Location: Corn, Soybean and Wheat Quality Research
2020 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
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
1. 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.
2. 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.
3. 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.
4. 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.
Review 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. https://doi.org/10.1111/ppa.13134.
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. https://doi.org/10.1094/pdis-08-19-1724-re.
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. https://doi.org/10.1094/pdis-09-19-2054-sc.
