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

Location: Molecular Plant Pathology Laboratory

2023 Annual Report

Objectives
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.

Approach
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 study on the discovery and molecular characterization of novel and emerging viruses infecting alfalfa. Viral infections of alfalfa are widespread in all major cultivation areas and their impact on alfalfa production is often underestimated. New symptomatic alfalfa samples from commercial production fields in Arizona have been received and are being investigated to diagnose the causes of diseases. Known, emerging and novel viruses of possible quarantine importance were identified. Alfalfa’s role as a reservoir for dissemination of viruses to other agriculturally important crops is being assessed. Progress directly relates to Objective 1. Initiated study on alfalfa seed virome. Seed transmission of plant viruses can be important due to the role it plays in their dissemination to new areas and subsequent epidemics. Except for a few individual seedborne viruses infecting alfalfa, its seed virome is poorly known. The goal of this research was to perform initial seed screenings on alfalfa germplasm accessions maintained by the USDA ARS National Plant Germplasm System in order to identify pathogenic viruses and understand their potential for dissemination. The research has been outlined in the publication documented in the Accomplishment 1. Progress directly relates to Objective 1, continued study on using nanopore sequencing for genomic and trabscriptomic research related to pathogens’ discovery and whole genome sequencing. Using nanopore sequencing, obtained a preliminary draft genome of alfalfa cyst nematode Heterodera medicaginis, an invasive species recently discovered in North America. Progress relates to Objectives 1 and 2. Initiated study on alfalfa pathobiome, a comprehensive biotic environment that not only includes a diverse community of all disease-causing organisms within the plant, but also defines their mutual interactions and resultant effect on plant health. To date, the concept of pathobiome as a major component in plant health and sustainable production of alfalfa (Medicago sativa L.), is non-existent. In a team work involving international collaboration, the ARS researchers revealed a remarkable abundance of different pathogenic communities associated with alfalfa in the natural ecosystem. Profiling of the alfalfa pathobiome is a starting point to assess known and identify new and emerging stress challenges in the context of plant disease management. In addition, it allows to address the complexity of microbial interactions within the plant host and their impact on the development and evolution of pathogenesis. The research resulted in a publication submitted to the peer-reviewed journal Frontiers in Microbiology. Progress relates to Objectives 1 and 2.

Accomplishments
1. Discovery of alfalfa seed virome to support distribution of healthy crop germplasm to ARS stakeholders. The current scope of knowledge on seed transmission of plant viruses in the agriculturally important forage crop alfalfa (Medicago sativa L.) is limited. Discovering the composition of the alfalfa seed virome and its implications for the distribution of pathogenic viruses has become increasingly important, because seed transmission can provide a source of primary infection and subsequent virus dispersal and disease epidemics in new areas. Therefore, it is critical to gather information on virus-free seed material. In this work, ARS researchers from Beltsville, Maryland, and Prosser, Washington, performed seed screenings of alfalfa germplasm accessions maintained by the USDA ARS National Plant Germplasm System (NPGS) to identify potentially pathogenic viruses and evaluate their prospects for dissemination. The screening showed that the crop’s mature seeds contain a broad range of viruses, some of which were not previously considered to be seed-transmitted. The information gathered will be used to make decisions on whether germplasm distributions need to be scrutinized more carefully and in developing policies that restrict possible dissemination of confirmed plant pathogenic viruses. This is the first study of the alfalfa seed virome carried out by HTS technology. The results were published in the peer-reviewed Virology Journal.

Review Publications
Postnikova, O.A., Irish, B.M., Eisenback, J., Nemchinov, L.G. 2023. Snake River alfalfa virus, a persistent virus infecting alfalfa (Medicago sativa L.) in Washington State, USA. Virology Journal. 20. Article 32. https://doi.org/10.1186/s12985-023-01991-7.
Nemchinov, L.G., Irish, B.M., Grinstead, S.C., Postnikova, O.A. 2023. Characterization of the seed virome of alfalfa (Medicago sativa L). Virology Journal. 20. Article 96. https://doi.org/10.1186/s12985-023-02063-6.