Location: Corn, Soybean and Wheat Quality Research
2019 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
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
1. 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.
2. 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.
3. 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.
4. 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.
Review 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. https://doi.org/10.1016/j.virusres.2018.10.005.
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. https://doi.org/10.1094/pdis-07-18-1282-RE.
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. https://doi.org/10.1007/s11032-019-0956-9.
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. https://doi.org/10.1007/s00122-019-03304-5
Schoelz, J.E., Stewart, L.R. 2018. The role of viruses in the phytobiome. Annual Review of Virology. 5:93-111. https://doi.org/10.1146/annurev-virology-092917-043421.
Redinbaugh, M.G., Stewart, L.R. 2018. Maize lethal necrosis: An emerging, synergistic viral disease. Annual Review of Virology. 5:301-322. https://doi.org/10.1146/annurev-virology-092917-043413.
