Location: Molecular Plant Pathology Laboratory
2018 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 research continues to yield unanticipated discoveries through genomic, physiological, and metabolic studies. Previously unknown phytoplasmas continue to be identified and classified; new molecular markers are identified for rapid detection of indigenous vs exotic phytoplasmas; complex phytoplasmal causes of important crop diseases are revealed; new knowledge is gained on how phytoplasmas and spiroplasmas cause disease and how plants react to infection; the research unveils early events in the evolutionary emergence of phytoplasmas, and new insights are gained into how a wall-less bacterial pathogen with a tiny genome can change the developmental direction of plant stem cells, causing abnormal plant growth and development. The research identifies key meristem switching genes and regulatory pathways involved in phytoplasma-induced reprogramming of stem cell destiny and plant morphogenesis. This latter line of research is important to eventual elucidation of molecular mechanisms of phytoplasma pathogenesis and will lead to new methods for early detection of diseases. Characterizations of ‘exotic’ phytoplasmas and naming of new ‘Candidatus Phytoplasma’ taxa are being carried out, continuously aiding implementation of quarantine measures to prevent their spread. The several lines of research progress will aid the cause of food security and sustainable agricultural production.
Work is in progress to refine the criteria for phytoplasma species delineation and for multi-locus genotyping-based phytoplasma classification.
In collaboration with scientists in Lithuania, Costa Rica, Italy, China, and Mexico, work continued to characterize potentially new phytoplasma subgroup lineages that infects 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.
In collaboration with scientists in Animal and Plant Health Inspection Service (APHIS) and Lithuania, work continued to characterize and decode the genomes of ‘Candidatus Phytoplasma pini’, a pathogen that causes serious disease in pine trees in Europe, and a closely related phytoplasma found in pine trees in Maryland. This work has successfully identified distinguishing characters suggesting that the two phytoplasmas represent mutually distinct species.
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.
In collaboration with scientists in China, Canada, and Mexico, work is in progress to characterize potentially new phytoplasma species and new phytoplasma subgroup lineages that infects 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 on identification of metabolites produced in response of plants to prokaryote infection, leading to new hypotheses aimed at devising novel technology for early pathogen detection.
We have been examining the early events that take place in the plant apoplast when a bacterial pathogen first encounters the plant cell. We have continued to support our finding that certain pathogens induce the plant to produce redox-active secondary metabolites can affect the redox environment and thus the direction of the interaction (resistance or disease). Recent methods that we developed have demonstrated that successful pathogens weaken the apoplast/symplast (A/S) barrier, allowing controlled flow/leakage of symplastic and vacuolar metabolites into the apoplast. Using a new technique, ‘sequential elution’, we are discovering different reservoirs of eluted materials in the apoplast indicating different regions of the apoplast respond differentially. Specifically, the apoplast cells that line the apoplast are in direct contact with the pathogens and the leakage of their A/S barrier that affects the outcome of the interaction. This could lead to new strategies for controlling disease by aerial application of metabolites that would buffer the apoplast redox potential at beneficial levels.
Accomplishments
1. Completed bioinformatic analysis and annotations of Spiroplasma kunkelii and Spiroplasma citri genomes. Spiroplasma kunkelii is a corn pathogen, and S. citri is a pathogen of citrus and other crop plants. Both cause serious economic losses. ARS scientists in Beltsville, Maryland, in cooperation with the University of Delaware and Salem University, sequenced and analyzed the complete genomes of these pathogens. This work allowed initiation of a comparative genomics study identifying common and unique genes in these serious plant pathogens. The genomes provide targets for molecular intervention in disease development and will aid the construction of synthetic spiroplasma genomes in efforts to combat phloem-inhabiting plant pathogens.
2. Completed description and naming of a new phytoplasma, ‘Candidatus Phytoplasma luffae,’ a pathogen affecting squash and other plants in Asia. A witches’ broom disease phytoplasma (a tiny bacterium lacking a cell wall) causes important losses in the economic luffa squash crop. This phytoplasma possesses unique features. ARS scientists in Beltsville, Maryland, characterized and named the phytoplasma. This work provided molecular characters for detection and identification, and a specific name for use in referring to this exotic pathogen. This is helping APHIS and other quarantine agencies prevent the international spread of the phytoplasma.
3. Decoded the genome of a phytoplasma responsible for yellows disease of lettuce. Phytoplasmas are pathogenic bacteria that cause diseases in more than 1,000 plant species worldwide. Diverse phytoplasmas are classified into groups and subgroups based on their genetic relatedness reflected in specific RNA genes, 16S ribosomal (16Sr). 'Candidatus Phytoplasma asteris’ strain NJAY belongs to subgroup 16SrI-A and causes severe yellowing diseases of lettuce in New Jersey. To understand the pathogenic nature of the phytoplasma, ARS scientists in Beltsville, Maryland, decoded its genome. This accomplishment revealed genes that are absent from other, previously sequenced phytoplasmas. The availability of the strain NJAY genome will facilitate the identification of genomic features responsible for pathogenicity. The information will aid disease control and will interest graduate students and scientists.
4. Unveiled diversity of phytoplasmas infecting a widespread ornamental in Costa Rica. Periwinkle (Catharanthus roseus) is widely grown as ornamental plants in subtropical regions. Natural infections by phytoplasma, a class of disease causing bacteria, are common in many countries, but prior to this work were not reported in Costa Rica. From 2012 to 2016 ARS scientists in Beltsville, Maryland, and colleagues in Costa Rica found phytoplasma-infected C. roseus plants growing in six of Costa Rica’s seven provinces. Phytoplasmas of six genomic subgroups belonging to five species level groups were identified. This work provided the first evidence that periwinkle plants in Costa Rica can harbor different phytoplasma species, the presence of phytoplasma species Group 16SrXIII in the country, and the first knowledge that C. roseus is naturally infected by six different phytoplasma species in Central America. This work will help APHIS and other quarantine agencies prevent the international spread of the phytoplasma.
5. Devised and improved sensitivity of technology for phytoplasma detection. Phytoplasmas, a class of pathogenic bacteria, cause serious crop diseases, but often found in low concentration in plants making it difficult to detect. In collaboration with the University of Florida, ARS scientists in Beltsville, Maryland, adapted a new technology known as digital PCR (dPCR), devised a protocol for its application as a superior approach for sensitive detection of palm infecting phytoplasmas, compared to other commonly used PCR platforms, and described the first use of the technology in phytoplasma detection. This work will facilitate the application of quarantine measures designed to prevent the spread of palm and other phytoplasmas across national borders. Such progress will aid the causes of food security, sustainable agricultural production, and stewardship of natural ecosystems. This report will interest diagnostics laboratories, research scientists, and farmers, as well as APHIS and international quarantine agencies.
6. Completed molecular genetic characterization of a phytoplasma lineage associated with sweet cherry virescence (SCV) disease in China and its possible insect vector. During a three-year period from 2013 to 2015, a disease repeatedly occurred in sweet cherry trees growing in Northern China and was characterized by developing malformed flowers with green pigmentation that failed to set fruit. In collaboration with researches in China, ARS scientists in Beltsville, Maryland, determined that the SCV disease was associated with infection by phytoplasma, an pathogenic class of bacteria belonging to subgroup B of the elm yellows phytoplasma group (16SrV-B). Further analysis of the regions in the genome that encode important phytoplasma cellular components revealed that the SCV phytoplasma was essentially indistinguishable from the phytoplasmas responsible for jujube witches’-broom (JWB) disease and other plant diseases. Evidence gathered in the present study indicated that SCV-JWB phytoplasma strains formed a highly homogenous ecological lineage and that a polyphagous erythroneurine leafhopper may play a role in spreading the SCV phytoplasma among sweet cherry plants. Findings of this study will help devise a practical approach to combat the SCV, JWB, and other diseases caused by the phytoplasma lineage. This information is also important to regulatory agencies for implementing quarantine measures to prevent the pathogen’s spread.
7. Identified a complex array of genes involved in reversing transport of nutrients in response to phytoplasma-infection in sweet cherry. Sweet cherry is a popular deciduous tree highly valued for its fruit and timber. However, its production is often hindered by diseases associated with a small bacterium called phytoplasma. A previous study revealed that photosynthetic activity had significantly declined in leaves of phytoplasma diseased trees. To gain a deeper understanding of the disease, ARS scientists in Beltsville, Maryland, and their colleagues in China jointly performed a large-scale gene expression study (transcriptomic analysis) comparing expression patterns of the entire set of host genes in leaves of diseased versus healthy sweet cherry trees and the study identified more than a thousand genes whose expression levels were altered in the diseased trees. These genes impact carbohydrate metabolism, amino acid metabolism, and hormonal balance. Findings from this study provide leads to elucidating host responses to the bacterial infection and mechanisms of disease induction. This report will interest research scientists who are studying pathogen-host interactions and the molecular basis of phytoplasmal diseases and this information is also important to diagnosticians and extension personnel concerned with phytoplasmal disease management.
8. Completed characterization of a new phytoplasma species in mixed infections that cause yellow decline disease in foxtail palm. Palm trees possess high economic and aesthetic values, but are often susceptible to infection by an array of small bacteria known as phytoplasmas. During the past few years, foxtail palms growing in Malaysia suffered a severe yellow decline disease. In collaboration with researchers in Malaysia and University of Florida, ARS scientists in Belstville, Maryland, found that the disease was associated with mixed infections. Results from DNA fingerprinting and gene ancestry analyses revealed that one of the co-infecting phytoplasmas was previously unknown and represents a new species. The research team unveiled distinguishing genetic features of the new phytoplasma species and identified molecular markers that can detect this phytoplasma. This accomplishment is important to regulatory agencies for implementing new quarantine measures to prevent spread of the new pathogen.
9. Identified new phytoplasmal disease symptoms and additional stages of pathogen-induced stem cell fate-derailment. During the life cycle of a flowering plant, stem cells in shoot tips undergo a graduated, multi-stage transition from vegetative to reproductive destiny. A previous study showed that phytoplasma, a tiny bacterium that parasitizes nutrient-conducting vessels in host plants, can disrupt such natural transition. ARS scientists in Beltsville, Maryland, showed that phytoplasma-induced abnormal morphogenesis of flowers reflects stage-specific derailment of shoot apical meristems from their genetically preprogrammed reproductive destiny. The current study unveiled new phytoplasmal disease symptoms that developed during the course of flower formation, fruit setting, and seed germination, pointing to derailment of the meristem fate at additional stages previously unknown. The study further highlights the ability of plant stem cells to form almost any tissue or cell, and provides a framework for identifying developmental stage-specific factors associated with infectivity. The information is important to scientists who are studying plant growth and development, pathogen-host interactions, and molecular basis of diseases. This article will also interest biotechnologists and agricultural economists concerned with phytoplasmal disease symptom management.
10. Completed identification and characterization of new ‘Candidatus Phytoplasma asteris’-related strains infecting grapevines in Pennsylvania. In this study, strains of phytoplasma, a unique class of bacteria, causing grapevine yellows disease in vineyards of Pennsylvania were subjected to genotyping based on analyses of 16S rRNA and secY genes, and to in silico 3-dimensional modeling of the SecY protein. This work determined that the studied plants were infected by a phytoplasma related to but distinct from the phytoplasma that causes the well-known aster yellows disease in herbaceous plants. The new information expands knowledge of plant diseases caused by phytoplasmas, and it provides molecular markers useful for detecting and identifying the phytoplasma, identifying its insect vector, and preventing its spread. The molecular markers provided will help identify specific insect vectors responsible for spreading the pathogen.
11. Identified two plant phenolics that are specifically induced upon bacterial infection of leaves. Bacterial plant diseases cause major damage to crops each year and the cost of controlling them adds greatly to production costs and often involves antibiotics which are a public health concern. 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 in Beltsville, Maryland, demonstrated that the concentration of the phenolics (provide definition) is either very low or non-detectable in healthy tissue. However, within two-three hours after inoculation with pathogenic bacteria, these phenolics increase several fold with the majority accumulating in the apoplast. This information will benefit plant scientists and breeders who are devising new strategies to improve disease resistance while decreasing the use of chemical pesticides and antibiotics.
Review Publications
Wang, J., Liu, Q., Wei, W., Davis, R.E., Tan, Y., Lee, I., Zhu, D., Wei, H., Zhao, Y. 2018. Multilocus genotyping identifies a highly homogeneous phytoplasma lineage associated with sweet cherry virescence disease in China and its carriage by an erythroneurine leafhopper. Crop Protection. 106:13-22.
Perez-Lopez, E., Wei, W., Wang, J., Davis, R.E., Luna-Rodriguez, M., Zhao, Y. 2017. Novel phytoplasma strains of X-disease group unveil genetic markers that distinguish North American and South American geographic lineages within subgroups 16SrIII-J and 16SrIII-U. Annals of Applied Biology. 171:405-416.
Bahder, B.W., Helmick, E.E., Harrison, N.A., Davis, R.E. 2018. Digital PCR technology for detection of palm infecting phytoplasmas belonging to group 16SrIV that occur in Florida. Plant Disease. 102:1008-1014.
Sparks, M., Bottner-Parker, K.D., Gundersen, D.E., Lee, I. 2018. Draft genome sequence of the New Jersey aster yellows strain of ‘Candidatus Phytoplasma asteris’. PLoS One. https://doi.org/10.1371/journal.pone.0192379.
Naderali, N., Nejat, N., Vadamalai, G., Davis, R.E., Wei, W., Harrison, N., Kong, L., Kadir, J., Tan, Y., Zhao, Y. 2017. ‘Candidatus Phytoplasma wodyetiae’, a new taxon associated with yellow decline disease of foxtail palm (Wodyetia bifurcata) in Malaysia. International Journal of Systematic and Evolutionary Microbiology. 67:3765-3772.
