Location: Molecular Plant Pathology Laboratory
2022 Annual Report
Objectives
Objective 1: Discover new genomic and physiological biomarkers potentially useful for improving detection and identification of phytoplasmas and plant pathogenic spiroplasmas [NP 303; C1, PS1]
• Sub-objective 1A: Identify genomic features correlated with divergent evolutionary trajectories of plant pathogenic spiroplasmas at differing levels of taxonomic rank.
• Sub-objective 1B: Identify multilocus genomic features and molecular markers of phytoplasma-plant host interactions correlated with phytoplasma genetic diversity at differing levels of taxonomic rank.
• Sub-objective 1C: Identify key primary and secondary metabolites involved in early stages of pathogenesis that may have global effects on disease resistance through either their bioactive nature or redox-status of the microbiome.
• Sub-objective 1D: Identify, and characterize multilocus genomic markers of, phytoplasmas carried by vectors and nonvector phloem-feeding insects in diverse agricultural and natural ecosystems.
Objective 2: Expand, refine, and advance gene-based phytoplasma and spiroplasma taxonomy and classification systems; evaluate new genomic and physiological biomarkers [NP 303; C1, PS1]
• Sub-objective 2A: Detect and identify new phytoplasmas associated with emerging diseases; update the ribosomal RNA gene-based phytoplasma classification scheme; enhance the functionality of the iPhyClassifier.
• Sub-objective 2B: Evaluate multilocus genomic features correlated with divergent evolutionary trajectories of phytoplasmas and spiroplasmas for enhanced detection, identification, and classification of exotic and emerging strains.
• Sub-objective 2C: Evaluate metabolic markers of pathogenesis for earlier detection, and enhanced identification, and classification of exotic and emerging phytoplasmas.
• Sub-objective 2D: Incorporate into the gene-based phytoplasma classification system additional molecular markers of evolutionarily conserved house-keeping genes.
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 is the final report for project 8042-22000-306-000D, "Genome-Based Strategies and Physiological Biomarkers for Detection and Identification of plant Pathogenic Phytoplasmas and Spiroplasmas” which ended March 25, 2022. New NP303 OSQR approved project 8042-22000-320-00D, entitled "Omics-Based Approach to Detection, Identification, and Systematics of Plant Pathogenic Phytoplasmas and Sprioplasmas" has been established. This five-year project continues to yield new findings that advance science and new technologies and improve pathogen detection and identification. Accomplishments of the project have made significant contributions to and have high impacts on the prevention and management of phytoplasma diseases that affect agriculture and enviroment. Novel phytoplasmas continue to be detected, identified and classified; The rapid and early detection methods were devised to dignose domestic and exotic phytoplasmas, especially quarantine pests, based on the new cutting edge technology such as CRISPR/Cas12a technology; new knowledge is gained about how phytopathogenic phytoplasmas and spiroplasmas cause diseases and how plants respond to pathogen infection. The research also elucidated how a bacterial pathogen modifies the preprogrammed fate of plant stem cells by altering the expression of meristem switch genes, leading to abnormal growth patterns and the architecture of host plants. This latter line of research is of great significance for understanding the pathogenesis of phytoplasmas and will provide new methods for disease diagnosis and management. The expertise, techniques and pathogen-host plant research model developed in pathogen-host interaction studies will benefit the future research and be applied to the new project plan (8042-22000-320-000D). Continued to characterize and decode the genomes of phytoplasmas, including potato purple top phytoplasma, which is the strain employed in pathogen-plant interaction study. The work will help to identify the potential virulence factors and elucidate the underlying pathogenic mechanisms. Work continued in comparative genomics to determine the differences and similarities between the plant pathogens Spiroplasma kunkelii and Spiroplasma citri, as well as distinguishing features of these plant pathogens compared to non-plant pathogenic species that occur in crustaceans including shrimps and crab and in diverse insects. The findings should contribute to improvement of diagnostic tools, and to understanding the evolutionary adaptation of spiroplasmas to diverse natural habitats. The characterizations of ‘exotic’ phytoplasmas and new phytoplasma species are ongoing and will continue to assist in the implementation of quarantine measures to thwart the spread of the associated diseases. This line of work requires national and international collaborations. In collaboration with scientists in Lithuania, Italy, Canada, Costa Rica, China, and Nigeria, work is in progress to identify and characterize new phytoplasmas that infect agriculturally and environmentally important plants. This work will lead to the identification of molecular markers and improved technologies for specific detection of exotic phytoplasmas and provide critical information to regulatory agencies for devising and implementing quarantine measures. The research advances will help maximize food production, enhance food security, protect agricultural health, and achieve sustainable agricultural development. Work continued to refine whole-genome information-based criteria for phytoplasma species delineation and for multi-locus genotyping-based phytoplasma classification. For examples, whole genome average nucleotide identity (ANI) threshold and coverage of draft genome for demarcating new species. The project team continued to collect, review, and disseminate new information regarding genetically diverse phytoplasmas and the plant diseases they cause, helping growers and field pathologists in disease diagnosis and management. For example, invited by APS compendium editors, the team contributed a chapter on “strawberry phytoplasma disease”. Work continued in the elucidation of phytoplasma-induced transcriptomic and metabolic reprogramming in host plants based on omics studies. Significant progress has been made in unveiling phytoplasma-induced metabolic reprogramming in host plants. New findings contribute to a better understanding of phytoplasma-plant interactions from a nutritional perspective. In collaboration with scientists at the University of Illinois, work is in progress to screen, identify and characterize phytoplasmas in leafhoppers collected from natural habitats worldwide. Multiple new phytoplasma subgroup lineages and geographic variants have been identified from leafhoppers collected from four different countries. Work continued in comparative genomics to determine the differences and similarities between three plant-pathogenic spiroplasmas, Spiroplasma kunkelii, Spiroplasma citri, and Spiroplasma phoeniceum, as well as distinguishing features of these plant pathogens and non-plant pathogenic species. The findings should contribute to the improvement of diagnostic tools and to understanding the evolutionary adaptation of spiroplasmas to diverse natural habitats. Studies reveraled several plant metabolites that were induced in tobacco and tomato in response to prokaryote infection. Progresses have been made to identify similar but mutually distinct metabolites that are induced by pathogens in other food crops. The similarity between the newly identified compounds and those previously found in tobacco and tomato suggests that these novel metabolites may also have bioactivity against pathogens. This indicates that the host metabolic response may be universal and will hopefully lead to new and novel technologies for early pathogen detection.
Accomplishments
1. Applied cutting edge genome editing technology to rapid and sensitive detection of potato purple top phytoplasma. ‘Candidatus Phytoplasma trifolii' is a small plant-pathogenic bacterium capable of infecting many agriculturally important crop species and causing significant economic losses. The bacterium is responsible for repeated outbreaks of potato purple top, potato witches'- broom, and other vegetable crop diseases that occurred along the Pacific Coast of the United States and Mexico. Early detection of the phytoplasma is key to effective management of these crop diseases. In collaboration with scientists at the Pennsylvania State University, ARS researchers at the Beltsville Agricultural Research Center applied cutting edge genome editing technology to phytoplasma detection for the first time. The team identified molecular target unique to 'Candidatus Phytoplasma trifolii' and developed a highly sensitive diagnostic tool for rapid and specific detection of the phytoplasma. This accomplishment provides a new diagnostic tool to research scientists, plant disease diagnosticians, and extension personnel for early detection of ‘Candidatus Phytoplasma trifolii'.
2. Revised the guidelines for naming phytoplasma species. The first guidelines for demarcating phytoplasma species were proposed by IRPCM [Phytoplasma/Spiroplasma Working Team – Phytoplasma taxonomy group] in 2004, which has served nearly 20 years. In recent years, with the rapid development of genomics and sequencing technology, important progress in bacterial taxonomy has been achieved. To embrace these new developments and to incorporate “whole genome” concept to phytoplasma taxonomy, an ARS scientist, in Beltsville, Maryland, in collaboration with worldwide scientists proposed revised guidelines. This accomplishment will provide a new standard for phytoplasma researchers to name new species.
3. Unveiled the underlying mechinism of phytoplasma infection induced witches broom symptom in diseased plants. Phytoplasma infected plants often exhibit witches broom (shoot branching caused by repeated initiation of lateral buds) at the later infection stage. However, it is unclear how these symptoms are caused or developed. ARS scientists in Beltsville, Maryland, discovered that inhibition of starch breakdown and degradation of dysfunctional chloroplasts in source leaves played the key role in premature leaf senescence. Impaired sugar metabolism and disrupted sucrose transport resulted in sucrose reallocation to new sink tissues; in addition to sucrose redistribution, levels of cytokinin (a plant hormone that promotes cell division) were also elevated, promoting the initiation of new lateral buds. The ARS scientists built a working model to explain how phytoplasma infection impairs sugar metabolism and transport in plants, leading to various symptoms including witches broom. This accomplishment advances our knowledge of the underlying mechanism of phytoplasma pathogenesis.
4. Contributed to the APS compendium on phytoplasma diseases in strawberry. Strawberries are a rich source of nutrients such as vitamins, antioxidants, and fiber and are widely cultivated around the globe. However, strawberries are susceptible to a variety of plant pathogens, including phytoplasmas, which are small and cell wall less bacteria. Although phytoplasmas can cause diseases that severely impact the quality and yield of strawberry production, they were not sufficiently described in the past. In the present work, invited by the editors of the Compendium of Strawberry Diseases, ARS scientists in Beltsville, Maryland, collected and assembled new information regarding emerging and reemerging strawberry diseases caused by phytoplasmas. Different strawberry phytoplasma diseases have been reported in sixteen countries. The associated phytoplasma strains are also very diverse, belonging to seven different classification groups. This accomplishment is vital for the extension personnel who are concerned with disease management, as well as for the regulatory agencies to strengthen border controls and prevent the spread of strawberry phytoplasma diseases.
5. Unveiled genetic diversity of phytoplasmas infecting pine trees in Curonian spit, Lithuania. Pine trees play a key role in sand dune stabilization and afforestation in Europe. Pine tree diseases caused by phytoplasmas have been reported in many European countries, however, the insect vectors that transmit the diseases have not been identified yet. ARS scientists, located in Beltsville, Maryland, in collaboration with Lithuanian scientists, collected insects from symptomatic pine trees and adjacent areas. Through insect identification, no known phytoplasma insect vectors were found. Furthermore, a literature search did not find any recognized phytoplasma insect vectors feeding on pine trees. But interestingly, phytoplasmas were detected in known and unknown insects including aphids. Some of newly identified phytoplasmas were genetically distinct from those previously reported, representing a new genetic lineage. This accomplishment will provide useful information for the researchers to identify potential insect vectors of pine phytoplasmas and study genetic diversity of phytoplasmas.
Review Publications
Ivanauskas, A., Valiunas, D., Rimsaite, J., Danilovas, J., Söderman, G., Šneideris, D., Genovaite Žižyte-Eid, M., Wei, W. 2022. New genetically distinct phytoplasmas and insect carriers associated with pine tree disease revealed by a survey in Curonian Spit, Lithuania. Canadian Journal of Forest Research. 52(2): 201-208. https://doi.org/10.1139/cjfr-2021-0152.
Wei, W., Inaba, J., Zhao, Y., Mowery, J.D., Hammond, R. 2022. Phytoplasma infection blocks starch breakdown and triggers autophagic degradation of chloroplasts, leading to premature leaf senescence, sucrose reallocation, and spatiotemporal redistribution of phytohormones. International Journal of Molecular Sciences. 23(3):1810. https://doi.org/10.3390/ijms23031810.
Bertaccini, A., Arocha-Rosete, Y., Contaldo, N., Duduk, B., Fiore, N., Guglielmi Montano, H., Kube, M., Kuo, C., Martini, M., Oshima, K., Quaglino, F., Schneider, B., Wei, W., Zamorano, A. 2022. Revision of the `Candidatus Phytoplasma' species description guidelines. International Journal of Systematic and Evolutionary Microbiology. 72:005353. https://doi.org/10.1099/ijsem.0.005353.
Gasparich, G.E., Bertaccini, A., Zhao, Y. 2020. Candidatus Phytoplasma. Book Chapter. https://doi.org/10.1002/9781118960608.gbm01259.pub3.
Wheatley, M.S., Wang, Q., Wei, W., Bottner-Parker, K.D., Zhao, Y., Yang, Y. 2022. CRISPR/Cas12a-based diagnosis of potato purple top disease complex associated with infection by 'Candidatus Phytoplasma trifolii'-related strains. Plant Disease. https://doi.org/10.1094/PDIS-09-21-2119-RE.
