Location: Molecular Plant Pathology Laboratory
2024 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
This is the final report for the Project 8042-21000-300-000D, which has been replaced by new Project 8042-21500-003-000D. For addtional information, see the new project report. Under the old project, an extensive knowledge on new and emerging organisms infecting alfalfa and on the genetic basis of host responses to some of them was generated. Multiple projects on global transcriptomic studies of alfalfa responses to stress were undertaken and completed. Viral infections of alfalfa are widespread in all major cultivation areas and their impact on alfalfa production is often underestimated. More than 45 distinct viruses have been identified in alfalfa field samples supplied by our collaborators, averaging 10 viruses per individual alfalfa plant. In addition to viruses that were known to cause diseases in alfalfa, such as alfalfa mosaic virus, pea streak virus, bean leafroll virus, etc, many potentially new viruses were identified and characterized, including emerging cyto-and nucleorhabdoviruses, Snake River alfalfa virus, alfalfa vein mottling virus, cherry virus Trakiya that was never reported in alfalfa prior to our research, lucerne transient streak virus that had not been described in alfalfa in the U.S., alfalfa-associated picorna-like virus 2 and many others. Field alfalfa samples were found to be co-infected with bacterial and fungal pathogens, some of which were not previously reported in the U.S. alfalfa, such as Alternaria alternata and Bipolaris spp. For the first time, alfalfa seed virome was deciphered using high throughput sequencing, revealing that the crop’s mature seeds contain a broad range of viruses, some of which were not previously considered to be seed-transmitted. For the first time, endogenous viral sequences present in alfalfa genome were discovered that could potentially acquire functional roles in alfalfa’s normal growth, organ development, and response to environmental stresses. Biological insights into mechanisms of alfalfa resistance to economically important root lesion nematode (RLN) Pratylenchus penetrans were proposed and gene-candidates identified. Virulence factors secreted by the root lesion nematode and burrowing nematode, Radopholus similis that are critical for the nematodes parasitism and could be used in biological control of these pathogens were identified. Novel, previously undescribed viral species infecting plant parasitic nematodes were discovered providing an entirely new resource for potential biological control applications. A comprehensive repertoire of more than eight thousand of alfalfa transcription factors was assembled, upgraded and is currently available as a web resource AlfalfaTFDB. These and other accomplishments listed below in more detail, resulted in over 20 research publications in peer-reviewed journals for the reporting period. The replacement project, which started on June 6, 2024, advances toward the next phase of this research: study of the alfalfa pathobiome, a comprehensive biotic environment that includes a diverse community of all disease-causing organisms within the plant, defines their mutual interactions and the resultant effect on plant health. Fulfilling Objectives of the new project will provide insights into the complexity of pathogenic communities associated with alfalfa, their collective impact on disease development, and the nature of the plant’s resistance to multiple interactive coinfections. Gaining this knowledge will result in recommendations for stakeholders, industry, and regulatory agencies for protecting the crop’s health. It will also lead to improved alfalfa fitness and enhance food and energy security while reducing the risks of crop failure.
Accomplishments
1. Identification of target genes for control of plant parasitic nematodes. Plant parasitic nematodes (PPN) are distributed worldwide and cause severe economic damage to thousands of plant species, including alfalfa. Little is known about the molecular mechanisms used by nematodes to infect such a wide range of host plants. ARS researchers in Beltsville, Maryland, identified gene-candidates associated with alfalfa resistance to Pratylenchus penetrans, a migratory species that attacks a broad range of crops. They proposed that significant accumulation of tannin-like deposits in root cells of the resistant alfalfa cultivars could be a key to nematode resistance. ARS researchers in Beltsville, Maryland, and a team of domestic and international scientists identified and functionally characterized a diverse repertoire of effector proteins involved in parasitism of two agriculturally important PPN: the burrowing nematode Radopholus similis and migratory root lesion nematode Pratylenchus penetrans. The research provided valuable data on nematode parasitism and potential strategies for the development of control measures using identified nematode effector genes as targets. The results of these studies have been a key contribution to the nematology research community and are currently used by scientists in the United States, United Kingdom, and Portugal.
2. Genome-wide identification of alfalfa transcription factors and an upgrade of a web resource center AlfalfaTFDB. 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 (https://plantpathology.ba.ars.usda.gov/alfalfa/table2.php). 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.
3. Discovery of RNA virome of nematodes and other soil-inhabiting organisms. Nematodes are among the most abundant animals on Earth. While the assemblage of viruses infecting many arthropods is becoming available, the virome associated with nematodes remains mostly unexplored. In this study, ARS researchers in Beltsville, Maryland, bridged this gap by discovering more than 140 new virus species and demonstrating a remarkable diversity of RNA viruses infecting nematodes and other soil-inhabiting hosts. These findings provided new insights into the manifold diversity of viruses associated with micro-scale soil environment and their important roles in the structural and evolutionary aspects of soil communities. Discovery of new viruses in nematodes can also lead to biotechnological applications for control of plant parasitic nematodes in alfalfa and other crops, and for genetic modification of PPN. As of today, genetic modification of plant-parasitic nematodes is in its infancy due to the large technical restrains.
4. Discovery and characterization of new and emerging viruses in alfalfa. Viral infections of alfalfa are widespread in all major cultivation areas and their impact on alfalfa production is often underestimated. ARS researchers in Beltsville, Maryland, Prosser, Washington, and Salinas, California, discovered characterized at the molecular level, and developed diagnostic methods for identification of many different viruses infecting alfalfa in the United States. International collaboration with leading plant virologists in France and Argentina delineated the latest data on etiology and distribution of new and emerging viral diseases in alfalfa. International collaboration with the Institute of Basic Biological Problems, Russian Academy of Sciences, identified in alfalfa genome multiple integrated sequences derived from plant pararetroviruses of the family Caulimoviridae. This discovery sheds light on the evolution of the host and indicates that endogenous viral sequences may potentially be a source and reservoir of virus infections in alfalfa. The research on alfalfa virome demonstrated that in agricultural settings alfalfa plants are coinfected with numerous viruses representing an omnipresent background for all other host-pathogen interactions. Molecular diagnostics tools and genomic data developed during the course of these studies are freely available for plant pathologists around the world and scientists working in the field of alfalfa improvement. The discovery of some viruses raised critical issues in quarantine regulations.
5. 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 in Beltsville, Maryland, and Prosser, Washington, performed seed screenings of alfalfa germplasm accessions 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.
6. Discovery of the composition of alfalfa pathobiome in commercial fields. ARS researchers in Beltsville, Maryland, initiated a study on the composition of the alfalfa pathobiome. Through the recent advances of modern high-throughput sequencing technologies, the "one microbe, one disease" dogma is being gradually replaced with the principle of the "pathobiome", a comprehensive biotic environment that includes a diverse community of all disease-causing organisms within the plant and defines their resultant effect on plant health. Prior to this work, the concept of pathobiome as a major component in plant health and sustainable production of alfalfa was non-existent. ARS researchers in Beltsville, Maryland, in collaboration with international partners, revealed a remarkable abundance of different pathogenic communities associated with alfalfa in the natural ecosystem. They also estimated host reaction to a consortium of pathogenic microorganisms by profiling gene expression patterns shaped by the plant pathobiome in the field environment. This research is a starting point leading to the exploration of alfalfa's microbial environment in the field. It provides an understanding of how the pathobiome promotes disease, creates an environment for synergisms between microbial pathogens, and contributes to the origination and distribution of novel pathogens.
Review Publications
Nemchinov, L.G., Postnikova, O.A., Wintermantel, W.M., Palumbo, J.C., Grinstead, S.C. 2023. Alfalfa vein mottling virus, a novel potyvirid infecting Medicago sativa L. Virology Journal. 20:284. https://doi.org/10.1186/s12985-023-02250-5.
Nemchinov, L.G., Grinstead, S.C. 2024. Complete genome sequence of alfalfa-associated picorna-like virus 2. Microbiology Resource Announcements. 13. Article e01052-23. https://doi.org/10.1128/mra.01052-23.
Nemchinov, L.G., Irish, B.M., Uschapovsky, I.V., Grinstead, S.C., Shao, J.Y., Postnikova, O.A. 2023. Composition of the alfalfa pathobiome in commercial fields. Frontiers in Microbiology. 14: Article e1225781. https://doi.org/10.3389/fmicb.2023.1225781.
Nemchinov, L.G., Irish, B.M., Grinstead, S.C. 2024. First report of novel Medicago trirhavirus 1 infecting alfalfa in Washington State, USA. Plant Disease. https://doi.org/10.1094/PDIS-05-24-1132-PDN.
