Location: Corn, Soybean and Wheat Quality Research
2022 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
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
1. 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.
2. 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.
Review 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.
