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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

2020 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
Continued studies on the discovery and identification of new pathogens infecting alfalfa. Alfalfa samples from commercial production fields with diseased symptomatology are being investigated to diagnose the causes of diseases. Progress directly relates to the Objective 1. Continued study on the identification and molecular characterization of novel and emerging viruses infecting alfalfa. Progress relates to the Objective 1. Study on alfalfa proteomic response to biotic stresses is advancing to the analytical stage. Progress directly relates to the Objective 2. Assembly and annotation of the full genome of the agriculturally important root lesion nematode species Pratulenchus penetrans is in progress. Progress relates to the Objective 2. Continued studies on the discovery and functional characterization of the virulence effectors of the root lesion nematode P. penetrans. Knowledge of the nematode parasitism genes can be applied toward alfalfa breeding programs or used in the development of new nematicides. Progress directly relates to the Objective 2.

1. A new alfalfa virus was identified. Alfalfa virus S (AVS) is a new species in the family Alphaflexiviridae that was identified in alfalfa plants originating from Sudan, Northern Africa, and in China in germinated fields were planted with alfalfa seeds originating from the U.S. ARS researchers from Beltsville, Maryland, reported the first detection of AVS in the United States as well as the complete nucleotide sequence and genome organization of the virus. This information can be used for the specific diagnosis and control of the virus in alfalfa plants. The research is significant because the biology and distribution of this emerging virus in U.S. alfalfa are not known, and results obtained in this study can fill this gap.

2. Determined a prevalence and distribution of the root lesion nematode virus in North American populations of the nematode Pratylenchus penetrans. In 2019, we discovered a novel virus that naturally infects the agriculturally important root lesion nematode Pratylenchus penetrans. Distribution of this virus in nematode populations remained unknown prior to this study. In this work, we have established that the virus is widespread in the U.S. and Canada and affects nematode populations collected from the diverse crop systems in North America. The research is important, because RLNV1 could represent an entirely new resource as a potential biological control agent for plant parasitic nematodes.

3. In collaboration with leading plant virologists in France and Argentina, analyzed the latest data on etiology and distribution of new and emerging viral diseases in alfalfa. Viral infections of alfalfa are often considered by breeders, growers, producers and researchers to be diseases of limited importance, although they are widespread in all major cultivation areas. Collaboratively with the scientists from the National Scientific and Technical Research Council of Argentina and the French Agricultural Research Center, assembled, analyzed and reviewed the latest research on new and emerging viruses of alfalfa that have a potential to cause serious yield losses in the crop. The study is significant because it called into question the assumed low economic impact of viral diseases in alfalfa and suggested their possible contribution to the severity of complex infections involving multiple pathogens. Further disregarding the role of viruses in alfalfa health could be irrational for alfalfa improvement and production.

4. Proposed biological roles of several novel effector proteins of root lesion nematode Pratylenchus penetrans in the nematode parasitism. Pratylenchus penetrans is a migratory species that attacks a broad range of crops, including alfalfa. Effectors or virulence factors, secreted by the root lesion nematode are critical for the nematode parasitism. Their genetic composition and roles are poorly known. In this study, we used virus-based transient expression assays to delineate interactions of several P. penetrans candidate effectors with the model host Nicotiana benthamiana. Phenotypically distinct plants expressing effector proteins were further investigated by the next generation sequencing to reveal changes in their transcriptome profiles triggered by the effector proteins. The research is significant because it proposed biological roles and perspective cellular targets of several pioneer effectors of P. penetrans and identified molecular mechanisms of host responses to these nematode-secreted proteins. This information can be used to develop novel nematode resistance strategies.

Review Publications
Vieira, P., Nemchinov, L.G. 2019. An expansin-like candidate effector protein from Pratylenchus penetrans modulates immune responses in Nicotiana benthamiana. Phytopathology.
Jiang, P., Shao, J.Y., Nemchinov, L.G. 2019. Identification of emerging viral genomes in transcriptomic datasets of alfalfa (Medicago sativa L.). Virology Journal. 16:153.
Vieira, P., Peetz, A.B., Mimee, B., Saikai, K., Mollov, D.S., Macguidwin, A., Zasada, I.A., Nemchinov, L.G. 2020. Prevalence of the root lesion nematode virus (RLNV1) in populations of Pratylenchus penetrans from North America. Journal of Nematology. 52:e2020-45.
Boutanaev, A.M., Nemchinov, L.G. 2020. Genome size dynamics within multiple genera of diploid seed plants. Russian Journal of Genetics: Applied Research. 56/667-676.