Location: Molecular Plant Pathology Laboratory
2021 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 generate new findings through genomic, molecular, and physiological studies. New genetically distinct native and exotic phytoplasmas in diseased plants and insects continue to be identified and classified; new genomic and physiological markers have been identified to improve the detection and identification of phytoplasmas 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 provided clues to how genomic fusion and subsequent DNA recombination events contribute to the evolution of phytoplasmas. 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 characterizations of ‘exotic’ phytoplasmas and new phytoplasma species are ongoing and will continue to assist in the implementation of quarantine measures to prevent their spread. The research advances will help protect agricultural health, enhance food security, and achieve sustainable agricultural production. Work continued to refine whole-genome information-based criteria for phytoplasma species delineation and for multi-locus genotyping-based phytoplasma classification. The project team continued to collect, review, and disseminate new information regarding invasive phytoplasma species and the plant diseases they cause, helping growers and field pathologists in disease diagnosis and management. 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. Work continued in phytoplasma and host plant interactions, identification of new symptoms, and elucidation of the underlying mechanisms. 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. 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. 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 indicates that the host metabolic response may be universal and will hopefully lead to new and novel technologies for early pathogen detection.
Accomplishments
1. Characterized and named a new phytoplasma species ‘Candidatus Phytoplasma tritici’. Wheat blue dwarf (WBD) is one of the most economically damaging cereal crop diseases in northwestern China. For a long time, the disease agent was considered a phytoplasma (cell wall free bacterium) belonging to a species termed 'Candidatus Phytoplasma asteris'. ARS scientists at the Beltsville Agricultural Research Center in Beltsville, Maryland, studied the biological and ecological features and a draft genome sequence of the WBD phytoplasma. Based on its unique vectorship, distinctive host response, and genome sequence features, the scientists determined that the WBD phytoplasma is clearly distinguished from 'Candidatus Phytoplasma asteris' and represents a novel phytoplasma species. The scientists gave the name ‘Candidatus Phytoplasma tritici’, to the new species and provided a 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.
2. Identified and characterized two membrane-interactive antimicrobial peptides against plant pathogenic spiroplasmas. Spiroplasma kunkelii and Spiroplasma citri are plant pathogenic bacteria responsible for corn stunt disease and stubborn citrus disease, respectively. Both diseases have high impacts on agriculture and the economy. ARS scientists at the Beltsville Agricultural Research Center in Beltsville, Maryland, identified and characterized two antimicrobial peptides (AMPs) that were effective in inhibiting the growth of S. kunkelii and S. citri; the efficacies of the AMPs were comparable to that of tetracycline, a potent antibiotic. The study also indicated that the AMPs could interact with the spiroplasma membrane and deform the bacterial cells. This report will be of interest to farmers and extension personnel who are concerned with spiroplasmal disease management. The findings will also be of interest to research scientists and agricultural biotechnologists for genetic engineering of disease resistance.
3. Identified new molecular markers for differentiating distinct phytoplasma lineages responsible for grapevine Bois noir disease. Bois noir is a grapevine disease associated with infection by a cell wall-less bacterium termed ‘Candidatus Phytoplasma solani’ (CaPsol). The disease is prevalent in many European countries and causes significant economic losses to the viticulture industry. In collaboration with scientists in Italy, ARS researchers at the Beltsville Agricultural Research Center conducted a multi-gene comparative analysis on CaPsol strains identified from vineyards in Italy and North Macedonia. The study found the presence of mutually distinct CaPsol lineages in the vineyards and identified new molecular markers that can tell the different lineages apart. This accomplishment will be of interest to research scientists, plant disease diagnosticians, and extension personnel interested in phytoplasma genetic diversity and disease management. This information is also important to regulatory agencies for implementing control measures to prevent the exotic phytoplasmas from coming into the U.S.
4. Identified a new phytoplasma that causes papaya bunchy top disease in Nigeria. Starting from 2018, a papaya disease characterized with symptoms including excessive shoot proliferation at the top of the crown, leaf mosaic and crinkling, and dieback occurred in Oyo State, Nigeria, and caused significant production losses. ARS scientists at the Beltsville Agricultural Research Center in Beltsville, Maryland, and Nigerian collaborators identified a phytoplasma as the etiological agent of papaya disease and determined the new phytoplasmas represented a new subgroup. This is the first time that papaya phytoplasma disease was reported in Nigeria. The findings will help extension personnel and farmers in developing disease management strategies. The information is also crucial for regulatory agencies to prevent exotic pathogens from entering the United States and other papaya producing countries. This accomplishment will benefit researchers and students interested in the diagnosis of plant pathogens.
Review Publications
Passera, A., Zhao, Y., Murolo, S., Pierro, R., Arsov, E., Mori, N., Moussa, A., Silletti, M.R., Casati, P., Panattoni, A., Wei, W., Mitrev, S., Materazzi, A., Luvisi, A., Romanazzi, G., Bianco, P.A., Davis, R.E., Quaglino, F. 2020. Multi-locus genotyping reveals new molecular markers for differentiating distinct genetic lineages among 'Candidatus Phytoplasma solani' strains associated with grapevine bois noir. Pathogens. 2020 9:(11)970 https://doi.org/10.3390/pathogens9110970.
Kazeem,Shakiru,A, Inaba, J., Zhao, Y., Zwolinska, A., Ogunfunmilayo, A.O., Arogundade, A., Wei, W. 2021. Molecular identification and characterization of `Candidatus Phytoplasma convolvuli'-related strains (representing a new 16SrXII-O subgroup) associated with papaya bunchy top disease in Nigeria. Crop Protection Journal. https://doi.org/10.1016/j.cropro.2021.105731.
Wei, W., Trivellone, V., Dietrich, C.H., Zhao, Y., Bottner-Parker, K.D., Ivanauskas, A. 2021 Identification of phytoplasmas representing multiple new genetic lineages from phloem-feeding leafhoppers highlights the diversity of phytoplasmas and their potential vectors Pathogens. 10(3):352. https://doi.org/10.3390/pathogens10030352.
Trivellone, V., Wei, W., Dietrich, C.H., Filippin, L. 2021 Screening potential insect vectors in a museum biorepository reveals undiscovered diversity of plant pathogens in natural areas Ecology and Evolution 10.1002/ece3.7502. https://doi.org/10.1002/ece3.7502
Tan, Y., Li, Q., Zhao, Y., Wei, H., Wang, J., Baker, C.J., Liu, Q., Wei, W. 2021 Integration of metabolomics and existing omics data reveals new insights into phytoplasma-induced metabolic reprogramming in host plants PLoS ONE. 16:e0246203. https://doi.org/10.1371/journal.pone.0246203.
Zhao, Y., Wei, W., Davis, R.E., Lee, I., Bottner-Parker, K.D. 2021. The agent associated with blue dwarf disease in wheat represents a new phytoplasma taxon, 'Candidatus Phytoplasma tritici'. International Journal of Systematic and Evolutionary Microbiology. 71:(1). https://doi.org/10.1099/ijsem.0.004604.
Zhao, Y., Wei, W. 2020. Candidatus Phytoplasma trifolii. Center for Agriculture and Biosciences International (CABI) Invasive Species Compendium. https://doi.org/10.1079/ISC.40854.20210200691.
