Research Project: Omics-Based Approach to Detection, Identification, and Systematics of Plant Pathogenic Phytoplasmas and Spiroplasmas

Location: Molecular Plant Pathology Laboratory

2023 Annual Report

Objectives
Objective 1: Enhance understanding of the genetic diversity of plant pathogenic mollicutes (phytoplasmas and spiroplasmas) and their interactions with host plants through genomic, transcriptomic, and metabolomic studies. (NP303, C1, PS1A, PS1B) Objective 2: Identify molecular markers involved in pathogen genetic diversity, niche adaptation, and pathogenicity. (NP303, C1, PS1A, PS1B) Sub-objective 2.A: Identify genus-, species-, and lineage-specific multi-locus genomic markers of diverse phytoplasmas associated with diseases of domestic and international importance. Sub-objective 2.B: Explore and evaluate redox, hormonal, and metabolic markers of pathogenesis for earlier detection and enhanced identification of diverse mollicutes. Objective 3: Devise new and improved diagnostic tools for the detection and identification of exotic, emerging, and evolving phytoplasmas. (NP303, C1, PS1A, PS1B) Sub-objective 3.A: Devise rapid and sensitive phytoplasma detection and identification protocols based on pathogen species- and lineage-specific genomic markers. Sub-objective 3.B: Devise biosensors for early disease diagnosis based on host redox, hormonal, and metabolic signals. Objective 4: Expand multi-locus and whole-genome sequence information-based classification and systematics of phytoplasmas and spiroplasmas. (NP303, C1, PS1A, PS1B) Sub-objective 4.A: Construct a multi-locus sequence typing (MLST)-based phytoplasma classification scheme and establish a whole-genome sequence information-based operational metrics for phytoplasma species delineation. Sub-objective 4.B: Identify genomic features correlated with divergent evolutionary trajectories of plant pathogenic spiroplasmas at differing levels of taxonomic rank.

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 yields new findings through molecular, physiological, microscopic, and omics studies. New genetically distinct phytoplasmas are discovered in diseased plants and potential insect vectors; new genomic and physiological markers are identified to improve the detection and identification of phytoplasmas responsible for emerging and re-emerging diseases. The research elucidates how a bacterial pathogen alters the growth patterns and the architecture of host plants, that is, pathogen-induced misregulation of the meristem switch genes leads to derailment of the genetically preprogrammed fate of plant stem cells. This line of research is of great significance for understanding the pathogenesis of phytoplasmas and provides a new concept for early disease diagnosis and symptom management. The research advances will help protect agricultural health, enhance food security, and achieve sustainable agricultural production. The project team seeks and compiles new information regarding emerging diseases in agronomically important crops including vegetables and fruits, as well as ornamentals and forest trees, helping growers and field pathologists in disease diagnosis and management. Work generates new findings in phytoplasma and host plant interactions and elucidation of the underlying mechanisms of how symptoms are caused by inhibited starch breakdown and degradation of dysfunctional chloroplasts. The genomes of several phytoplasmas (including potato purple top phytoplasma) are sequenced and assembled. In collaboration with scientists in Lithuania, Italy, Canada, Costa Rica, China, Nigeria, Poland, and Taiwan work is in progress to identify and characterize novel phytoplasmas that infect agriculturally and environmentally important plants. Initiated a new line of research aiming at AI-based diagnosis of phytoplasma diseases. The first phase of the new research has focused on the construction of a comprehensive database consisting of images of phytoplasma-infected and healthy plants. The symptom images are annotated with metadata including disease names, host plant species, geographic distributions, and symptom types as well as severity levels. Initiated a new study to improve the speed and accuracy of grapevine flavescence dorée phytoplasma identification. Work continues exploring cutting-edge technologies like CRISPR/Cas12a for specific detection and differentiation of exotic phytoplasmas. Advances in the research will provide critical information and diagnostic tools to regulatory agencies for devising and implementing quarantine measures. Work continues to refine whole-genome information-based criteria for phytoplasma species delineation and for multi-locus genotyping-based phytoplasma classification. Sequencing and comparative genomic analysis of complete phytoplasma genomes are in progress. The ongoing work has significantly improved the genome assembly and differentiation of closely related phytoplasma strains in the elm yellows phytoplasma group (16SrV). Investigated the impact of potato purple top (PPT) phytoplasma infection on the endoplasmic reticulum (ER), ER-resident proteins, and the unfolded protein response (UPR) triggered by ER stress in tomato plants.

Accomplishments
1. Developed machine-learning models to predict and select antimicrobial peptides against plant pathogenic bacteria. Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics for combating plant pathogenic bacteria in agriculture and the environment. ARS researchers in Beltsville, Maryland, together with scientists at George Mason University, located in Fairfax, Virginia, took a bioinformatics approach utilizing machine learning models based on N-gram representations of peptide sequences to predict and select AMPs against plant pathogenic bacteria. The models were applied to predict putative AMPs encoded by DNA sequences of unknown functions in the citrus genome. Several of such predicted AMPs were tested in the laboratory and confirmed to have growth-inhibitory effects on Spiroplasma citri, a bacterial pathogen of citrus trees. This accomplishment provides a valuable tool for identifying and selecting potential AMPs and contributes to the future development of effective strategies for plant disease management.

2. Completed a major update on iPhyClassifier, the online phytoplasma taxonomy/classification tool. Phytoplasmas are cell wall-less bacteria that parasitize the nutrient-conducting vessels of vascular plants and are responsible for numerous diseases in agriculturally and environmentally important plants worldwide. Since phytoplasmas cannot be cultured in laboratories and their phenotypic characters are inaccessible, DNA fingerprinting is the best way to identify phytoplasmas. iPhyClassifier is a user-friendly platform for real-time identification and classification of known and discovery of new phytoplasmas. The online tool performs computer-simulated DNA fingerprinting, compares the result with a database of known phytoplasma DNA fingerprint patterns, and assists users to make informed decisions regarding the identity of any phytoplasmas under their study. ARS researchers in Beltsville, Maryland, made a major update on the phytoplasma DNA database, expanded the existing classification scheme, and set the stage for implementing the new phytoplasma taxonomy guidelines set forth by the international committee that governs the phytoplasma taxonomy.

3. Unveiled phytoplasma infection may change the protein glycosylation profiles of host plants. Phytoplasmas are plant pathogenic bacteria responsible for numerous diseases in agriculturally important crops. Phytoplasma infection can change many aspects of the biochemical and physiological processes in host plants. ARS researchers in Beltsville, Maryland, unveiled that phytoplasma infection may alter the glycosylation profiles of host plant cells. Since glycosylation is a common posttranslational modification that contributes to the activity and stability of diverse eukaryotic proteins, this accomplishment will help elucidate the role of protein glycosylation in phytoplasma pathogenesis and host defense response.

4. Characterized and named ‘Candidatus Phytoplasma prunorum’, a new phytoplasma species affecting North American pine trees. Pine trees possess significant ecological value and serve as vital resources for timber, resin, and aesthetic landscaping. However, they face a high susceptibility to infection by phytoplasmas, a group of cell wall-less bacteria that impose severe threats to their host plants. While 'Candidatus Phytoplasma pini' has been a known phytoplasma species predominantly identified in European pine trees, recent research conducted by ARS researchers in Beltsville, Maryland, in collaboration with APHIS scientists, unveiled a novel phytoplasma species affecting North American pine trees. Initially presumed to be related to 'Ca. P. pini', this newly discovered phytoplasma species underwent a thorough examination of its distinct molecular, genomic, and ecological traits. The comprehensive analysis conducted by the research team conclusively determined it to be a distinct phytoplasma species, thus naming it 'Candidatus Phytoplasma prunorum'. The characterization and nomenclature of 'Candidatus Phytoplasma prunorum' carry significance for stakeholders involved in the study of phytoplasmas and disease management. Findings from this research provide valuable insights for research scientists, plant disease diagnosticians, and extension personnel, particularly in comprehending the genetic diversity among bacterial pathogens and devising effective strategies for managing plant diseases.

5. Identified potential plant hosts of phytoplasmas from their insect carriers by employing DNA-barcoding-based gene marker. Phytoplasmas, bacterial pathogens infecting various plant species, form a complex pathosystem involving phytoplasma, insect vectors, and plant hosts. Our previous studies revealed the genetic diversity of phytoplasmas carried by leafhoppers, potential insect vectors collected from natural habitats. To unravel the potential plant hosts and ecological impacts, scientists from ARS in Beltsville, Maryland, collaborated with researchers from the University of Illinois, Champaign, Illinois. Using DNA barcoding, a rapid and precise identification method, 14 plant species were identified from the examined leafhopper specimens. These included previously undocumented/potential new plant hosts of phytoplasmas, alongside recognized hosts like tomato, alfalfa, and maize. Notably, even well-managed croplands cultivated tomatoes and maize were found to serve as food sources for leafhoppers collected in natural habitats. This suggests a potential spillover/spillback risk of phytoplasmas between crop and non-crop regions. These findings hold significant importance for professors, researchers, and students interested in understanding the intricate interplay between pathogens, vectors, and plants. Moreover, the study's outcomes will aid in phytoplasma disease forecasting and future surveillance efforts.

6. Investigated volatile organic compound (VOC) changes in tomato plants infected with potato purple top phytoplasma. VOCs emitted by plants play a crucial role in their interactions with plant pathogens. This study examined the alterations in VOCs released by tomato plants infected with potato purple top phytoplasma. ARS scientists in Beltsville, Maryland, employed gas chromatography-mass spectrometry to compare the VOC profiles of infected plants with healthy ones. The analysis revealed significant differences in VOCs between the infected and control tomato plants. Notably, one VOC, a-copaene, demonstrated varying emission levels throughout different stages of the disease. The findings highlight the potential of VOC production changes as a diagnostic tool for identifying phytoplasma-caused diseases. Moreover, these results will lay the foundation for the development of AI-based E-nose detection systems, enabling early and accurate detection of phytoplasma diseases. These insights are highly valuable to researchers, professors, and students interested in comprehending the intricate interplay between pathogens and plants, ultimately advancing our understanding of plant health and disease management.

7. Unraveled the effect of phytoplasma infection on lipid composition, chloroplast degradation, and senescence in host plants. Phytoplasmas, minute and unculturable bacteria, cause various plant diseases. In the present study, ARS scientists in Beltsville, Maryland, examined the effects of phytoplasma infection on polar lipid composition, crucial molecules in plant functions. The infected plants showed reduced levels of certain polar lipids, potentially leading to yellowing symptoms and decreased photosynthesis efficiency. Additionally, the infection triggered chloroplast protein degradation and activated autophagy in the chloroplasts, a process of cellular component breakdown and recycling. These findings highlight the significant disruption of plant functioning and the development of disease symptoms due to phytoplasma infection. Understanding the mechanisms of phytoplasma infection could lead to the development of effective strategies for managing plant diseases. This study provides valuable insights for researchers, students, and professors studying plant-pathogen interactions.

Review Publications
Pierro, R., Bottner-Parker, K.D., Panattoni, A., Wei, W., Marcone, C., Rizzo, D., Materazzi, A., Quaglino, F., Zhao, Y. 2022. Multilocus sequence typing of phytoplasmas associated with Flavescence dorée disease in Tuscany vineyards identifies a highly homogeneous lineage in the subgroup 16SrV-C. Crop Protection. 163. Article 106114. https://doi.org/10.1016/j.cropro.2022.106114.
Wei, W., Shao, J.Y., Bottner-Parker, K.D., Zhao, Y. 2022. Draft Genome Sequence Resource of CBPPT1, a 'Candidatus Phytoplasma trfolii'-related strain associated with potato purple top disease in the Columbia Basin, USA. Plant Disease. https://doi.org/10.1094/PDIS-08-22-1788-A.
Wei, W., Ait Barka, E., Eichmeier, A. 2023. Editorial: Recent advances in crop diseases associated with plant vascular-colonizing bacteria. Frontiers in Plant Science. 14. Article e1171973. https://doi.org/10.3389/fpls.2023.1171973.
Inaba, J., Shao, J.Y., Trivellone, V., Zhao, Y., Dietrich, C.H., Bottner-Parker, K.D., Ivanauskas, A., Wei, W. 2023. Guilt by association: DNA-barcoding based identification of potential plant hosts of phytoplasmas from their insect carriers. Phytopathology. 113(3):413-422. https://doi.org/10.1094/PHYTO-09-22-0323-R.
Ivanauskas, A., Zhang, A., Zhao, Y., Wei, W. 2023. Exploring changes in Volatile Organic Compound (VOC) profiles of tomato plants infected with phytoplasma. Phytopathogenic Mollicutes. 13(1):5-6. https://doi.org/10.5958/2249-4677.2023.00003.8.
Inaba, J., Kazeem, S.E., Zhao, Y., Zwolinska, A., Ogunfunmilayo, A.O., Arogundade, O., Wei, W. 2023. Tomato and Jute Mallow are two new hosts of papaya bunchy top phytoplasma, a 'Candidatus phytoplasma convolvuli'-related strain in Nigeria. Plant Disease. https://doi.org/10.1094/PDIS-09-22-2192-PDN.
Kim, B., Inaba, J., Zhao, Y., Wei, W. 2023. Lectin binding assay reveals phytoplasma infection-induced alteration of plant host protein glycosylation. Phytopathogenic Mollicutes. 13(1):7-8. https://doi.org/10.5958/2249-4677.2023.00004.X.
Inaba, J., Kim, B., Zhao, Y., Wei, W. 2023. Phytoplasma infection alters polar lipid composition and triggers chloroplast autophagy in host plants. Phytopathogenic Mollicutes. 13(1):3-4. https://doi.org/10.5958/2249-4677.2023.00002.6.