Project : USDA ARS

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

Location: Molecular Plant Pathology Laboratory

2025 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 reports on research findings derived from molecular, physiological, microscopic, and omics approaches to better understand plant diseases caused by phytoplasmas and spiroplasmas. Work across all objectives has advanced knowledge of pathogen diversity, interactions with hosts, diagnostic capabilities, and classification systems that are critical for managing emerging and re-emerging crop diseases. Objective 1: Research continued to enhance understanding of the genetic diversity of plant pathogenic mollicutes and their interactions with host plants through genomic, transcriptomic, and metabolomic studies. Sequencing multiple phytoplasma genomes revealed important details about their genetic composition, structural features, and potential virulence factors, including a phytoplasma-encoded lipase. Studies have also examined how phytoplasmas disrupt normal plant development by misregulating meristem switch genes, resulting in altered stem cell fate and abnormal growth. These insights enhance our understanding of how phytoplasmas manipulate host physiology to establish infections and cause disease. Comparative analyses focused on distinguishing strains within the Elm Yellows phytoplasma group (16SrV) and the Ash Yellows group (16SrVII), providing a clearer picture of lineage differentiation, host specificity, and disease potential. International collaborations with scientists from Lithuania, Italy, Canada, Costa Rica, China, Nigeria, Poland, Taiwan, and Jordan supported efforts to characterize and document novel phytoplasma strains affecting vegetables, fruits, ornamentals, and forest trees. This work makes a direct contribution to improved disease management and sustainable agriculture worldwide. Objective 2: Research continued to identify molecular markers involved in pathogen genetic variation, niche adaptation, and pathogenicity. Genetically distinct phytoplasma strains were detected in diseased plants and potential insect vectors, facilitating the discovery of novel genomic and physiological markers with diagnostic and epidemiological relevance. These markers enhance the accuracy and reliability of detecting and identifying phytoplasmas responsible for emerging and recurring plant diseases. Comparative genomic analyses identified patterns of gene gains, losses, and rearrangements associated with the evolutionary emergence of major spiroplasma and phytoplasma lineages. This work provides a framework for linking genetic variation to ecological adaptation and pathogenic potential, ultimately strengthening surveillance and control strategies. Objective 3: Research continued to devise new and improved diagnostic tools for the detection and identification of exotic, emerging, and evolving phytoplasmas. Artificial Intelligence (AI)-based diagnostic tools targeting phytoplasma strains associated with cranberry false blossom disease were advanced, building on previous experimental models using tomato plants infected by potato purple top phytoplasma. These innovative technologies aim to enable rapid and precise diagnosis, supporting early intervention and reducing the economic and agronomic impact of infection. Development progressed on a real-time PCR assay capable of simultaneously detecting ‘Candidatus Liberibacter asiaticus’, Spiroplasma citri, and diverse phytoplasma species in citrus. This multiplex approach is designed to enhance the efficiency of pathogen surveillance in both host plants and insect vectors, supporting more timely and effective management of citrus diseases. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a-based methods are also being developed to improve the speed, sensitivity, and specificity of phytoplasma and spiroplasma detection. Volatilomic analysis was conducted to characterize the spectrum of volatile organic compounds (VOCs), highlighting key compounds that indicate metabolic activity and act as potential physiological markers. Objective 4: Research continued to expand multi-locus and whole-genome sequence information-based classification and systematics of phytoplasmas and spiroplasmas. Efforts focused on integrating whole-genome data to refine species definitions, clarify evolutionary relationships, and improve taxonomic frameworks. Sequencing and analysis of complete phytoplasma genomes provided valuable reference data for comparative studies and classification. This work supports more accurate identification and tracking of plant pathogenic lineages and helps establish standardized criteria for species delimitation based on genomic evidence. Collectively, the FY25 research progress contributes to safeguarding agricultural health, improving food security, and promoting sustainable farming practices worldwide.

Accomplishments
1. Developed an artificial intelligence (AI) tool for the identification of phytoplasma infections. Phytoplasmas are small, unculturable bacteria that infect key crops, resulting in substantial agricultural losses worldwide. Traditional diagnostic methods are slow, require specialized expertise, and often delay necessary actions, highlighting the need for more efficient and user-friendly tools for farmers and growers. To address this, ARS scientists in Beltsville, Maryland, developed an AI-based diagnostic tool for early detection of tomato plants infected with potato purple top (PPT) phytoplasma using Convolutional Neural Networks (CNNs). A dataset of 8,000 images (4,000 healthy and 4,000 infected) was used for training the model, along with an additional 1,600 images (800 healthy and 800 infected) for testing unseen cases. The training images were used in TensorFlow to build five CNN models: four pre-trained architectures (VGG-16, Google Inception V3, NASNet, and DenseNet201) via transfer learning, and one custom CNN. The pre-trained models achieved accuracy around 95%, while the custom model reached approximately 90%. Validation with a separate test set confirmed strong performance, and ensemble learning approaches are being explored to improve accuracy further. This work demonstrates how AI can revolutionize phytoplasma disease diagnosis by providing faster, more accurate, and accessible detection for farmers. The findings benefit researchers, students, and agricultural experts committed to advancing plant pathology and precision agriculture.

2. Developed CRISPR/Cas-based detection of Spiroplasma citri for citrus stubborn disease management. Spiroplasma citri is a small, pathogenic bacterium that causes citrus stubborn disease, a major threat to citrus farming worldwide. To enhance detection methods, ARS scientists in Beltsville, Maryland, have developed a CRISPR/Cas12a-based DETECTR assay (a precision DNA detector) targeting a unique genetic fingerprint for the rapid identification of S. citri. It can quickly indicate whether bacteria are present, either by using a machine that detects a glowing signal or by employing a simple paper strip test similar to those used in COVID-19 testing. The test can tell S. citri apart from other similar bacteria that infect plants. It performs well on real-world samples from the field and remains accurate even when using a crude extraction protocol, maintaining detection sensitivity and facilitating portability and field applicability. This detection platform enables prompt diagnosis, supporting early intervention strategies essential for controlling citrus stubborn disease. The findings provide citrus producers, inspectors, biosecurity agencies, and researchers with a reliable tool to prevent outbreaks, reduce economic losses, and protect the citrus trade by minimizing disease spread.

3. Identified new phytoplasma diseases in bitter melon (China), bindweed (Jordan), and buckwheat (Taiwan). In response to unusual plant disease symptoms observed in China, Jordan, and Taiwan, ARS scientists in Beltsville, Maryland, collaborated with local researchers to investigate the role of phytoplasmas, bacterial pathogens known to alter plant development and threaten crop health. In China’s Yunnan Province, bitter melon plants exhibiting stem fasciation and excessive tendril growth were diagnosed with a 16SrXXXII subgroup phytoplasma, the first such report in this host. In Jordan, field bindweed with little leaf and stunted growth was linked to a novel 16SrXXIX-C subgroup strain. In Taiwan, buckwheat plants showing phyllody and virescence were found to be infected with a phytoplasma strain belonging to the 16SrII-A subgroup. These studies represent the first documented cases of phytoplasma infection in each of these plant species within their respective countries. Together, these findings expand the known host range and geographic distribution of phytoplasmas, offering crucial insights into their genetic variations and transmission. They are of particular importance to plant pathologists, extension agents, and farmers working to monitor, manage, and mitigate the spread of these pathogens in diverse agricultural systems.

4. Identified and characterized a novel Ash Yellows group phytoplasma infecting peach trees in Pennsylvania. Peach trees in a Pennsylvania orchard exhibited unusual symptoms such as leaf yellowing, reddening, and distortion, which suggest a possible phytoplasma infection. Phytoplasmas are a group of bacteria known to cause serious declines in fruit trees, but the identity and origin of the pathogen involved were previously unknown. Researchers from the Pennsylvania Department of Agriculture collected leaf and stem samples from both symptomatic and healthy trees. ARS scientists in Beltsville, Maryland, performed molecular analyses and sequencing of housekeeping genetic markers from the collected samples. The research team compared the sequences obtained from the infected samples with known phytoplasma strains using tools like iPhyClassifier, the ARS curated sequence database for phytoplasma classification. The analyses revealed that the strains are related to ‘Candidatus Phytoplasma fraxini’ but represent a genetically distinct lineage. These findings suggest the presence of a novel, peach-associated phytoplasma strain belonging to the Ash yellows group that differs substantially from any known phytoplasma species documented to date. This is the first report of a ‘Ca. P. fraxini’-related strain infecting peach trees in Pennsylvania, raising concerns for the stone fruit industry in the region and beyond. Early detection and accurate identification are critical for growers, plant disease diagnosticians, and extension personnel for managing the spread of emerging phytoplasma diseases. The findings underscore the importance of ongoing surveillance and genetic characterization efforts to protect agricultural production and biosecurity.

5. Identified two new grapevine-associated phytoplasma strains in Minnesota. Grapevine yellows is a plant disease that impacts grapevines by causing yellowing leaves, stunted growth, and reduced fruit yield. The disease is triggered by phytoplasmas, small bacteria transmitted by insect vectors. Identifying and understanding phytoplasmas in specific regions is crucial for effective disease management and grapevine protection. ARS scientists in Beltsville, Maryland, collaborated with researchers from the Minnesota Department of Agriculture and identified two types of phytoplasmas linked to North American Grapevine Yellows (NAGY) in Minnesota for the first time. These strains are related to ‘Candidatus Phytoplasma pruni’ and ‘Candidatus Phytoplasma asteris’. This discovery marks the first confirmed presence of these pathogens in Minnesota vineyards, adding valuable insight into the phytoplasmas that could pose a threat to grape production in the area. The findings are important for grape growers and the broader agricultural community in Minnesota and surrounding regions. By identifying the specific phytoplasmas involved in NAGY, researchers and farmers can better assess disease risk and implement more targeted management practices. This work supports efforts to maintain healthy grapevines, promote sustainable grape production, and protect the wine and grape industries.

U.S. DEPARTMENT OF AGRICULTURE

Molecular Plant Pathology Laboratory: Beltsville, MD