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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Research Project #434720

Research Project: Regulation of Gene Expression in Alfalfa Development and Stress Tolerance

Location: Molecular Plant Pathology Laboratory

2019 Annual Report

1. Improve the efficiency of developing alfalfa with greater tolerance to biotic and abiotic stresses by characterizing gene-stress responses and pathways. Biotic and abiotic stresses cause significant yield losses in alfalfa and greatly reduce the crop’s productivity. Understanding the molecular mechanisms of stress tolerance and the ways by which stress-responsive genes are regulated is essential for improvement of alfalfa adaptability and breeding programs. 2. Aid plant breeders in improving alfalfa productivity and adaptability by implementing genetic and genomic approaches to improve traits related to biotic and abiotic stress tolerance, including, but not limited to, root-knot nematodes and salinity tolerance. Data on stress-responsive genes obtained in this study and other information on alfalfa genomics will be used to identify molecular markers associated with resistance and adaptation to abiotic and biotic stresses in alfalfa.

The research project will identify stress-responsive gene-candidates in alfalfa and assign them to cognate functional groups related to specific stress responses. It will quantify and confirm roles of the selected genes in adaptation to abiotic and biotic stresses and in regulation of stress responses. Sequence polymorphism in genes underlying stress tolerance will be delineated and molecular markers associated with resistance and adaptation of alfalfa to biotic and abiotic stresses developed. Markers will be validated through cooperative research collaborations.

Progress Report
This progress report documents research performed during the bridge period between termination of the old project on April, 2018 and start of the replacement project on March 2019. Parts of this report were included in the final report on the terminated project, submitted on June 2018. A separate Annual Report has been submitted on the replacement project. We continued research on alfalfa interaction with agriculturally important root lesion nematode Pratylenchus penetrans. Significant progress has been made toward understanding molecular mechanisms of alfalfa resistance to the nematode. As part of the transition to the new project, we expanded research on the discovery and characterization of new and emerging pathogens, particularly viruses that were not previously known to infect alfalfa. We continued our research on the development of molecular markers linked to salinity tolerance in alfalfa. This is a long-term genome-wide association study (GWAS), which is focused on associations between molecular markers such as single-nucleotide polymorphisms (SNPs) and trait of seed germination under salt stress.

1. Condensed tannins play an important role in alfalfa resistance to root lesion nematode. Condensed tannins (CT) are flavonoid oligomers that contribute to many agronomically important plant traits, including disease resistance. CT may provide a way to beneficially manipulate protein digestion and prevent pasture bloat in ruminants. Leaves of alfalfa, a major forage crop, contain no detectable tannins that could protect ruminant animals from potentially lethal pasture bloat. Researchers in Beltsville, Maryland, have been working for years to produce tannins in the leaves and stems of the alfalfa plant to prevent pasture bloat in dairy and beef cattle and to suppress internal parasites. In this research, we demonstrated that levels of condensed tannins significantly increased in alfalfa roots upon infection with root lesion nematode. We anticipate that the same mechanisms that contribute to the CT gain in alfalfa roots during nematode infection, can be used to increase tannin concentrations in alfalfa leaves and develop tannin-rich alfalfa cultivars.

2. For the first time, identified and experimentally confirmed a presence of amalgavirus in U.S. alfalfa (Medicago sativa L.) germplasm. Alfalfa productivity is often limited by various biotic and abiotic components in the ecosystem, including viruses. Amalgaviruses are a newly discovered seed transmitted pathogens that are broadly found in plants. Prior to this research, amalgaviruses have never been experimentally diagnosed in the U.S. alfalfa. In this study, amalgavirus sequences were retrieved from alfalfa transcriptomic datasets and assembled into a complete genome. The virus identity was confirmed experimentally by PCR amokification, cloning and sequencing. This research is significant because the amalgaviruses are known to be vertically transmitted through seeds and biological impact of the amalgaviruses on alfalfa productivity is unknown.

3. Demonstrated that alfalfa varieties in the U.S. could be massively infected with cryptic viruses. The partitiviruses are associated with persistent infections of fungal, protozoan and plant hosts. Their relationship with plant hosts is not well understood. While examining transcriptomic data derived from two U.S. alfalfa check cultivars, we found that 95% of alfalfa plants were infected with partitivirus recently reported from Korea. The complete nucleotide sequence of the U.S. isolate of the virus was obtained. Impact: Since partitiviruses are vertically transmitted via seeds, the seeds of these standard check U.S. cultivars commonly used in research and breeding, were most probably infected with the virus. Biological significance or any negative effects of the virus on alfalfa are currently unknown and require further investigation. This is a first identification of the virus in alfalfa samples in the U.S. (manuscript in preparation).

4. Discovered and characterized a new enzyme produced by root lesion nematode. The plant cell wall plays an important role in various fundamental physiological processes of plant growth and development, such as maintaining the integrity of cellular content, morphogenesis, and cell signaling. In addition, the cell wall is the primary interface for most plant-pathogen interactions, since it is the first physical barrier against invasion and infection. Root lesion nematodes (RLNs) are migratory, endoparasitic nematodes that are able to parasitize a broad host range and cause extensive root damage to plant hosts. The successful invasion of RLN depends in part on the battery of cell wall-degrading enzymes (CWDEs) that are secreted into host tissues. In collaborative research with Virginia Technical University, ARS scientists at Betlsville, Maryland, have discovered and characterized a new gene, encoding CWD enzyme pectin methylesterase (PME) in root lesion nematode Pratylenchus penetrans. It is the first evidence of the PME-encoding gene found within the phylum Nematoda. The research provides new insights into biology of the nematode and may result in the development of a novel class of plant protective nematicide.

Review Publications
Vicente, C.S., Nemchinov, L.G., Mota, M., Eisenback, J.D., Kamo, K.K., Vieira, P. 2019. Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans. PLoS One. 14 (2):e0212540.
Nemchinov, L.G., Lee, M., Shao, J.Y. 2018. First report of alphapartitiviruses infecting alfalfa (Medicago sativa) in the United States. Genome Announcements.