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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Research Project #436115

Research Project: Emerging Stress Challenges and Functional Genomics of Stress Responses in Alfalfa

Location: Molecular Plant Pathology Laboratory

2021 Annual Report

1. Identify and develop molecular characterizations of new and emerging disease-causing pathogens in alfalfa to prevent potential threats to alfalfa production. Many known bacterial, fungal, oomycete, nematode, mollicute or viral diseases represent causes of concern for alfalfa industry. In addition, new, emerging and invasive pathogens of uncertain impact pose a serious challenge to the alfalfa improvement. Rapid identification of the causal agents, their characterization at the molecular level and development of sensitive diagnostic assays will reduce yield losses and prompt new insights into practices of alfalfa disease management. 2. Identify genes involved in stress responses in alfalfa to define the genetic basis of resistance and accelerate breeding programs. Emerging disease challenges demand novel approaches to maintain and improve alfalfa production. Understanding molecular mechanisms of stress tolerance is an essential requirement for improvement of alfalfa adaptability and acceleration of breeding programs in increasingly less favorable environmental conditions.

To fulfill the main goal of Objective 1, the Project will pursue rapid identification of the causal agents, their characterization at the molecular level, and development of sensitive diagnostic assays, aiming to reduce yield losses and to prompt new insights into practices of alfalfa disease management. The approach and research methodology for the detection and/or discovery of new biological and environmental stressors influencing alfalfa quality and productivity will include the following steps critical for the success of the Project: • Specimens collection: alfalfa samples delivery will be negotiated with colleagues, collaborators, alfalfa extension specialists, commercial growers, industry professionals and with diagnostic laboratories-participants of the National Plant Diagnostic Network. Samples will also be collected during on-site visits to alfalfa fields for detection of plant pathogens. • Diagnostics and identification: alfalfa samples will be evaluated by visual assessment, microscopic tools, molecular detection methods (PCR/RT-PCR, LAMP and others), serological assays, and next generation sequencing. • Molecular characterization: identified plant pathogens will be further characterized at the molecular level using comprehensive bioinformatics, molecular and phylogenetic tools. • Development of specific diagnostics tools for pathogen detection, such as pathogen-specific PCRs (conventional, RT-PCR, quantitative PCR, nested and multiplex PCR), molecular hybridization techniques, and serological assays. • Field pathogenomics: integration of genomic data into traditional pathogen surveillance activities. To fulfill the main goal of Objective 2, the Project will use modern experimental and genomic tools combined with computational analysis and systems biology research. Consecutively applied toward each of the plant-pathogen interaction studies, these state-of-the-art methodologies will enable identification and characterization of the genes, involved in stress responses in alfalfa. • Experimental approaches will primarily include the latest high-throughput sequencing methodologies to capture and quantify transcripts present in an RNA extract. • Computational approach will include transcript quantification (estimation of gene and transcript expression); differential gene expression analysis (comparison of expression values among different samples); and functional profiling of RNA-seq data (characterization of the molecular functions or pathways in which differentially expressed genes (DEGs) are involved) • Systems biology research will integrate quantitative metagenomics data into descriptions of genes, pathways, cellular processes and networks to uncover biological insights of alfalfa adaptive responses. • To supplement high-throughput transcriptomics data, the project will attempt to employ global proteomic profiling to identify and characterize proteins involved in alfalfa responses to stress.

Progress Report
Genome-wide identification of alfalfa transcription factors and an upgrade of a web resource center AlfalfaTFDB, (NP215 Component 2A). For years, only a scattered knowledge on a few individual transcription factors (TFs) - proteins that govern organismal development and response to the environment - was available for alfalfa, one of the most widely planted forage legume in the world. Taking advantage of the most recent developments in the field of alfalfa genomics, ARS researchers at Beltsville, Maryland, have identified and assembled a comprehensive repertoire of more than eight thousand of alfalfa TFs. Phylogenetic analysis of the largest alfalfa TF families showed that their composition, although similar to the related species M. truncatula, is substantially more diverse. ARS researchers have also upgraded a quality resource hub AlfalfaTFDB ( The resource center is publicly available at the MPPL’s web site. The results of this study demonstrated that TF families in alfalfa sustained a pronounced expansion that may indicate sophisticated mechanisms of adaptive responses to environmental stresses. The results are significant because they will further contribute to the fundamental questions pertaining to biology of alfalfa and other legumes and expand our knowledge of TF families in the plant kingdom. Continued studies on the discovery and identification of new pathogens infecting alfalfa. New alfalfa samples from commercial production fields with diseased symptomatology are being investigated to diagnose the causes of diseases. Progress directly relates to Objective 1. Continued study on the identification and molecular characterization of novel and emerging viruses infecting alfalfa. Progress relates to Objective 1. Initiated study on the genome-wide identification of endogenous viral sequences in alfalfa. The endogenization was proposed to contribute toward the evolution of living organisms via horizontal gene transfer of novel genetic material and resultant genetic diversity. No endogenous viral elements were found in alfalfa prior to our work. Progress directly relates to Objective 1. Continued study on the quantity, diversity and biological roles of alfalfa transcription factors - proteins that regulate gene expression and mediate various plant responses to stress. Progress directly relates to Objective 2. Together with Non-Assistance Cooperative Agreement (NACA) collaborator, continued studies on the discovery and functional characterization of the virulence effectors of the root-lesion nematode P. penetrans and the burrowing nematode Radopholus similis, economically important pathogens that inflict damage and yield loss to a wide range of crops. Knowledge of the nematode parasitism genes can be applied toward alfalfa breeding programs or used to develop new nematicides. Progress directly relates to Objective 2. Initiated metatranscriptomic study on the viral communities of nematodes and other soil-inhabiting organisms. Progress directly relates to the Objective 1.

1. Discovery and characterization of novel RNA viruses in nematodes and other soil-inhabiting organisms. Thus far, no comprehensive research focusing exclusively on nematode virome has been carried out. Breeding for resistance to nematodes can be difficult and disease management practices are limited to sanitation and the use of nematicides. Nematode-infecting viruses can represent an entirely new resource as a potential biological control agents. ARS researchers in Beltsville, Maryland, performed an extensive metatranscriptomic analysis of soil samples, collected in different regions of the United States and deciphered viral communities associated with soil-inhabiting organisms, particularly free-living and plant parasitic nematodes. The study revealed a number of novel, previously undescribed viral species and characterized their phylogenetic relationships, which will expand our knowledge on the diversity, significance, ecology and interactions of RNA viruses. These are essential steps toward understanding major factors that promote abundance and roles of RNA viral communities in natural environment.

2. Genome-wide identification of endogenous viral sequences in alfalfa. Endogenous viral elements are partial or entire viral genes or genomes integrated into host chromosomes and inherited as alleles. They are instrumental in a gene flow between viruses and eukaryotes and may have a substantial role in the evolution of their hosts. None have been reported in alfalfa prior to this work. ARS researchers at Beltsville, Maryland, performed a comprehensive, genome-wide screening of tetraploid and diploid alfalfa genomes and identified multiple integrated sequences derived from plant pararetroviruses of the family Caulimoviridae. Distribution and numbers of the endogenous viral sequences indicated that the integration events most likely occurred before the whole genome duplication. The same regions of homology with these viruses were also found in the genome of Medicago truncatula, a close alfalfa relative, implying that integration events could have happened at least ~5.3 million years ago, before alfalfa diverged from M. truncatula. The study is significant, because viral insertions could acquire functional roles in alfalfa’s normal growth, organ development, metabolism, and response to environmental stresses.

3. Identification of novel candidate effector genes for the burrowing nematode, Radopholus similis. The burrowing nematode is an economically important pathogen that inflicts damage and yield loss to a wide range of crops. It is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops. This study has been conducted via multiple collaborative efforts that included ARS and academic researchers at different locations in the U.S., as well as researchers in UK and France. The study revealed that burrowing nematode holds a diverse and emergent repertoire of effectors - factors that facilitate nematode invasion. Their functional roles have been suggested and characterized and the evolutionary histories that shaped the effector repertoire proposed. Considering the economic damage caused by the borrowing nematode, the study is significant because the information obtained provides valuable data to elucidate the mode of parasitism of this nematode. It also offers critical suggestions for the potential use of R. similis-specific effector genes to control this important pathogen.

Review Publications
Vieira, P., Vicente, C.S., Branco, J., Bauchan, G.R., Mota, M., Nemchinov, L.G. 2021. The root lesion nematode effector Ppen10370 is essential for parasitism of Pratylenchus penetrans. Molecular Plant-Microbe Interactions.
Nemchinov, L.G., Mollov, D.S., Grinstead, S.C. 2020. Identification of a novel isolate of Alfalfa virus S from China suggests a possible role of seed contamination in the distribution of the virus. Plant Disease.
Nemchinov, L.G., Grinstead, S.C., Irish, B.M., Shao, J.Y. 2020. Identification of complete genome sequencing of alfalfa virus S diagnosed in alfalfa plants (Medicago sativa L.) from Washington State, USA. Plant Disease.
Bejerman, N., Roumagnac, P., Nemchinov, L.G. 2020. High-throughput sequencing for deciphering the virome of alfalfa (Medicago sativa L.). Frontiers in Microbiology.