Location: Molecular Plant Pathology Laboratory
2024 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 reports research discovered through new findings in molecular, physiological, microscopic, and omics studies. Genetically distinct phytoplasmas are discovered in diseased plants and potential insect vectors, leading to the identification of new genomic and physiological markers that enhance the detection and identification of phytoplasmas responsible for emerging and re-emerging diseases. The research reveals how bacterial pathogens alter growth patterns and the architecture of host plants, with pathogen-induced mis regulation of meristem switch genes causing deviations from the genetically preprogrammed fate of plant stem cells. This study advances our understanding of phytoplasma pathogenesis by uncovering the molecular mechanisms behind pathogen manipulation of plant development. It also introduces new concepts for early disease diagnosis and symptom management, offering potential improvement in control strategies. The research advancements will protect agricultural health, enhance food security, and promote sustainable agricultural production. The project team gathers and compiles new information on emerging diseases in agronomically important crops, including vegetables, fruits, ornamentals, and forest trees, aiding growers and field pathologists in disease diagnosis and management. The genomes of several phytoplasmas, including the potato purple top phytoplasma, are sequenced and assembled. In collaboration with scientists from Lithuania, Italy, Canada, Costa Rica, China, Nigeria, Poland, Taiwan, and Jordan, ongoing efforts focus on identifying and characterizing novel phytoplasmas that infect agriculturally and environmentally essential plants. The project also explores cutting-edge technologies like CRISPR/Cas12a to detect and differentiate exotic phytoplasmas. Research advancements will provide critical information and diagnostic tools to regulatory agencies, aiding in the development and implementation of quarantine measures. Efforts continue to refine whole-genome information-based criteria for phytoplasma species delineation and multi-locus genotyping-based phytoplasma classification. Sequencing and comparative genomic analysis of complete phytoplasma genomes are in progress. The genome assembly and differentiation of closely related phytoplasma strains in the elm yellows phytoplasma group (16SrV) are being optimized. Artificial Intelligence (AI)-based diagnosis of phytoplasma diseases is in progress. This detection tool will help growers diagnose and manage phytoplasma diseases in a timely manner, minimizing yield losses and securing farmers' livelihoods.
Accomplishments
1. Web-based tool, iPhyDSDB, for the early detection and management of phytoplasma diseases. Phytoplasmas are intracellular bacteria that cause significant economic losses by infecting numerous plant species. Early and rapid diagnosis is crucial to prevent disease spread, primarily through early symptom recognition by farmers and growers. To aid in this effort, ARS scientists in Beltsville, Maryland, retrieved nearly 35,000 phytoplasma sequence entries from the NCBI nucleotide database, identifying about 950 plant species associated with phytoplasma diseases. Symptomatic images of these plants were manually curated to create the Phytoplasma Disease and Symptom Database (iPhyDSDB), implemented with a web-based interface. The database/website features nearly 1200 symptomatic images, links to original sources, and descriptions of typical phytoplasma symptoms. iPhyDSDB allows efficient querying by plant host and symptom type, aiding users in comparing, identifying, and diagnosing phytoplasma-related diseases. This resource enhances understanding and management of phytoplasma infections, benefiting farmers, growers, researchers, and educators by improving early detection and disease management.
2. Morphological, physiological, and transcriptomic alterations that drive the formation of little leaves in phytoplasma-infected plants. Phytoplasmas are small plant bacteria that cause a variety of symptoms in different plant species, including witches'-broom (WB), which is characterized by clusters of small, densely packed leaves. However, the exact mechanism behind these little leaf formations remains unknown. ARS scientists in Beltsville, Maryland, and researchers in Tai'an, Shandong, China, conducted morphological, physiological, and transcriptomic analyses to study the effects of phytoplasma infection in sweet cherry trees. The findings revealed premature leaf senescence, disrupted sugar metabolism, imbalanced hormone levels, and impaired ribosome biogenesis, all contributing to little leaf development. Additionally, the upregulation of marker genes that induce premature leaf senescence and downregulation of genes involved in ribosome biogenesis highlighted the complex relationship between leaf development and pathogen-induced stress responses. The study provides valuable insights into the molecular mechanisms behind little leaf formation and suggests potential strategies for managing phytoplasma diseases in sweet cherry cultivation. This research will be of interest to scientists and students focused on pathogen-induced plant stress, pathogen-host interactions, and high-throughput omics studies.
3. Endoplasmic reticulum as a key battleground between phytoplasma aggression and host plant defense. Phytoplasmas are small and intracellular bacteria that depend on host nutrients for survival and replication. The endoplasmic reticulum (ER) plays a crucial role in cellular processes and is a prime target for many intracellular pathogens. ARS scientists in Beltsville, Maryland, revealed that phytoplasma infection disrupts ER balance, causing abnormal accumulation of ER-resident proteins and morphological changes in the ER network. The finding suggests an interplay between ER stress, the unfolded protein response (UPR), and phytoplasma infection. The ER serves as a battleground where phytoplasmas exploit plant resources while the plant activates defense mechanisms. Understanding these interactions can help develop new strategies to manage phytoplasma diseases. These insights are valuable for researchers in plant pathology, molecular biology, and crop protection, particularly those studying phytoplasma diseases and plant-pathogen interactions.
4. Differential symptomology, susceptibility, and titer dynamics in periwinkle and tomato plants infected with the same phytoplasma strain. Phytoplasmas are pathogenic bacteria that infect various plants, causing significant global economic losses. Understanding symptom severity, disease progression, and phytoplasma concentration is crucial for effective disease management. ARS scientists in Beltsville, Maryland, employed seedling grafting to study the infection dynamics of potato purple top (PPT) phytoplasma in periwinkle and tomato. Higher phytoplasma concentrations were observed in the early infection stage, characterized by abnormal reproductive growth, rather than during the later stage with excessive vegetative growth. Additionally, periwinkles showed significantly higher phytoplasma loads compared to tomatoes, highlighting distinct plant responses and varying susceptibility to infection. These findings can enhance agricultural disease management and our understanding of plant-pathogen interactions, benefiting researchers in plant pathology, physiology, and horticulture.
5. Third-generation sequencing-based diagnostic tool for rapid and accurate detection and identification of grapevine flavescence dorée phytoplasma. Flavescence dorée (FD) is the most severe and incurable disease affecting European grapevines. The FD phytoplasma (belonging to the 16SrV classification group) is listed as a quarantine pathogen in many countries due to its importance for the economy and international trade. However, efficient diagnostic tools are lacking for the rapid and accurate detection and identification of FD phytoplasma. As a result, FD-infected vines are rarely intercepted at country ports of entry. ARS scientists in Beltsville, Maryland, investigated the feasibility and utility of the MinION Nanopore high-throughput sequencing technology as a diagnostic tool and developed a multi-locus sequence typing scheme based on four housekeeping genes. Using the MinION sequencing platform, European FD strains and closely related non-FD domestic strains within the 16SrV group were sequenced. Despite their close phylogenetic relationship, exotic FD and domestic non-FD strains were successfully distinguished. The findings highlight the potential of this methodology for enhanced phytoplasma monitoring in viticulture and agriculture. Findings from this research provide valuable insights into research scientists, plant disease diagnosticians, and extension personnel. Moreover, the study's outcomes can be extended to identifying other exotic phytoplasmas that could adversely affect U.S. agriculture.
6. Novel 'Candidatus Phytoplasma sacchari’-related strain affecting silver bluestem perennial grass in Texas. In 2002, silver bluestem plants exhibiting significant yellowing of leaf blades and general decline were observed in a grassland near Pipe Creek, Texas. This prompted an investigation conducted by an ARS scientist in Beltsville, Maryland, and collaborators from Texas A&M University, San Antonio, Texas. Molecular identification revealed the diseased silver bluestem plants were infected by a phytoplasma strain closely related to 'Candidatus Phytoplasma sacchari', which is associated with sugarcane grassy shoot disease, one of the most destructive diseases of sugarcane. The finding marks the first report of such infection in silver bluestem grass, making it a new host for this phytoplasma strain and the first occurrence in the Americas. This discovery holds implications for sugarcane producers, research scientists, and plant disease management professionals, highlighting the need to study the epidemiology of this disease, including insect vector transmission and genetic diversity of phytoplasma strains.
7. Elm yellows phytoplasma-infected Virginia creeper vines in Maryland. In 2002, Virginia creeper vines displaying yellowing and premature leaf reddening were found in Lanham, Maryland. An elm yellows phytoplasma strain (16SrV) associated with symptomless Virginia creepers was previously identified in southern Florida. Even though Virginia creepers are not economically significant, these vines can harbor phytoplasmas that might spread to other agronomically important crops via insect vectors. ARS scientists in Beltsville, Maryland, in collaboration with researchers in APHIS, detected the presence of phytoplasma in diseased Virginia Creeper plants and molecularly characterized the pathogen, which belongs to the 16SrV group. The current research revealed that this Maryland phytoplasma strain, while distinct, shares molecular traits with strains associated with elm trees in the northeastern United States, alder trees in Washington, and grapevines in Europe. This research provides a foundation for future studies and will be valuable for research scientists, plant disease diagnosticians, and extension personnel involved in plant disease management.
8. Phytoplasma classification and identification tools. Phytoplasmas are cell wall-less bacteria that infect vascular plants and are responsible for numerous diseases in agriculturally and environmentally important plants worldwide. Since phytoplasmas cannot be cultured in laboratories, their characterizations mainly rely on DNA fingerprinting. iPhyClassifier is a user-friendly platform for real-time identification and classification of known and discovery of new phytoplasmas. The online tool performs computer-simulated DNA fingerprinting and genetic identity analyses, assisting users in making informed decisions regarding the identity and classification status of phytoplasmas under their study. The virtual RFLP gallery provides DNA fingerprinting profiles of established phytoplasma groups and subgroups for comparative studies of diverse phytoplasmas. ARS scientists in Beltsville, Maryland, made a major update to the phytoplasma DNA database, expanded the existing classification scheme, and enhanced the functions of phytoplasma species assignment and group/subgroup classification.
9. Developments in phytoplasma taxonomy, including identification, classification, and nomenclature. Phytoplasmas are small bacteria that infect nearly one thousand plant species and are responsible for numerous diseases affecting agriculture and the environment. These disease-causing bacteria lack a cell wall, live inside the phloem of infected plants, and are spread among vulnerable plants by insects. Precise identification and classification of widely divergent phytoplasmas are essential to accurate disease diagnosis and proactive epidemic management. Since phytoplasmas cannot be cultivated in the laboratory, genomic sequence analysis (DNA fingerprinting) is the best way to distinguish them from one another. ARS scientists in Beltsville, Maryland, critically reviewed the recent advances and challenges in phytoplasma taxonomy, which deals with identification, classification, and nomenclature (providing proper scientific names to each species). This accomplishment provided plant doctors and regulatory agencies with updated information regarding phytoplasma detection, identification, and classification and addressed important issues for further improvement of the existing phytoplasma taxonomic system.
Review Publications
Shao, J.Y., Zhao, Y., Wei, W., Vaisman, I. 2024. AGRAMP: Machine learning models for predicting antimicrobial peptides against phytopathogenic bacteria. Frontiers in Microbiology. 15:1304044. https://doi.org/10.3389/fmicb.2024.1304044.
Ivanauskas, A., Inaba, J., Zhao, Y., Bottner-Parker, K.D., Wei, W. 2024. Differential symptomology, susceptibility, and titer dynamics manifested by phytoplasma-infected periwinkle and tomato plants. Plants. 13:787. https://doi.org/10.3390/plants13060787.
Inaba, J., Kim, B., Zhao, Y., Jansen, M.A., Wei, W. 2023. The endoplasmic reticulum is a key battleground between phytoplasma aggression and host plant defense. Cells. https://doi.org/10.3390/cells12162110.
Bottner-Parker, K.D., Qiao, K., Huang, W., Zhao, Y., Yang, Z., Cai, H., Wei, W. 2024. Momordica charantia is a novel host of 'Candidatus Phytoplasma malaysianum'-related strains associated with bitter melon stem fasciation disease in China. Plant Disease. https://doi.org/10.1094/PDIS-05-24-0971-PDN.
Rios, D., Ueckert, J., Ong, K., Barillas, J.R., Costanzo, S. 2024. First report of a ‘Candidatus Phytoplasma sacchari’-related strain associated with yellowing and decline of Silver Bluestem in Texas, U.S.A. Plant Disease. https://doi.org/10.1094/PDIS-03-24-0524-PDN.