Location: Molecular Plant Pathology Laboratory
2022 Annual Report
Objectives
Objective 1: Enhance understanding of the genetic diversity of plant pathogenic mollicutes (phytoplasmas and spiroplasmas) and their interactions with host plants through genomic, transcriptomic, and metabolomic studies. (NP303, C1, PS1A, PS1B)
Objective 2: Identify molecular markers involved in pathogen genetic diversity, niche adaptation, and pathogenicity. (NP303, C1, PS1A, PS1B)
Sub-objective 2.A: Identify genus-, species-, and lineage-specific multi-locus genomic markers of diverse phytoplasmas associated with diseases of domestic and international importance.
Sub-objective 2.B: Explore and evaluate redox, hormonal, and metabolic markers of pathogenesis for earlier detection and enhanced identification of diverse mollicutes.
Objective 3: Devise new and improved diagnostic tools for the detection and identification of exotic, emerging, and evolving phytoplasmas. (NP303, C1, PS1A, PS1B)
Sub-objective 3.A: Devise rapid and sensitive phytoplasma detection and identification protocols based on pathogen species- and lineage-specific genomic markers.
Sub-objective 3.B: Devise biosensors for early disease diagnosis based on host redox, hormonal, and metabolic signals.
Objective 4: Expand multi-locus and whole-genome sequence information-based classification and systematics of phytoplasmas and spiroplasmas. (NP303, C1, PS1A, PS1B)
Sub-objective 4.A: Construct a multi-locus sequence typing (MLST)-based phytoplasma classification scheme and establish a whole-genome sequence information-based operational metrics for phytoplasma species delineation.
Sub-objective 4.B: Identify genomic features correlated with divergent evolutionary trajectories of plant pathogenic spiroplasmas at differing levels of taxonomic rank.
Approach
The proposed project unites physiology, molecular biology, and genomics in synergistic multidisciplinary research. The goal is to discover and utilize new knowledge to devise and develop new, improved technologies to detect, identify, and classify wall-less bacteria (mollicutes), (noncultivable) phytoplasmas and (cultivable) spiroplasmas that cause economically important plant diseases. The project will discover gene markers of previously unknown phytoplasmas; new strains will be incorporated into our classification scheme, forming new phylogenetic groups, and we will describe/name the new taxa. Small genomes, and evolutionary loss of metabolic functions, make mollicutes ideal models for comparative genomics. Comparative genomics will elucidate genotypic events in the evolution of phytoplasmas and spiroplasmas, and will help establish molecular markers at differing levels of taxonomic rank. Spiroplasma genus-universal and species-specific gene markers will be identified to facilitate spiroplasma identification, and established spiroplasma species will serve as models to distinguish putative species and genera of phytoplasmas. Investigation of physiological and metabolic signals, and gene pathways regulating the oxidative (redox) and hormonal status, will open new avenues for early phytoplasma disease diagnosis - possibly before symptoms appear - and for control of redox sensitive plant pathogenic mollicutes. We will devise a scheme of combined rRNA-ribosomal protein-secY gene sequences to classify closely related phytoplasma strains, and will expand our online program for computer-assisted phytoplasma classification to accommodate automated analysis of diverse functional classes of genes. The new knowledge gained and technologies and tools devised will advance fundamental science, strengthen applied research, enhance disease management, and improve implementation of quarantine regulations worldwide.
Progress Report
This project yields new findings through molecular, physiological, microscopic and omics studies. New genetically distinct phytoplasmas are discovered in diseased plants and potential insect vectors; new genomic and physiological markers are identified to improve the detection and identification of phytoplasmas responsible for emerging and re-emerging diseases. The research elucidates how a bacterial pathogen alters the growth patterns and the architecture of host plants, that is, pathogen-induced misregulation of the meristem switch genes leads to derailment of the genetically preprogrammed fate of plant stem cells. This line of research is of great significance for understanding the pathogenesis of phytoplasmas and provides new concept for early disease diagnosis and symptom management. The research advances will help protect agricultural health, enhance food security, and achieve sustainable agricultural production. The project team seeks and compiles new information regarding emerging diseases in agronomically important crops including vegetables and fruits, as well as ornamentals and forest trees, helping growers and field pathologists in disease diagnosis and management. Work generates new findings in phytoplasma and host plant interactions, and elucidation of the underlying mechanisms how symptoms are caused by inhibited starch breakdown and degradation of dysfunctional chloroplasts. The genomes of several phytoplasmas (including potato purple top phytoplasma) are being sequenced and assembled. In collaboration with scientists in Lithuania, Italy, Canada, Costa Rica, China, Nigeria, and Poland, work is in progress to identify and characterize novel phytoplasmas that infect agriculturally and environmentally important plants. New cutting-edge technologies like CRISPR/Cas12a are employed for specific detection of exotic phytoplasmas. Advances of the research will provide critical information and diagnostic tools to regulatory agencies for devising and implementing quarantine measures. In collaboration with scientists at the University of Illinois, work is conducted to screen, identify and characterize phytoplasmas in potential disease-transmitting insects (especially leafhoppers) collected from natural habitats worldwide; and also, Illumina and Sanger sequencing are being performed to identify the potential plant hosts of leafhopper harboring phytoplasmas. Work continues to refine whole-genome information-based criteria for phytoplasma species delineation and for multi-locus genotyping-based phytoplasma classification.
Accomplishments
1. Decoded the genome of a phytoplasma responsible for Columbia Basin potato purple top disease. The Columbia Basin potato purple top (CBPPT) phytoplasma is a cell wall-less bacterium responsible for the potato purple top that repeatedly occurred in the Pacific Northwest region of the United States. The CBPPT phytoplasma has a broad plant host range and is capable of infecting potato, tomato, broccoli, carrot, radish, and many other vegetable crops, inflicting economic losses. In order to understand how the CBPPT phytoplasma causes diseases in host plants, ARS researchers at the Beltsville Agricultural Research Center decoded the genome of the agricultural pest. This accomplishment will help answer what pathogenic factors the CBPPT phytoplasma possesses and how these factors weaken plant defense, leading to diseases. The accomplishment will also help identify molecular targets for disease control.
2. Identified molecular markers for differentiating a distinct phytoplasma lineage responsible for grapevine Flavescence Dorée disease in Central Italy. Flavescence Dorée (FD) is the most threatening grapevine yellows disease in Europe. Despite strict control measures, alarming signs of the spread of the disease in viticultural areas continue to be detected. FD is attributed to infection by a cell wall-less bacterium termed FD phytoplasma. In collaboration with scientists in Italy, ARS researchers at the Beltsville Agricultural Research Center conducted multi-gene comparative analysis on FD phytoplasma strains identified from vineyards in Central Italy. The study found that the FD phytoplasma strains in the region constitute a highly homogeneous lineage whose collective genotype of the lineage is different from previously characterized FD phytoplasmas in Europe. Molecular markers were identified for distinguishing this lineage from other FD phytoplasma lineages. Such molecular markers provide clues to understanding the genetic relationships among different FD phytoplasma lineages present in Europe and are useful for searching potential vectors and reservoirs involved in the spread of the pathogen. This accomplishment is important to viticulture industry and plant pathologists for grapevine disease diagnosis and management. The accomplishment is also important to regulatory agencies for implementing control measures to prevent exotic phytoplasmas from coming into the U.S.
3. Proposed new and improved criteria for Candidatus Phytoplasam species and reviewed the history of phytoplasma taxnomy. In 2022, the 2004 guidelines for demarcating phytoplasma species were revised and published in International Journal of Systematic and Evolutionary. However, some provisions in the revised guidelines lack clarity and precision. ARS scientists, located in Beltsville, Maryland, proposed new and improved criteria for naming new phytoplasma species and provided detailed rules and clear instructions for researchers to follow. In addition, the ARS scientists also reviewed the latest progress in phytoplasma nomenclature, classification, and identification, the three major aspects of phytoplasma taxonomy. This accomplishment will not only help the regulatory agencies to implement border controls and prevent the spread of quarantine pests, but also help the researchers to diagnose, characterize, and identify phytoplasmas in a timely and accurate manner.
