Location: Corn, Soybean and Wheat Quality Research2017 Annual Report
1. Monitor and identify emerging insect-transmitted pathogens of maize and soybean, and identify management strategies. 2. Determine whether multiple virus resistance in maize inbred lines is the result of pleiotropic or closely linked genes, and develop and release virus-resistant germplasm to breeders. a) Determine whether resistance to potyviruses is pleiotropic in Pa405. b) Mapping multiple virus resistance in Oh1VI. c) Develop and release virus resistant germplasm. 3. Develop genetic and genomic information on two insect vectors, including the molecular response to feeding on virus-infected plants. 4. Identify virus components important for pathogenesis, insect transmission, and host interactions, and develop virus systems for gene discovery and functional analysis in maize. a) Assess viral protein complements, expression strategies, and functions in maize. b) Develop maize virus-based forward and reverse genetics systems. c) Characterize virus and insect factors needed for virus transmission, and develop methods to study these processes.
1. A sequence-independent approach (SIA) for amplification of viral genome sequences will be used for initial identification of viruses in suspected, symptomatic plants. Mollicutes will be identified using PCR with genus-specific ribosomal DNA (rDNA) primers. The identity of known pathogens will be confirmed with a combination of microscopic, serological and molecular assays. New viruses will be cultured in susceptible plants and characterized. As possible under permit conditions, we will test known vectors of maize and soybean diseases for their ability to transmit pathogens. Mechanical or vector transmission of pathogens will be used to screen maize or soybean germplasm for resistant genotypes. 2. To determine whether the Wsm1 and Wsm2 genes for WSMV resistance confer resistance to multiple potyviruses, to isolate or fine map these two genes in Pa405, and to develop germplasm to fine map or isolate Wsm3. The putative insertional mutants Wsm1µ and Wsm2µ plants identified in the current project will be tested for chromosomal deletions on chr. 6 and 3, respectively, prior to testing for pleiotropic gain of susceptibility to potyviruses. We will clone sequences flanking the insertion sites to identify candidate genes. Genes and cDNAs encoding Wsm1 and Wsm2 will be cloned, and sequences will be used in loss and gain of function assays to confirm gene identity. Because of the risk associated with identifying insertional mutations in Wsm1 and Wsm2, we will continue efforts to develop a fine map Wsm1 and Wsm2, using available recombinant plants and populations. Additional markers will be identified in SNP and microarray analyses. We will develop germplasm to identify mutator insertions and fine map Wsm3. 3. Use second-generation sequence analysis to build and analyze EST libraries for two important vectors of soybean and maize viruses: A. glycines and G. nigrifrons. The vectors will be fed on plants infected with viruses that are transmitted in a non-persistent (SMV), semi-persistent (MCDV), persistent-circulative (SbDV) or persistent-replicative (MFSV) manner. EST libraries will be made with RNA from: 1) A. glycines biotypes 1 and 2 fed on healthy, and SMV or SbDV-infected soybean, and 2) G. nigrifrons fed on healthy, and MCDV- and MFSV-infected maize. Libraries will be sequenced, assembled and annotated. Differential EST expression between different treatments will be verified with quantitative real-time RT-PCR (RT-qPCR), and sequences from A. glycines and G. nigrifrons will be compared with those of other vector genomes. 4. An in vivo protease assay will be used to determine MCDV polyprotein cleavage sites by co-expressing active viral protease with epitope-tagged MCDV polyprotein regions and determining sizes of cleavage products. Antibodies made against predicted small ORF-encoded proteins will be used to test for protein expression in infected plants. MCDV proteins will be tested for subcellular localization and virus protein-protein interactions, and MCDV and MFSV proteins will be tested for their ability to suppress gene silencing in N. benthamiana.
This project expired and has been replaced with 5082-22000-001-00D. Over the life of the project substantial progress was made in all objectives, although some Obj. 2 experiments were were shifted to meet a critical need to identify and understand maize resistance and tolerance to maize chlorotic mottle virus (MCMV). We identified and characterized populations of known, emerging and previously unknown viruses infecting maize, wheat and weeds in Ohio and East Africa using next generation sequencing. Highly diverse populations of wheat mosaic virus were identified for the first time in Ohio. Populations of MCMV from across East Africa were similar, as expected from a recent introduction. In contrast, populations of potyviruses, which together with MCMV cause maize lethal necrosis (MLN), were highly diverse and included Johnsongrass mosaic virus in addition to sugarcane mosaic virus. Screening methods for resistance to MLN viruses were developed and MCMV-tolerant germplasm was identified. Loci for resistance to nine viruses from five virus families was characterized in the multi-virus resistant maize line Oh1VI, and found to be associated with clusters of loci on chromosomes 2, 3, 6 and 10. Genetic tolerance to MCMV was mapped in five biparental populations, and indicated that, in contrast to those previously mapped for other viruses, loci conferring tolerance are germplasm specific. Transcriptomes for leafhopper (Graminella nigrifrons) and aphid (Aphis glycines) vectors of maize and soybean viruses were developed. The vectors had common and distinct molecular and biological responses to feeding on virus-infected plants. Transcriptome data for the leafhopper vector, Dalbulus maidis, were developed data to assess the transcriptome responses of D. maidis and G. nigrifrons to temperature were collected. Virus-derived tools for gene silencing and basic virology research were developed utilizing soilborne wheat mosaic virus and maize necrotic streak virus. We developed methods to quantify maize fine streak virus gene expression quantification in maize and leafhopper hosts and demonstrated interactions between the viral N and P and 3 and 4 proteins. Maize fine streak virus titer in the insect was found to be a determinant of vector capability. The suppressor of the plant silencing defenses, p51, was identified for maize chlorotic dwarf virus, along with its viral protease cleavage site for maturation from the viral polyprotein. This is the first identification of a silencing suppressor for the genus waikavirus.
1. Identification and characterization of new and emerging corn viruses in East Africa. Next generation sequencing (NGS) is an effective method to rapidly identify emerging viruses and assess existing virus populations, even for previously unknown viruses. ARS researchers at Wooster, Ohio and collaborators surveyed and used NGS with in-house developed bioinformatics pipelines to identify and characterize maize and wheat virus genome sequences. Samples collected from East African countries where Maize lethal necrosis (MLN) has emerged were analyzed using NGS to characterize the virus population diversity, and significant variability in sugarcane mosaic virus genome sequences that impact the effectiveness of diagnostic protocols were identified. An East African Johnsongrass mosaic virus isolate that contributes to MLN was also discovered and diagnostic amplification primers developed. Knowledge of this virus and its contribution to disease is now aiding MLN containment and management efforts, and has provided diagnostic tools to detect and contain this virus. Containment and management protects international and U.S. agriculture by identifying and providing means to limit spread of pathogens.
2. Discovery of a plant defense-blocking virus protein. Most plant viruses encode proteins that block host defense systems in order to initiate and sustain infection. However, the functions of many corn virus-encoded proteins are not currently known. ARS researchers at Wooster, Ohio identified a protein encoded by maize chlorotic dwarf virus (MCDV) that suppresses plant RNA interference (RNAi) or ‘silencing’, a crucial plant antiviral defense system. RNAi (ribonucleic acid interference) or RNA silencing is an important plant defense mechanism that targets and destroys invading viruses in a sequence-dependent manner. Similar silencing suppression function was further identified in several related viruses, with differences in degrees of efficacy that could be important in determining virus virulence. This discovery is a significant step in our understanding of gene function and pathogenicity of a major U.S. maize virus and related viruses that infect other crops.
3. Maize lethal necrosis (MLN) spread in Tanzania. Maize lethal necrosis (MLN) is a devastating virus disease that has emerged in East Africa beginning in 2011. The MLN outbreak that was first discovered in Kenya has intensified and spread to surrounding countries. MLN has been found in regions of northern Tanzania abutting Kenya, but its distribution across major corn-growing regions of the country was not known. With collaborators, ARS researchers in Wooster, Ohio conducted surveys from southern, central, coastal, and northern Tanzania and identified potyviruses that contribute to MLN across the country. However, the second virus required for MLN, maize chlorotic mottle virus, is not yet widespread in southern and central Tanzania; however, it was detected in samples collected from central and southern regions where the disease is not yet observed or recognized. These results inform outreach and grower education efforts as well as MLN research and containment efforts, providing a protection against global spread, and also provide sequence, biological, and diagnostic data to identify viruses present globally including in the U.S.
4. Model for maize lethal necrosis (MLN) disease development. In sub-Saharan Africa, maize is a staple crop used as food and a cash crop by small farmers. Since 2011, farmers in the region have been dealing with a debilitating outbreak of MLN, which has now been reported from Ethiopia, South Sudan, Kenya, Uganda, Tanzania, Rwanda, Burundi and the Democratic Republic of the Congo. MLN is caused by infection of maize with two maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV). Since the outbreak occurred, significant gains in understanding the distribution of the viruses causing MLN, and developing disease tolerant cultivars and hybrids have been made. However, the roles of grassy weeds, other crops, seed, soil and insect vectors in the development of disease are not well understood. To better understand the potential benefits of various control measures, ARS researchers and collaborators developed a model for the spread of MLN viruses within and between growing seasons. The model incorporates virus transmission within a field via vectors, soil and seeds, and includes contributions from outside the field. Using existing scant information on the rates of MLN virus introduction through vector, soil and seed, the model predicted different contributions to disease from each factor. The model will be used to prioritize future research and to evaluate the relative costs and benefits of control measures such as crop rotation.
5. Soybean resistance to brown marmorated stink bug. The brown marmorated stink bug (BMSB) is an invasive insect pest native to Asia that was introduced to the Atlantic Seaboard of the United States. in 1996. BMSB has quickly become a severe agricultural pest from Tennessee to New York. BMSB are active from late spring to early fall when they feed on numerous fruits, vegetables, ornamentals, and crops. On many crops the feeding pressure can significantly reduce yield, but the insects also give off a persistent foul smell that can spoil fruits and vegetables. BMSB cause feeding damage on developing soybean pods that can range from holes in seed to complete loss of seed, reducing the value of soybeans for products like tofu. Host plant resistance is an effective and environmentally sound approach for controlling the damage done by pests like BMSB, but methods for identifying resistance in soybeans and resistant soybean lines were not known. To identify sources of resistance, ARS scientists grew soybean plants in insect proof field cages into which BMSB were released and allowed to feed during pod development. Over repeated tests in 2014 and 2015, two soybean lines from Asia were found that had lower frequency and severity of BMSB damage. The identification of these resistant lines provides a starting point to identify genes for BMSB resistance and the development of resistant soybean cultivars, but additional research is needed before the resistant lines can be used to reduce BMSB populations under field conditions.
Stewart, L.R., Jarugula, S., Zhao, Y., Qu, F., Marty, D. 2017. Identification of a maize chlorotic dwarf virus silencing suppressor protein. Virology. 504:88-95.
Stewart, L.R., Willie, K.J., Wijeratne, S., Redinbaugh, M.G., Massawe, D., Niblett, C.N., Asiimwe, T. 2017. Johnsongrass mosaic virus contributes to maize lethal necrosis in East Africa. Plant Disease. 101(8):1455-1462. https://doi.org/10.1094/PDIS-01-17-0136-RE.
Redinbaugh, M.G., El Desouky, A. 2016. Chapter 19: Maize Rhabdovirus-Vector Transmission. In: J. Brown editor. Vector-Mediated Transmission of Plant Pathogens. St. Paul, MN:APS Press. p. 277-287.