Location: Molecular Plant Pathology Laboratory
2019 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 new discoveries through genomic, physiological, and metabolic studies. New phytoplasmas associated with emerging and re-emerging crop diseases continue to be identified and classified; new genomic markers are identified for rapid detection of and differentiation between indigenous vs exotic phytoplasmas; new hypotheses are formed and knowledge is advanced on how phytoplasmas and spiroplasmas cause disease and how plants react to infection; the research unveils early genomic events in the evolutionary emergence of phytoplasmas, and new insights are gained into how a bacterial pathogen can change the developmental direction of plant stem cells, causing abnormal plant growth and development. 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 new phytoplasma species are being carried out, continuously aiding 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, Costa Rica, Italy, China, Mexico, and Canada, 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.
We have found several new plant metabolites that are produced in response to prokaryote infection. We have been exploring other food crops and found that similar but different phenolic metabolites are induced by pathogens. The similarity of these new compounds to those we previously found in tobacco and tomato, suggests that they may also have bioactivity against pathogens. This suggests the phenomenon may be universal and will hopefully lead to new and novel technologies for early pathogen detection.
Accomplishments
1. Completed bioinformatic analysis and annotation of Spiroplasma phoeniceum genome that ARS scientists had completely sequenced and assembled. Spiroplasma phoeniceum is one of the three known plant pathogenic spiroplasmas. In previous work, ARS scientists in Beltsville, Maryland, in cooperation with the University of Delaware and Salem University sequenced the genomes of two other plant-pathogenic spiroplasmas that cause diseases in corn, citrus, and many other plants. The genome information serves as a resource for comparative analyses that will lead to identification of common and unique genes in these serious plant pathogens and identification of molecular targets for disease control.
2. Developed a new diagnostic tool for sensitive and rapid detection of `Candidatus Phytoplasma pini'. `Candidatus Phytoplasma pini' is a phytoplasma that seriously damages pine trees and was first discovered in Europe. In this work, ARS scientists in Beltsville, Maryland, and collaborators in Lithuania analyzed DNA samples from diseased pine trees growing in forests in Lithuania. The research team determined the nucleotide sequences of a cluster of essential genes and identified unique nucleotide sequence blocks that can be used as molecular markers for specific detection and identification of the pine phytoplasma. Based on such unique molecular markers, the team devised a new diagnostic tool that can quickly detect the pine-infecting phytoplasma and distinguish the phytoplasma from other known phytoplasmas. Findings from the work expands knowledge of distinct genomic features of the pine phytoplasma and provides a new and improved tool for detection of the pathogen. The findings are important to scientists and extension personnel who are concerned with phytoplasmal disease diagnosis and management. The information is also critical to regulatory agencies for preventing exotic pathogens from being introduced into the U.S.
3. Completed description and naming of a new phytoplasma species, ‘Candidatus Phytoplasma wodyetiae’, a cell wall-less bacteria associated with yellow decline disease in foxtail palm in Malaysia. In collaboration with researchers in Malaysia and University of Florida, ARS scientists in Beltsville, Maryland, found that this phytoplasma possesses unique genetic features not seen in other phytoplasmas and recognized it as a new species. The unique genetic features of the phytoplasma can be used as molecular markers to detect this species and to distinguish this exotic pathogen from other pathogens, aiding APHIS and other quarantine agencies to prevent international spread of the pathogens.
4. Completed molecular genetic characterization of a phytoplasma associated with the first occurrence of sugarcane yellow leaf disease in Mexico. Phytoplasma infection in sugarcane can seriously reduce the commodity quality as well as yield. During the 2015-2016 growing season, sugarcane plants exhibiting leaf discolorations indicative of sugarcane yellow leaf disease were observed in a sugarcane field in Mexico. Using DNA fingerprinting genetic profiling technology, ARS scientists in Beltsville, Maryland, concluded that the sugarcane disease was caused by a phytoplasma closely related to that responsible for yellows diseases in aster and many other herbaceous plants. This is the first report of phytoplasmal sugarcane yellow leaf disease in Mexico. Findings from the study underscore a need for disease surveillance of sugarcane in neighboring countries, since insect vectors capable of spreading aster yellows phytoplasma strains are known to be present over wide areas, including the Caribbean countries and the United States. This information is important to farmers and extension personnel who are concerned with phytoplasmal disease diagnosis and management.
5. Completed a study that advanced our knowledge of plant stem cell plasticity under pathological conditions. Over the life cycle of a flowering plant, stem cells in shoot tips undergo a graduated, multi-stage transition from vegetative to reproductive destiny. Through a multi-year study, ARS scientists found that such natural transition can be disrupted by phytoplasma, a tiny bacterium that parasitizes nutrient-conducting vessels of host plants. ARS scientists in Beltsville, Maryland, demonstrated that phytoplasma-induced abnormal morphogenesis of flowers and vegetative growth patterns reflects stage-specific derailment of shoot apical meristems from their genetically preprogrammed reproductive destiny. The study unveiled a total of eight phytoplasmal disease symptoms that developed over the course of vegetative development, flower formation, fruit setting, and seed germination, each pointing to a stage-specific derailment of the genetically preprogrammed fate of stem cells. The study reveals the pluripotency and plasticity of plant stem cells under pathological conditions. 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 be of interest to biotechnologists and agricultural economists who are concerned with phytoplasmal disease symptom management.
6. Identified a complex array of differentially expressed genes and differentially accumulated metabolites in leaves of phytoplasma-infected sweet cherry trees. Sweet cherry is highly valued for its fruit and timber. However, its production can be hindered by diseases associated with phytoplasma infections. ARS scientists in Beltsville, Maryland, and their colleagues in China jointly performed a combined transcriptome and metabolome study. The study identified more than a thousand genes and hundreds of metabolites whose expression levels and abundance were altered in diseased trees. Results indicate that phytoplasma infection causes large-scale transcriptional reprogramming and disturbs carbohydrate metabolism, amino acid metabolism, and hormonal balance. The gene expression and metabolic changes are accompanied by a source-to-sink shift in leaves of the infected plants. Findings from this study provide leads to elucidating host responses to the bacterial infection and mechanisms of disease induction. This report will be of interest to research scientists who are studying pathogen-host interactions and molecular basis of phytoplasmal diseases. The information is also important to diagnosticians and extension personnel who are concerned with phytoplasmal disease management.
7. Discovered that plant phenolics induced by invading bacteria can interact to regulate the redox potential of the apoplast. 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. We have demonstrated that certain phenolics are induced in the apoplast within 2 to 3 hours after inoculation with pathogenic bacteria. We have now shown that the oxidation of these phenolics leads to intermediates that interact and can intensify the oxidative environment creating a transient condition which blocks bacterial growth. 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
Davis, R.E., Shao, J.Y., Zhao, Y., Wei, W., Bottner-Parker, K.D., Silver, A.B., Stump, Z.A., Gasparich, G.E., Donofrio, N. 2019. Complete genome sequence of Spiroplasma phoeniceum strain P40T, a plant pathogen isolated from diseased plants of Madagascar periwinkle (Catharanthus roseus (L.) G. Don). Microbiology Resource Announcements. https://doi.org/10.1128/MRA.01612-18.
Wei, W., Davis, R.E., Bauchan, G.R., Zhao, Y. 2019. New symptoms identified in phytoplasma-infected plants reveal extra stages of pathogen-induced meristem fate-derailment. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-01-19-0035-R.
Perez-Lopez, E., Wei, W., Davis, R.E., Wang, J., Zhao, Y. 2019. First report of sugarcane yellow leaf disease in Mexico and detection of `Candidatus Phytoplasma asteris'-related strains in affected plants. Plant Disease. https://doi.org/10.1094/PDIS-09-18-1591-PDN.
Davis, R.E., Dally, E.L., Zhao, Y., Wolf, T.K. 2018. Genotyping points to divergent evolution of ‘Candidatus Phytoplasma asteris’ strains causing North American grapevine yellows and strains causing aster yellows. Plant Disease. 102:1696-1702. https://doi.org/10.1094/PDIS-10-17-1690-RE.
Davis, R.E., Dally, E.L., Zhao, Y., Webb, C., Appel, J.A. 2019. First report of North American grapevine yellows (NAGY) in Kansas: Association of ‘Candidatus Phytoplasma pruni’- and ‘Ca. Phytoplasma asteris’-related strains with the disease. Plant Disease. https://doi.org/10.1094/PDIS-05-18-0869-PDN.
Villalobos, W., Bottner-Parker, K.D., Lee, I., Albertazzi-Castro, F., Montero-Astua, M., Coto, T., Sandoval, I., Garita, L., Moreira, L. 2019. Catharanthus roseus naturally infected with diverse phytoplasmas in Costa Rica. Journal of Tropical Biology. 67:(1) pp. 321-336.
Martini, M., Bottner-Parker, K.D., Lee, I. 2018. PCR-based sequence analysis on multiple genes other than 16S rRNA gene for differentiation of phytoplasmas. In: Musetti, R., Pagliari, L. Phytoplasmas: Methods and Protocols. Basel, Switzerland: Springer. 1875:97-115.
Baker, C.J., Mock, N.M., Averyanov, A.A. 2019. Redox- and bio-activity of Apocynin (acetovanillone) in plants, a plant phenolic that alleviates. Free Radicals in Biology and Medicine. https://doi.org/10.1016/j.pmpp.2019.01.005.
