the trait and evaluated for MCMV tolerance prior to release. This germplasm is important because maize in East Africa is predominantly white endosperm and used for human consumption. A MCMV resistance source that is white endosperm will speed the breeding process. For Sub-objective 3.B., to characterize interactions among potyviruses, MCMV and virus resistance/tolerance in maize, progress is delayed due to inability to separate and isolate potyvirus variants from MCMV coinfections in source and vacant personnel. Originally, we planned to passage coinfected source material through sorghum, reported to be a non- host of MCMV but a host of SCMV. However, virus passaging and MCMV screening of the sorghum association mapping populations (AMPs) both showed that sorghum is a host of MCMV, although titers are highly variable and possibly suppressed. Thus, another approach is needed to separate highly infectious MCMV coinfections from SCMV potyvirus isolates and clearly assess the interaction of SCMV isolates with quantitative maize resistance traits. However, near isogenic lines (NIL) for potyvirus resistance genes on chromosomes 3, 6, and 10 exhibiting various levels of potyvirus resistance have been developed by crossing the resistance gene donor parents Pa405, Oh1VI, and FAP1360A with a common recurrent parent Oh28 6 times, followed by two generations of self pollination. The lines underwent one year of field screening with three potyvirus species. These NILs will allow study of virus x gene interactions and the role of resistance genes in controlling diseases caused by synergy of two viruses. The 233 entry Germplasm Enhancement of Maize (GEM) release was screened with MCMV and SCMV in the field. Data for SCMV and MCMV (2017) were compiled and submitted. The goal of this project is to broaden the germplasm base of the U.S. corn crop, and ARS researchers at Wooster, Ohio, are contributing virus resistance characterization. Sub-objective 3.C., characterize and map novel soybean traits for host plant resistance to BMSB. In order to develop an environmentally sustainable and effective way to reduce losses from these insects, our project sought to identify certain lines of soybeans that show resistance to BMSB. This year, we cultivated soybean plants from promising lines in greenhouse tests, and scored/weighed/tested seeds for damage. In addition, we scored/weighed soybean seeds this year from past field experiments. Objective 4: For this objective, to characterize pathogen vectoring relationships using comparative genetic and genomic analyses, a major project was completed and two manuscripts drafted. Comparison of transmission rates and vector transcriptional response to virus-infected versus healthy plants were compared for Dalbulus maidis, and a transcriptome of this leafhopper vector was developed. The effect of temperature on transmission rates and transcriptomic response was also compared. Accomplishments 01 Vector and symptom determination of global maize polerovirus. In recent years, sequences of many previously unknown viruses have been discovered in maize, but basic biology such as how these viruses are transmitted and whether they cause disease was not known. A new maize- associated polerovirus, often termed maize yellow mosaic virus (MaYMV) was recently discovered and found to be highly prevalent globally (Asia, Africa, South America) and present at very high incidences, but its biology and ability to cause disease was unknown. ARS researchers at Wooster, Ohio, isolated this virus from other coinfecting viruses in source plants and completed the first biological characterization of this virus. Koch⿿s postulates experiments (pathogen isolation, inoculation of healthy plants, and recapitulation of disease symptoms), which established the causative relationship between a pathogen and a disease, were completed to show infection of (not just detection in) maize, leaf reddening symptoms in a diverse panel of maize genotypes, and transmission by two aphid species. Demonstrated prevalence of this virus in East Africa, including in detail in Tanzania and Rwanda. This research leads characterization of potential pathogenicity of a newly identified global virus infecting maize. Virus isolation and transmission to maize opens research avenues to understand disease impact and epidemiology. 02 Basic research tools were developed to understand maize viruses. Infectious clones are a foundational research tool to understand virus biology including maize virus gene function and the infection interactions that lead to disease. The ability to genetically manipulate and replicate viruses in the laboratory is essential for basic virology research and can also be utilized to understand maize host plant gene functions. However, these tools are lacking for many maize viruses. ARS scientists at Wooster, Ohio, developed new and improved methods virology research and virus-based functional genomics with maize viruses. They improved infectivity and sequence stability of infectious laboratory cloned maize dwarf mosaic virus (MDMV), a major U. S. maize-infecting virus; developed infectious clones and an effective delivery methodology for difficult-to-deliver maize viruses including the globally distributed maize yellow mosaic virus (MaYMV), and a major U.S. maize virus, maize chlorotic dwarf virus (MCDV); and developed maize rayado fino virus (MRFV) clones to very efficiently silence host maize genes and study their functions. Infectious clones of MaYMV, MDMV, and MRFV and derivative tools have been requested by multiple national and international laboratories for research purposes and a subset are currently shared with national laboratories to better understand plant or virus biology and disease. 03 Survey of wheat viruses. In grass crops including corn and wheat, 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, ARS researchers at Wooster, Ohio, working with Ohio State University personnel, identified known and previously undescribed viruses in Ohio wheat, and tested their incidences across the state in multiple years. Sequence-based diagnostics were developed and reported for these viruses, which are now available to researchers and diagnosticians. Researchers and diagnosticians now utilize these data and tools to identify viruses contributing to disease. 04 Maize chlorotic mottle virus resistance introgression into useful genetic backgrounds for agriculture. Maize lethal necrosis (MLN) is a globally emergent virus complex severely impacting yield and food security. It is caused by co-infection of any endemic maize potyvirus with the emergent maize chlorotic mottle virus (MCMV). Resistance was previously identified for maize potyviruses, but tolerance traits in corn were only recently identified for MCMV by ARS researchers at Wooster, Ohio. To further enable utility of this identified resistance, advancing its introgression from tropical source germplasm to usable breeding germplasm is ongoing. The resistance traits will be fixed in desirable germplasm and evaluated for MCMV tolerance prior to release. This germplasm is important because maize in East Africa is predominantly white endosperm and used for human consumption. A MCMV resistance source that is white endosperm will speed the breeding process.
Impacts
(N/A)
Publications
- Asiimwe, T., Stewart, L.R., Willie, K.J., Massawe, D., Kamatanesi, J., Redinbaugh, M.G. 2019. Maize lethal necrosis viruses and other maize viruses in Rwanda. Journal of Plant Pathology. 69(3):585-597.
- Hodge, B.A., Paul, P.A., Stewart, L.R. 2020. Occurrence and high throughput sequencing of viruses in Ohio wheat. Plant Disease. 104(6):1789- 1800.
- Stewart, L.R., Todd, J.C., Willie, K.J., Massawe, D., Khatri, N. 2020. A recently discovered maize polerovirus causes leaf reddening symptoms in several maize genotypes and is transmitted by both the corn leaf aphid (Rhopalosiphum maidis) and the bird cherry-oat aphid (Rhopalosiphum padi). Plant Disease. 104(6):1589-1592.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Scientists in Wooster, Ohio, have made significant progress on all four major objectives of the project in its second 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, analyses of Rwanda samples tested in project year one were completed and a manuscript reporting viruses discovered and analyzed was submitted (now accepted with revisions). Diagnostic analyses of samples collected in Tanzania also progressed in year two and diagnostic tests were completed, with statistical and sequence analysis remaining to complete. Significant progress on a multistate U.S. survey for maize viruses was made. Although maize samples from Hawaii could not be obtained as planned, maize samples from Washington, New Mexico, Louisiana, Pennsylvania, Tennessee, Kentucky, Iowa, and Indiana were obtained and tested using ELISA diagnostic panels. RNA from these samples was extracted, quality control steps completed, and pooled multiplexed RNA was deep sequenced to provide datasets to search for known and previously unknown viruses. Field experiments testing the impact of brome mosaic virus (BMV), a well-known virus infecting both maize and wheat along with many other species, which has long been considered agriculturally insignificant but without experimental support, was discovered in multiple years in Ohio wheat and demonstrated to cause up to 60% yield loss in soft red winter wheat varieties grown in Ohio. This year resulted in culmination of these findings and tests over years demonstrating pathogenicity of BMV in wheat and publication of this work. 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). Basic virology research, encompassed by Objective 2, had major progress in this second year. Maize dwarf mosaic virus (MDMV) was robustly engineered for simultaneous marker gene expression and multi- gene targeting silencing was developed and tested. Constructs were built to test sequence determinants of resistance breaking for MDMV and SCMV isolates using infectious clones constructed in the first project year. MDMV and SCMV are potyviruses, and are the most ubiquitous viral pathogens of maize in the United States and globally, thus the importance of this progress is high. In addition to potyvirus research, the first infectious clones of an extremely challenging and manipulation- recalcitrant virus, maize chlorotic dwarf virus, were also generated this year. These are the first viruses of the genus that have been successfully developed as infectious clones, despite decades of attempts by multiple labs. Constructs to test further polyprotein cleavage sites in MCDV were built, which will serve as tools to further characterize the mature protein complement generated by the virus-encoded protease. Finally, work to better understand how viruses impact the behavior of their insect vectors progressed. After comparing feeding behaviors of soybean aphids (Aphis glycines) using the electrical penetration graph technique on uninfected soybeans, plants infected with Soybean mosaic virus, which is aphid-transmitted, and plants infected with Bean pod mottle virus, which is beetle transmitted, data were annotated and statistical analyses initiated. A first draft of results has been prepared for future publication development. Using the electropenetrography (EPG) technique, the reactions of 2 vector leafhoppers (Graminella nigrifrons and Dalbulus maidis) to maize infected with maize rayado fino virus were compared and data annotated in preparation for statistical analyses. The response of G. nigrifrons to maize infected with Maize chlorotic dwarf virus (MCDV), which is semi- persistently transmitted, and Maize fine streak virus (MFSV), which is persistently transmitted, has been annotated and prepared for statistical analyses. For Objective 3, to identify and characterize virus resistance loci, research focused on resistance in maize and sorghum to MCMV, a major emerging pathogen driving the problematic maize lethal necrosis (MLN) global epiphytotic, and for which no complete resistance has yet been reported. Arrays of homozygous F3 crossover lines for the N211 and KS23-6 x Oh28 populations were identified and screened with MCMV to fine map the location of MCMV resistance quantitative trait loci (QTL). These lines are being crossed to Oh28 to generate populations of recombinants within the crossover region. Near Isogenic Lines (NILs) for combinations of three potyvirus resistance loci using Pa405, Oh1VI, and FAP1360A as donor parents and Oh28 as the recurrent parent were completed. Fifteen elite inbred releases from CIMMYT for use in East Africa were begun being backcrossed by MAS using the MCMV resistant inbred N211, KS23-5, and KS223-6 as donor parents. N211, KS23-5, and KS23-6 have been converted to white endosperm (preferred in East Africa) and are being made homozygous after six backcrosses. A first year of screening of the 233 line GEM release with MDMV and SCMV was completed. Two replicates of screening 11 sorghum lines from the sorghum association mapping population (AMP) were screened for MCMV reaction using ELISA, with a third in progress. Objective 4 research to characterize vector relationships using genomic analyses made significant progress, as analyses of experiments comparing the responses of two leafhoppers that transmit maize rayado fino virus (MRFV), D. maidis and G. nigrifrons, were nearly complete and a Dalbulus maidis transcriptome assembled. Two manuscripts are in preparation from this work. Accomplishments 01 Brome mosaic virus can cause significant yield loss in U.S. wheat. Brome mosaic virus (BMV) is a well-known ⿿lab-rat of virology, known to be ubiquitous and known to infect many plants including wheat and other grain crops, but has long been held by dogma to be unimportant as a pathogen in crops. Following repeated detection of BMV in multiyear Ohio statewide surveys of wheat from 2012-2017 at incidences of up to 25%, BMV was field tested for impact on Ohio soft red winter wheat cultivars. Data over field seasons and greenhouse studies showed that all tested Ohio-grown wheat cultivars were susceptible to infection, and that inoculation with BMV at any of four tested growth stages resulted in up to 60% yield losses. Some wheat cultivars showed tolerance to the virus, such that cultivar selection may minimize grower losses. 02 Brown marmorated stink bug resistant QTLs discovered in soybeans. Host plant resistance is one of the most effective and environmentally friendly means to reduce losses from insect pests. Halyomorpha halys, the brown marmorated stink bug is an invasive insect pest that is emerging in the Midwest soybean belt. Using advanced generation progeny and single nucleotide polymorphisms genotyping, ARS researchers in Wooster, Ohio, have identified resistance quantitative trait loci associated with stink bug feeding incidence and severity. This discovery is an important step in understanding host plant resistance to this invasive polyphagous pest and enhancing the sustainability of soybean yields in the Midwest U.S. 03 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 in East African maize. 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 National Center for Biotechnology Information, to identify viruses contributing to disease. 04 Survey of damage caused by different stink bugs. Several stink bug species pose a serious threat to numerous crops, vegetable, and fruit commodities produced in the U.S. Stink bug species can also transmit pathogens that may cause further crop damage. We have cultured Eremothecium coryli, a fungal pathogen causing soybean yeast spot disease, from several stink bug species. The fungal isolates of different sting bug species all appeared identical in sequence and disease damage to soybean seeds. However, differences in feed damage to soybean caused by each stink bug species were observed. 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.
Impacts
(N/A)
Publications
- Meulia, T., Stewart, L.R., Goddin, M. 2018. Short Communication: Sonchus yellow net virus nucleocapsids form on nuclear rings enriched in phosphoprotein. Virus Research. 258:64-67.
- Hodge, B.A., Salgado, J.D., Paul, P., Stewart, L.R. 2019. Characterization of an Ohio isolate of brome mosaic virus and its impact on growth, development, and yield of soft red winter wheat. Phytopathology. 103(6) :1101-1111.
- La Mantia, J.M., Mian, R.M., Redinbaugh, M.G. 2019. Genetic mapping of soybean aphid biotype 3 and 4 resistance in PI 606390A. Molecular Breeding. 39:53.
- Lee, S., Van, K., Sung, M., McHale, L., Nelson, R.L., La Mantia, J.M., Mian, R.M. 2019. Genome-wide association study of seed protein, oil, and amino acid contents in soybean from maturity groups I to IV. Journal of Theoretical and Applied Genetics. 132(6):1639⿿1659.
- Schoelz, J.E., Stewart, L.R. 2018. The role of viruses in the phytobiome. Annual Review of Virology. 5:93-111.
- Redinbaugh, M.G., Stewart, L.R. 2018. Maize lethal necrosis: An emerging, synergistic viral disease. Annual Review of Virology. 5:301-322.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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.
Impacts
(N/A)
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.
- 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.
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