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 Corn, Soybean and Wheat Quality Research: Wooster, OH
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ARS Home Midwest Area Wooster, Ohio Corn, Soybean and Wheat Quality Research Research





- Ohlson, Erik
- Wilson, Jennifer - Jenny
- 2023
- 2022
- Publications
- Plant Diseases (303)

Research Project: Detection and Characterization of Genetic Resistance to Corn and Soybean Viruses **Location:
                            Corn, Soybean and Wheat Quality Research** 2023 Annual Report **Objectives**
                                

Objective 1:  Identify and characterize endemic and emergent viruses in corn and soybean, and develop sequence and detection resources. 
Sub-objective 1: Identify, diagnose, and characterize insect-transmitted pathogens of maize and soybean. 

Objective 2: Develop genetic markers and germplasm associated with corn virus resistance genes, and transfer information for practical management solutions. 
Sub-objective 2.A: Confirm the identity of HPWMoV resistance loci in maize and evaluate the effects of disease resistance on seed contamination and transmission. 
Sub-objective 2.B: Identify and characterize loci conferring tolerance/resistance to maize yellow mosaic virus. 
Sub-objective 2.C: Identify bean pod mottle virus resistant Glycine accessions and incorporate resistance into a cultivated soybean background. 

Objective 3: Fine map, clone, and characterize virus resistance genes, facilitating the investigation of host-pathogen interactions. 
Sub-objective 3.A: Fine map the WSMV resistance gene, wsm3, and examine pathogenesis of WSMV in resistant and susceptible maize. 
Sub-objective 3.B: Fine map MCMV resistance loci in maize inbred lines and develop near isogenic lines to study host-pathogen interactions. **Approach**
                                

Corn and soybean production in the United States is valued at more than $80 billion annually. Pathogens, including plant viruses, constitute a major component of crop loss, reducing U.S. corn and soybean yields by approximately 15%. Furthermore, pathogen contamination of grain poses major phytosanitary concerns for international trade. The spread of invasive pests and pathogens due to increased global trade and changing habitats, necessitates continued monitoring and identification of emerging viruses and their vectors to inform appropriate disease management strategies. The overarching goals of our research are to detect and characterize important viruses of corn and soybean, identify and develop virus resistant germplasm, map resistance loci, and elucidate mechanisms of host-vector-virus interactions. Using serological, molecular, and next generation sequencing techniques, we will identify key endemic and emerging maize and soybean viruses, evaluate virus population structures, and develop diagnostic assays. Host range and insect-vectors will be determined to reveal factors important for pathogenesis and transmission. We will identify resistant germplasm and develop molecular breeding tools to combat emerging maize viruses, such as high plains wheat mosaic virus (HPWMoV) and maize yellow mosaic virus (MaYMV). Secondary and tertiary soybean gene-pools will be evaluated to identify bean pod mottle virus (BPMV) resistant germplasm and resistance will be incorporated into cultivated soybean lines. Fine mapping and map-based cloning approaches will used to delineate the genomic loci associated with maize chlorotic mottle virus (MCMV) and potyvirus resistance genes, helping to determine mechanisms of host-resistance and to broaden our overall understanding of virus resistance in plants. Seed producers, breeders, researchers, and farmers will benefit from new disease diagnostic and molecular breeding tools, leading to improved corn and soybean yields. **Progress Report**
                                

We made significant progress on all three project objectives in the first full year. 

Objective 1, Identify, diagnose, and characterize insect-transmitted pathogens of maize and soybean. An experimental host and vector range of the emerging maize yellow mosaic virus was established leading to the discovery that oats, barley, rye, and foxtail millet are susceptible cereal crops to this virus. Four other grass species that include ryegrass, switchgrass, green foxtail and sand lovegrass were also found to be susceptible to the virus. Furthermore, a third vector was discovered in the greenbug aphid, in addition to two previously reported aphid species. Moreover, after developing a purification method for maize yellow mosaic virus, we developed the first serological diagnostic test for this virus. We also supported diagnostics for High Plains wheat mosaic virus by developing and delivering new diagnostic primers as well as providing technical guidance and positive control tissue to industry and academic diagnostic labs. Field surveys were conducted to collect leaf samples infected with High Plains wheat mosaic virus with collaborators in the Pacific Northwest. These new virus isolates were propagated and characterized to better understand the diversity of the virus. This growing isolate collection will be used to further develop and validate diagnostic tests for High Plains wheat mosaic virus. We completed the seventh and final year of yield trials to evaluate the impact of sugarcane mosaic virus on commercial corn hybrids. We discovered that on average, sugarcane mosaic virus reduced corn yields by 10% and as much as 30% in the most susceptible varieties. All hybrids were found to be susceptible to the virus. Remote sensing studies were conducted to identify multispectral and hyperspectral features associated with virus infection and yield. Eighteen significant spectral features were detected between infected and uninfected treatments among the multispectral data. Hyperspectral analyses are ongoing. Moreover, diverse sugarcane mosaic virus isolates were characterized and found to be highly virulent on corn and able to overcome several potyvirus resistance genes. This increased virulence did not contribute to more severe disease when plants were co-infected with the hypervirulent sugarcane mosaic virus isolates and maize chlorotic mottle virus. For soybean viruses, a seed sample displaying virus-like symptoms was tested and diagnosed as being infected with bean pod mottle virus. Management recommendations for the virus were passed along to the grower through university collaborators. Technical guidance was also given to the university Soybean Extension Plant Pathologist to establish soybean virus testing protocols in his laboratory.

Objective 2, Develop genetic markers and germplasm associated with corn virus resistance genes, and transfer information for practical management solutions. Inoculation protocols using wheat curl mites were optimized to study the recalcitrant High Plains wheat mosaic virus. Using these protocols, the potyvirus resistant donor corn inbred line, Pa405, was found to be asymptomatic to High Plains wheat mosaic virus infection. However, diagnostic testing indicated that the virus was able to infect Pa405, demonstrating incomplete resistance. A potyvirus resistance locus, Scmv1, was found to mask symptoms caused by the virus but did not completely eliminate infection. Twenty-eight additional corn inbred lines including sweet, field, and popcorns were inoculated with High Plains wheat mosaic virus and transplanted to the field for seed to be used in subsequent seed transmission studies. A maize diversity panel was screened for resistance to maize yellow mosaic virus. Seven lines tested negative for virus presence based on reverse transcription – polymerase chain reaction (RT-PCR), but confirmation is required. Approximately 75% of the population was found to exhibit leaf reddening that is characteristic of infection. However, no symptoms were present on the remaining lines, suggesting possible resistance. The tropical inbred line, Ki3, was found to have 100-fold reduced virus titer and no symptoms compared to the susceptible control, suggesting a high level of resistance. A recombinant inbred maize population developed from the inbred lines Ki3 and B73 was acquired and seed was multiplied for approximately 200 lines for subsequent mapping experiments to identify resistance genes. Fifteen soybean inbred lines and 37 wild soybean lines were evaluated for resistance to bean pod mottle virus. Three soybean lines were found to have improved disease resistance, with disease severities less than half of those found among the susceptible controls. Two wild soybean (Glycine tomentella) lines were found to have complete resistance. None of the wild soybean G. soja lines were found to have complete resistance, though two lines had significantly higher disease resistance than the susceptible controls. Germplasm and mapping populations derived from the most resistant G. max and G. soja lines are being developed.

Objective 3, Fine map, clone, and characterize virus resistance genes, facilitating the investigation of host-pathogen interactions. Eighty-five F3 families segregating for the potyvirus resistance genomic locus, wsm3, totaling 1,700 plants, were evaluated for resistance to wheat streak mosaic virus. These data were combined with the parental F2 genotype data to narrow down the wsm3 locus to a 119 kilobase genomic interval which contains four candidate resistance genes. Full length genomic sequences from the Oh28 (susceptible) and Pa405 (resistant) parental lines were generated for two of the four candidate genes and high-fidelity amplicons for the other two candidate genes will be obtained and sequenced in FY23. A targeted reverse-transcription polymerase chain reaction (RT-qPCR) approach is being taken to quantify expression levels of the four candidate genes, supplanting the broadscale RNAseq approach that was originally planned since fine mapping delimited the wsm3 resistance gene to just four candidates. We have designed RT-qPCR primers for all four candidate genes and relative expression levels between the resistant and susceptible lines are being determined. To characterize the mechanism by which the wsm3 resistance gene operates, a fluorescently tagged wheat streak mosaic virus infectious clone was used to track virus movement in Oh28 and near-isogenic lines carrying the wsm3 locus in homozygous and heterozygous genotypes. We were able to successfully track virus movement in the susceptible and heterozygous lines, though no fluorescent signal was observed in the homozygous resistant near isogenic line due to the complete resistance conferred by this line. In the susceptible Oh28, virus presence was dispersed throughout the leaf which was consistent with the mosaic symptoms typically associated with the virus. However, in the heterozygous form the wsm3 gene limited the virus to smaller lesions that were sporadically present across the leaf. Interestingly, autofluorescence observed under the microscope near the margins of virus lesions was consistent with dead cells, suggesting that wsm3 virus resistance may operate by a hypersensitive-like response. Fine mapping of maize chlorotic mottle virus resistance is ongoing in two populations derived from the MCMV resistant parents N211 and CML333 and the susceptible parent Oh28. Due to the higher level of resistance present in N211 than in CML333 and our ahead of schedule population development, the N211 populations have been prioritized for fine mapping and gene identification. Backcross three (BC3) selfed (S1) families have been developed and planted in the field. Genotyping will be performed in summer FY23 and individuals that recombine between two genetic markers flanking each of the chromosome 3 and chromosome 5 resistance loci will be selfed to generate BC3S2 families for subsequent phenotyping and fine mapping. The CML333 fine mapping population development is ongoing, and additional generations of backcrossing were performed prior to fine mapping to ensure good genetic background uniformity. BC2 lines were backcrossed to B73 to generate BC3 lines and selected for the chromosome 10 resistance locus. The BC3 lines will be self-pollinated in summer FY23 to generate BC3S1 populations for subsequent fine mapping. **Accomplishments**
                                

1. Maize chlorotic mottle virus has low transmission rates through corn seed and drying seed reduces virus spread. Maize lethal necrosis is a devastating virus disease of corn caused by simultaneous infection by two viruses, one of which is maize chlorotic mottle virus. Maize lethal necrosis epidemics can occur when maize chlorotic mottle virus is introduced to a new region, which can lead to hundreds of millions of dollars in lost yields. Understanding how the virus spreads and the risks associated with seed trade is critical for preventing the spread of maize lethal necrosis. ARS researchers in Wooster, Ohio, discovered that maize chlorotic mottle virus is transmitted through seed at extremely low rates of less than 0.01%. The virus was found only in dead outer seed tissues that are less able to maintain viable virus particles than internal living seed tissues such as the embryo. When seed was dried to 15% or lower moisture content, no transmission of the virus was found, which indicates proper seed drying can reduce the risk of virus spread through seed. This information is valuable for the corn seed industry and plant health regulatory agencies for reducing the spread of maize chlorotic mottle virus through seed, informing seed trade policies, and preventing the massive economic losses associated with the introduction of maize lethal necrosis to new geographic regions. 2. Diagnostic primers for a virus of corn that is an emerging trade issue potentially impacting billions of dollars in economic value. High Plains wheat mosaic virus is widespread in the United States, especially in the Midwest and Pacific Northwest where corn seed is produced. Recently imposed trade restrictions prevent the export of corn seed infected with the virus, greatly impacting the ability of U.S.-based seed companies to export seed to key trading partners. Reliable diagnostic tests for the virus are desperately needed, to certify corn seed that is clean of the virus and safe to export. ARS researchers in Wooster, Ohio, developed five new diagnostic primers for High Plains wheat mosaic virus that are being used by university and industry researchers to test for the virus. These new primers have broad recognition of different variants of the virus. These diagnostic primers will benefit diagnostic labs that test for the virus, corn seed industry stakeholders that depend on these test results, and regulatory authorities that certify seed as safe for export, facilitating safe seed trade with other countries. 3. New crop hosts and insect vectors identified to help manage an impactful emerging virus in corn. Maize yellow mosaic virus is an emerging virus of corn and other cereal crops that has been recently found in North America and can cause up to 30% yield loss. Since this virus was only recently discovered, it is not yet known which crops it can infect and which insects can transmit it. ARS researchers in Wooster, Ohio, have found that, in addition to corn, wheat, sorghum, and sugarcane, the virus can infect four additional crops: oats, barley, rye, and foxtail millet. They also identified four common weedy grass species that can be infected by the virus and hence, may serve as a reservoir for virus when the crop hosts are not present. Furthermore, they discovered it can be transmitted by an insect known as the greenbug aphid, in addition to the two insects already known to transmit the virus. Understanding the plant hosts and insect vectors of this virus informs not only which crops may be at risk, but also how growers and extension researchers can best manage the disease using appropriate crop rotations and pest control. **Review Publications**
                            *Ohlson, E.W., Timko, M.P. 2022. Mapping and validation of Alectra vogelii resistance in the cowpea landrace B301. Agronomy. 12(11). Article #2654. https://doi.org/10.3390/agronomy12112654.*
                                *Bernardo, P., Barriball, K., Frey, T.S., Meulia, T., Wangai, A., Suresh, L., Heuchelin, S., Paul, P., Redinbaugh, M.G., Ohlson, E.W. 2023. Transmission, localization, and infectivity of seedborne maize chlorotic mottle virus. PLOS ONE. 18(2). Article #e0281484. https://doi.org/10.1371/journal.pone.0281484.*
                                *Schiltz, C.J., Wilson, J.R., Hosford, C., Adams, M.C., Preising, S.E., Deblasio, S.L., Maccleod, H.J., Ven Eck, J., Heck, M.L., Chappie, J.S. 2022. Polerovirus N-terminal readthrough domain structures reveal novel molecular strategies for mitigating virus transmission by aphids. Nature Communications. 13:6368. https://doi.org/10.1038/s41467-022-33979-2.*
                                *Olmedo-Velarde, A., Wilson, J.R., Stallone, M., Deblasio, S.L., Chappie, J.S., Heck, M.L. 2023. Potato leafroll virus molecular interactions with plants and aphids: gaining a new tactical advantage on an old foe. Physiological and Molecular Plant Pathology. https://doi.org/10.1016/j.pmpp.2023.102015.*


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