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United States Department of Agriculture

Agricultural Research Service

Research Project: Genome Sequence-Based Strategies for Detection & Identification of Plant Pathogenic Phytoplasmas & Spiroplasmas, & Vascular Walled Bacteria

Location: Molecular Plant Pathology

2013 Annual Report

1a.Objectives (from AD-416):
Objective 1: Identify molecular biomarkers useful for detection and identification of phytoplasmas and plant pathogenic spiroplasmas at clade, group, subgroup, species, pathotype, and strain levels.

Objective 2: Expand and refine the current gene-based phytoplasma classification system.

Objective 3: Establish a framework toward DNA barcoding of plant pathogenic mollicutes, a system for multilocus genotyping, strain description, and eventual formal molecular taxonomy of spiroplasmas and phytoplasmas.

1b.Approach (from AD-416):
The goal of this project is to discover and utilize new knowledge to devise and develop new, improved technologies to detect, identify, and classify phytoplasmas and spiroplasmas (mollicutes) that cause economically important plant diseases. We will identify highly conserved genes, moderately sequence-variable genes, and rapidly evolving genes, across phylogenetically divergent lineages. Small genomes, and evolutionary loss of genes and metabolic functions, make mollicutes ideal models for comparative genomics. Comparative genomics will elucidate genotypic events in evolutionary emergence of the phytoplasma clade, and will help establish molecular markers for genus-level identification and criteria for formal genus Phytoplasma taxonomy. Established species of spiroplasmas will serve as models for assessing inter- and intra-species sequence variability and for delineating gene sets to be evaluated as a conceptual framework to distinguish putative species and genera of phytoplasmas. Phytoplasmal genetic SNPs and sequences of rapidly evolving genes, including lineage-specific pathogenicity genes, will provide unique molecular biomarkers for improved detection and identification. A previously developed online program for computer-assisted phytoplasma classification will be expanded to accommodate automated analysis of diverse functional classes of genes. Subsets of multiple gene sequences will be assembled to configure “constellations” of diverse molecular biomarkers for use in constructing DNA barcodes for phytoplasma identification, for detection and classification of new phytoplasmas in emerging diseases, and for use as molecular descriptors in a formal Phytoplasma spp. taxonomy. The new knowledge gained and the technologies and tools devised will advance fundamental science, strengthen applied research, enhance disease management, and improve implementation of quarantine regulations worldwide.

3.Progress Report:
While bringing to light previously unknown bacterial pathogens and pinpointing molecular markers for their detection and identification, we gained new knowledge that is opening unexpected insights into how bacteria cause disease and how plants react to infection, The former work is needed to produce disease-free germplasm and construct and implement new quarantine regulations, and the latter is needed to devise effective methods and tools to control diseases that damage or threaten to damage U.S. agriculture. Our discovery this year that bacterial infection can subvert the normal fate of plant stem cells led us to new concepts and theories concerning plant responses to bacteria, and to visualization of completely new avenues for protecting plants against the effects of bacterial infections. Plans were formulated to follow these leads next year as resources permit. Phytoplasma classification/taxonomy is in early stages of development and needs better criteria for recognizing both domestic and exotic species. Using new taxonomic concepts that we conceived and proposed in naming ‘Candidatus Phytoplasma pruni’, cause of X-disease of stone fruits, we described and named the exotic, ‘Ca. Phytoplasma solani’, perhaps the most economically damaging phytoplasma in Europe. We continued improving phytoplasma classification by analyzing three different genes in phytoplasmas related to ‘Ca. Phytoplasma pruni’, and have applied the new knowledge to discover, identify, and classify phytoplasmas causing serious diseases of potato in Russia and determining their insect vectors, as well as to unravel diseases of cranberry and blueberry in eastern U.S. We gained new information through next generation sequencing and decoding the genomes of multiple phytoplasma species. These data will be used to identify better molecular markers of species, to learn how phytoplasmas cause disease, to understand phytoplasma-plant interactions, and to devise targeted molecular methods for reducing disease severity and stemming disease spread. To further advance emerging phytoplasma taxonomy, we tested our hypothesis that certain protein-coding genes played key roles in evolutionary emergence of new species. The findings are elucidating genomic factors significant in phytoplasma pathogenicity and host range. These findings are being used to understand the North American grapevine yellows (NAGY) disease problem, which we discovered in six states and hypothesize is present from coast to coast. The work is providing tools for assessing disease spread, devising control measures, and aiding identification and targeted control of insect vectors. Our work on decoding the spiroplasma genome progressed to near completion and should have impacts beyond plant pathology, since others have searched for spiroplasmas in vertebrates, and researchers in Asia have found spiroplasmas causing diseases in crustaceans (shrimps and crab). We continued improving web-based customer service and outreach through MPPL’s publically accessible web site, Phytoplasma Resource Center, which was enhanced and expanded this year.

1. Discovered that bacterial infection can derail normal plant development by diverting stem cells from their normal destiny. In higher plants, apical meristems (stem cells) are major determinants of plant morphotype and fertility. We found that a bacterial infection can divert the meristems from their genetically-programmed destiny, thereby altering the pattern of plant growth and development. We identified an array of symptoms in infected plants, and found that each symptom corresponds to a distinct phase in derailment of meristem fate. We unveiled molecular events beneath the pathogen-induced meristem fate derailment and proposed a model to explain the phenomenon. Our findings contribute to understanding plant physiology (growth and development) under both normal and pathological conditions and will aid progress toward effective control of bacterial diseases.

2. Gained insight into the effect of phytoplasma infection on host gibberellin (GA) homeostasis. Many agriculturally important crops are vulnerable to infection by phytoplasmas, which significantly alter the physiology of the plants, which often exhibit symptoms indicative of hormonal disorder. We found that phytoplasma infection in tomato reduces internal levels of gibberellic acid, a naturally-occurring plant hormone, and found that the reduced level was due to suppression of key genes responsible for the hormone’s synthesis. We discovered that phytoplasma infection triggered de-sensitization of the GA biosynthesis negative feedback regulation. External application of the hormone at early stages of phytoplasma infection compensated for the hormonal loss and reduced severity of disease. The findings offer clues for devising novel practical approaches to control these bacterial diseases.

3. Elucidated the role of gibberellic acid in plant defense against a bacterium, phytoplasma. Based on our observation that phytoplasma infection causes disruption of GA homeostasis in tomato and that can be partially reversed by exogenous application of GA, we hypothesized that GA plays a role in plant defense. By examining expression profiles of genes involved in GA signaling, we discovered that GA promotes production of other hormones that work together to enhance the plant’s defense system. Following GA application and inoculation of plants, there was coordinated down-regulation of the GA signaling and growth repressor gene (GAI) and up-regulation of genes involved in salicylic acid synthesis (ICS1), signaling (NIM1), and downstream defense responses (PRP-1). We further found that the differential gene regulation was correlated with increased activities of defense-related enzymes ß-1,3-glucanase (GLU) and chitinase (CHI). The findings present new opportunities for understanding plant defenses against bacteria, through studies of the GA signaling network.

4. New concepts and theories on the evolutionary emergence, lineage radiation, and pathogenicity of phytoplasmas. Discovered genes and genomic features that distinguish phytoplasmas from other cell-wall less bacteria. Knowledge from previous analyses opened only a small window on the uniqueness of the phytoplasma genome and its functional implications. Following our earlier discovery of unique genome architecture, we pinpointed phytoplasma-unique genes and genes that are essential to a minimal free-living bacteria but absent in phytoplasma genomes. We elucidated metabolic pathways that are undergoing either lineage-specific acquisition or loss, and provided molecular evidence to support our concept of raising the phytoplasma clade to a taxonomic level of family. We presented new concepts and theories on mechanisms giving rise to genetic diversity and the changing landscape of host range and pathogenicity, opening unexpected possibilities for devising novel disease control measures having significance for and beyond phytoplasmas.

5. Completed characterization and molecular differentiation of phytoplasmas causing with disease of blueberry in New Jersey. Recently, the recurrence of blueberry stunt disease became evident in many farms in New Jersey. The phytoplasmas causing the disease had not been previously characterized. A survey, throughout New Jersey, identified two distinct phytoplasmas, one previously unknown, in diseased blueberries in New Jersey. The information and molecular tools developed have facilitated studies on investigation of insect vectors and disease diagnosis and spread. This work will benefit students, research scientists, diagnostic laboratories and extension personnel engaged in disease management, and government agencies that implement quarantine regulations to prevent disease spread domestically and internationally.

6. Completed characterization and classification of phytoplasmas causing diseases of potato in Russia. Potato purple top diseases are widespread in major potato growing regions in the world and cause significant economic damage in potato production, but the identities of the causal pathogens have not been definitively determined. Following seven years of research in several potato growing regions in Russia, we found that the diseases were caused by at least five different phytoplasma species. The disease of potato crops in Russia is due to a complex of diverse phytoplasmas, likely spread by different insect vector species. The findings provide needed new understanding and molecular markers for quarantine agencies to prevent introduction of the exotic phytoplasmas into new geographic regions including the U.S., and should aid in the formulation of strategies to minimize potato crop losses wherever the diseases occur.

7. Web-based customer service and outreach. We reconfigured the interactive iPhyClassifier program, MPPL’s publically accessible online tool for phytoplasma classification and taxonomy. Now, the dynamically drawn images superimpose the in silico calculated RFLP patterns of both sequence heterogeneous rRNA operons. We also expanded the underlying database. We added new information concerning grapevine diseases, which have become of worldwide concern. The online functions, assembled under MPPL’s Phytoplasma Resource Center, are used by scientists, students, professors, quarantine agencies, and diagnostics companies to aid their identification and classification of phytoplasmas worldwide.

Review Publications
Davis, R.E., Zhao, Y., Dally, E.L., Lee, I., Jomantiene, R., Douglas, S.M. 2013. 'Candidatus Phytoplasmas pruni', a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes. International Journal of Systematic and Evolutionary Microbiology. 63:766-776.

Lee, I., Bottner-Parker, K.D., Zhao, Y., Bertaccini, A., Davis, R.E. 2012. Differentiation and classification of phytoplasmas in the pigeon pea witches'-broom group (16SrIX): an update based on multiple gene sequence analysis. International Journal of Systematic and Evolutionary Microbiology. 62:2279-2285.

Zhao, Y., Wei, W., Lee, I., Shao, J.Y., Suo, X., Davis, R.E. 2012. The iPhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment. Methods in Molecular Biology. 938:329-338.

Bagadia, P.G., Polashock, J.J., Bottner-Parker, K.D., Zhao, Y., Davis, R.E., Lee, I. 2013. Characterization and molecular differentiation of 16SrI-E and 16SrIX-E phytoplasmas associated with blueberry stunt disease in New Jersey. Molecular and Cellular Probes. 27:90-97.

Ding, Y., Wu, W., Wei, W., Davis, R.E., Lee, I., Hammond, R., Sheng, J., Shen, L., Jiang, Y., Zhao, Y. 2013. Potato purple top phytoplasma-induced disruption of gibberellin homeostasis in tomato plants. Annals of Applied Biology. 162:131-139.

Ding, Y., Wei, W., Wu, W., Davis, R.E., Jiang, Y., Lee, I., Hammond, R., Sheng, J., Shen, L., Zhao, Y. 2013. Role of gibberellic acid in tomato defense response to potato purple top phytoplasma infection. Annals of Applied Biology. 162(2)191-199.

Nejat, N., Vadamalai, G., Davis, R.E., Harrison, N.A., Sijam, K., Dickinson, M., Abdullah, S., Zhao, Y. 2012. Candidatus Phytoplasma malaysianum, a novel taxon associated with virescence and phyllody of Madagascar periwinkle (Catharanthus roseus). International Journal of Systematic and Evolutionary Microbiology. 63:540-548.

Martini, M., Lee, I. 2012. PCR and RFLP analyses based on the ribosomal protein operon. In: Dickinson, M., Hodgetts, J., editors. Springer Protocols: Methods in Molecular Biology. New York, NY: Humana Press. p. 173-188.

Duduk, B., Paltrinieri, S., Lee, I., Bertaccini, A. 2012. Nested PCR and RFLP analysis based on the 16S rRNA gene. In: Dickinson, M., Hodgetts, J., editors. Springer Protocols: Methods in Molecular Biology. New York, NY: Humana Press. p. 159-172.

Last Modified: 4/19/2014
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