Location: Molecular Plant Pathology Laboratory
2020 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 project continues to yield new discoveries through genomic, molecular, and physiological studies. New lineages of indigenous and exotic phytoplasmas associated with various crop diseases continue to be identified and classified; new genomic and physiological markers have been identified for improved detection and identification of phytoplasmas responsible for emerging and re-emerging diseases. New hypotheses and knowledge have been developed about how phytoplasma and spiroplasma cause diseases and how plants respond to infection. The research sheds new light on the role of genomic fusion and subsequent DNA recombination events in evolution of phytoplasmas. New insights are gained into how a bacterial pathogen can alter the expressions of meristem switching genes thereby the developmental direction of plant stem cells, leading to abnormal plant growth patterns and architecture. This latter line of research is important to eventual elucidation of underlying mechanisms of phytoplasma pathogenesis and will lead to new methods for disease diagnosis and management. The characterizations of ‘exotic’ phytoplasmas and new phytoplasma species are ongoing and continue to aid in implementation of quarantine measures to prevent their spread. The research progress will aid the cause of protecting agricultural health, enhancing food security, and achieving sustainable agricultural production. Work continued to refine whole-genome information-based criteria for phytoplasma species delineation and for multi-locus genotyping-based phytoplasma classification. In collaboration with scientists in Lithuania, Italy, Canada, Costa Rica, and China, work is in progress to identify and characterize new phytoplasmas that infect agriculturally and environmentally important plants. This work will lead to 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. 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 that occur in crustaceans including shrimp 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. This research has revealed several plant metabolites that were produced in tobacco and tomato in response to prokaryote infection. Work is in progress 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 suggests that the host metabolic response may be universal and will hopefully lead to new and novel technologies for early pathogen detection.
Accomplishments
1. Completed an investigation into spatial and temporal distribution of onion yellows (OY) phytoplasma in its leafhopper vector. Phytoplasmas are cell wall-less, plant pathogenic bacteria transmitted by insects; but their movement and distribution pattern in insect vector remained poorly understood. An ARS scientist at the Beltsville Agricultural Research Center in Beltsville, Maryland, together with collaborators in Japan, employed fluorescent tracing, three-dimensional imaging, and real-time gene quantification technologies to investigate the spatial and temporal distribution and accumulation of OY phytoplasma in its leafhopper vector. The research team identified two major sites of phytoplasma infection and proposed a model for understanding spatiotemporal dynamics of phytoplasmas in insect vectors. The findings from this study will help elucidating transmission mechanism of insect-borne plant pathogens. The information is important to research scientists, students, and university professors who are studying insect-borne pathogens, pathogen-host interactions, and plant disease management.
2. Identified a new phytoplasma that causes floral deformation in bougainvillea plants. Bougainvillea is a popular ornamental plant widely cultivated in tropical, subtropical, and temperate areas around the world. In 2016, blooming bougainvillea plants with abnormal floral arrangement were observed in Cuba. In response to a collaboration request by a scientist at the University of Saskatchewan, Canada, ARS scientists at the Beltsville Agricultural Research Center in Beltsville, Maryland, conducted DNA fingerprint analysis on samples from affected plants and found that the diseased plants were infected by a mycoplasma-like bacterium termed 'Candidatus Phytoplasma asteris'. The research team determined that the disease symptom was a result of pathogen-induced excessive branching and floral bracket proliferation. Phytoplasmal diseases in bougainvillea were previously reported in other countries but the unique bracket proliferation symptom was first noted in bougainvillea. The study described in this communication will be of interest to farmers and extension personnel who are concerned with phytoplasmal disease diagnosis and management.
3. Unveiled both phytoplasma inoculum titer and inoculation timing could significantly influence the symptom development in infected plants. Phytoplasmas are miniature, cell wall-less bacteria capable of inducing an array of symptoms and changing plant morphology. In previous studies, a team of ARS researchers at the Beltsville Agricultural Research Center in Beltsville, Maryland, identified multiple sequentially developed symptoms in phytoplasma-infected tomato plants. In the present work, the team studied the effects of phytoplasma inoculum titer and inoculation timing on host response. The researchers unveiled that both phytoplasma inoculum titer and inoculation timing could significantly change the course of symptom induction and result in different combinations of symptoms. Findings from the study will help understand interactions between host plant and phytoplasmas. The information is important to research scientists, students, and university professors who are studying plant growth and development, pathogen-host interactions, and disease symptom management.
4. Identified a new species within the aster yellows phytoplasma group. Aster yellows (AY) phytoplasma is a collective name of a large group of plant pathogenic bacteria with a broad host range and wide geographic distribution. It has long been suspected that AY phytoplasma group may consist of more than one species. ARS scientists at the Beltsville Agricultural Research Center in Beltsville, Maryland, studied the ecological and genomic features of a wheat-infecting phytoplasma and found the wheat phytoplasma is clearly distinguished from other members of the AY phytoplasma group in transmitting vector, in host response, and in genomic sequence. Results from the study indicate the wheat phytoplasma represent a novel phytoplasma species and provide molecular marker for specific detection of the new species. The findings are important to scientists and extension personnel who are concerned with phytoplasmal disease and are also critical to regulatory agencies for preventing the exotic phytoplasma from being introduced into the U.S.
5. Reviewed and provided new information regarding phytoplasmas and phytoplasma research to scientific community. Phytoplasmas are small, cell wall-less bacteria that cause numerous diseases in agriculturally and economically important plants worldwide. These bacteria invade nutrition-conducting tissues of affected plants and are spread by a special group of insects. Together with a scientist at the Salem State University in Massachusetts and a scientist in Italy, an ARS scientist at the Beltsville Agricultural Research Center in Beltsville, Maryland, reviewed and updated the current status of important aspects of phytoplasma research, including taxonomy, classification, the diseases phytoplasmas cause, the interactions the pathogens have with their hosts, and the molecular makers for phytoplasma disease diagnosis. The updated information will benefit research scientists, plant disease diagnosticians, and extension personnel who are concerned with plant disease management and pathogen genetic diversity. The information is also important to regulatory agencies for enhancing border control to prevent spread of phytoplasmas of international importance.
6. Completed an in-depth examination of issues critical to taxonomic grouping and naming of mycoplasma species. Mycoplasmas and mycoplasma-like organisms are a large group of small bacteria that lack a cell wall, many of which are serious pathogens in agriculture and medicine. Naming and taxonomic grouping of mycoplasma species are governed by the International Code of Nomenclature of Prokaryotes (the Code). Together with 20 other members of the International Committee on Systematics of Prokaryotes' Subcommittee on the taxonomy of Mollicutes, an ARS scientist at the Beltsville Agricultural Research Center in Beltsville, Maryland, examined issues critical to taxonomic grouping and naming of mycoplasma species and joined a consensus to uphold the current mycoplasma nomenclature standard. Compliance with the Code and maintaining nomenclature consistency are crucial to disease diagnosis, pathogen identification, and quarantine implementation. This information will benefit agricultural and medical researchers who are involved in disease diagnosis and pathogen identification.
7. Decoded the genome of a pine-infecting phytoplasma. ‘Candidatus Phytoplasma pini’ is a small bacterium that infects pine trees, causing abnormal shoot proliferation, dwarfing and decline. The bacterium lacks a rigid cell wall, occupies nutrient-conducting vessels of affected plants, and is spread by insect vectors. Initially found in Europe, ‘Candidatus Phytoplasma pini’ was recently found in North America. To learn how the bacterium causes disease in pine, ARS scientists at the Beltsville Agricultural Research Center in Beltsville, Maryland, teaming up with scientists in USDA Animal and Plant Health Inspection Service (APHIS), decoded most of the genes in a North American strain of the bacterium. The findings will facilitate comparative analysis of the bacterium strains present in Europe and North America. The information will interest quarantine agencies, diagnostics laboratories, microbiologists, plant pathologists, entomologists, and the forest industry.
8. Elucidated the role of plant phenolics in redox modulation and plant defense. The plant leaf apoplast, which is the cell wall region just outside the plant cell itself, is the first line of defense against most aerial pathogens. The early plant responses to pathogen infection involve oxidants and antioxidants (reductants) that have major effects on the 'redox' potential of the apoplast. To understand the effects of these responses, ARS scientists at the Beltsville Agricultural Research Center in Beltsville, Maryland, studied two pathogen-induced phenolics, acetovanillone (AV) and acetosyringone (AS), and found they play an important role in regulating the redox potential. Control of the redox potential can determine the outcome of pathogen-host interaction and resistance or susceptibility of the plant tissue. This information will benefit plant scientists and breeders who are devising new strategies to improve disease resistance in plants as well as decrease the use of chemical pesticides and antibiotics.
9. Discovered a universal host metabolic response to pathogen infection. Plant bacterial diseases cause major damage to crops each year, and disease management greatly increases production costs. The plant leaf apoplast, which is the cell wall region just outside the plant cell itself, is the first line of defense against most aerial pathogens. ARS scientists at the Beltsville Agricultural Research Center in Beltsville, Maryland, demonstrated that production of a class of phenolic compounds, first described as a newly discovered plant defense mechanism in tobacco, also occurs in pepper and may represent an inherent property of various crop plants. This mechanism could be used for disease diagnosis prior to symptom development; components of the mechanism could also be enhanced for disease resistance. This information will benefit plant scientists and breeders who are devising new strategies to improve disease resistance in plants as well as decrease the use of chemical pesticides and antibiotics.
Review Publications
Balish, M., Bertaccini, A., Blanchard, A., Brown, D., Browning, G., Chalker, V., Frey, J., Gasparich, G., Hoelzle, L., Zhao, Y. 2019. Recommended rejection of the recently proposed sweeping changes to nomenclature of members of the Mycoplasmatales. International Journal of Systematic and Evolutionary Microbiology. 69/3650-3653. https://doi.org/10.1099/ijsem.0.003632.
Wei, W., Perez-Lopez, E., Zhao, Y. 2020. First report of bougainvillea floral bract proliferation disease in Cuba and its association with phytoplasmal infection. Plant Disease. 104/967. https://doi.org/10.1094/PDIS-09-19-2052-PDN.
Cai, W., Shao, J.Y., Zhao, Y., Davis, R.E., Costanzo, S. 2020. Draft genome sequence of `Candidatus Phytoplasma pini'-related strain MDPP: a resource for comparative genomics of gymnosperm-infecting phytoplasmas. Plant Disease. 104/1009-1010. https://doi.org/10.1094/PDIS-10-19-2127-A.
Wei, W., Zhao, Y., Davis, R.E. 2019. Phytoplasma inoculum titre and inoculation timing influence symptom development in newly infected plants . Phytopathogenic Mollicutes. 9/115-116. https://doi.org/10.5958/2249-4677.2019.00058.6.
Valiunas, D., Jomantiene, R., Ivanauskas, A., Sneideris, D., Zizyte-Eidetiene, M., Shao, J.Y., Zhao, Y., Costanzo, S., Davis, R.E. 2019. Rapid detection and identification of ‘Candidatus Phytoplasma pini’-related strains in Lithuania based on genomic markers present in 16S rRNA and tuf genes. Forest Pathology. 49:e12553. https://doi.org/10.1111/efp.12553.
Baker, C.J., Smith, J.M., Rice, C. 2020. Apoplast redox metabolism: Effect of acetovanillone (apocynin) and acetosyringone, on their in vitro co-oxidation and redox properties. Physiological and Molecular Plant Pathology. 110:101481. https://doi.org/10.1016/j.pmpp.2020.101481.
Baker, C.J., Smith, J.M., Yarberry, A.J., Rice, C. 2020. Induction of apoplast phenolics in pepper (Capsicum annum) leaves in response to pathogenic bacteria. Physiological and Molecular Plant Pathology. 109/101453. https://doi.org/10.1016/j.pmpp.2019.101453.
Koinuma, H., Maejima, K., Tokuda, R., Kitazawa, Y., Nijo, T., Wei, W., Kumita, K., Miyazaki, A., Namba, S., Yamaji, Y. 2020. Spatiotemporal dynamics and quantitative analysis of phytoplasmas in insect vectors. Scientific Reports. 10:1-13. https://doi.org/10.1038/s41598-020-61042-x.
