Location: Emerging Pests and Pathogens Research
2025 Annual Report
Objectives
Objective 1: Identify genomic resources for development of diagnostics and detection tools for emerging and re-emerging bacterial plant pathogens.
Sub-objective 1.A: Perform comparative genomics of bacterial pathogens.
Sub-objective 1.B: Investigate diversity of soft rot Pectobacteriaceae (SRP).
Objective 2: Characterize biology and virulence factors of bacterial plant pathogens and identify their targets in host plants.
Sub-objective. 2.A: Discover and characterize genes that contribute to disease and/or host adaptation of bacterial soft rot pathogens.
Sub-objective 2.B: Discover and characterize genes involved with interactions between bacterial species.
Sub-objective 2.C: Determine the mechanistic basis of socially affected behaviors in bacteria.
Sub-objective 2.D: Determine the contribution of AlgU to virulence and factors affecting AlgU activity.
Sub-objective 2.E: Characterize the signaling pathways that impact expression of HiVir gene cluster in Pantoea.
Objective 3: Investigate sustainable strategies for control of bacterial plant diseases.
Sub-objective. 3.A: Investigate the role of antimicrobials in tolerance to bacterial soft rot pathogens.
Sub-objective 3.B: Identify genetic markers of soft rot disease tolerance in US Potato Genebank germplasm.
Approach
Soft rot bacteria, such as Dickeya, Pectobacterium, and Pantoae are among the most important pathogens of vegetables, fruits, and ornamentals. Bacterial diseases of potato and onion alone cause more than $60M in losses annually in the U.S. Despite the extensive amount of research available on bacterial plant pathogens, there is a lack of understanding about how bacterial plant pathogens enter and move within crop production systems and to what degree these diseases are caused by endemic populations. Furthermore, some bacterial species are endemic and, in some environments, a single bacterial species can be represented by a number of different strains, some of which are pathogens, and some of which are non-pathogens with beneficial biocontrol activities. Therefore, determining which bacteria are responsible for disease and furthermore how certain bacterial strains become pathogenic is an area of research that warrants further study. To address this, we will use genome sequencing methods to characterize populations bacteria present in diseased crops. The patterns that emerge at the intersection of pathogen diversity and geographical location will provide key insights on disease emergence as well as identify diagnostic markers able to distinguish pathogens from non-pathogens. For some plant diseases, such as bacterial soft rot (potato: Dickeya spp. and Pectobacterium spp.; onion: Pantoea spp.) many bacterial species may be involved, with the pathogens being members of a broader community of plant-associated microbes. Little is known about the relationships and interactions of plant pathogens with the host, the microbial community, and the environment and the impact on disease outcome. We will investigate bacterial communication mechanisms involved in pathogen fitness and formation of complex communities in plants to identify factors critical for disease. For many bacterial soft rot diseases there are no effective management options. For example, there are no commercially available potato or onion cultivars with soft rot resistance, thus management options for these pathogens are very limited. Additionally, there is little known about the specific mechanisms involved in host tolerance or susceptibility. To address this, we will first identify and characterize factors that bacteria use to cause disease and then use that information to guide discovery of bacterial control strategies. Second, we will identify and characterize sources of natural resistance in wild crop relatives to provide information for breeders as well as a source germplasm for breeding resistant varieties. All together this research will lead to improved fundamental understanding of bacterial soft rot disease dynamics and reveal vulnerabilities that can be exploited for control of bacterial plant diseases, helping us work towards the goal of sustainable plant disease management.
Progress Report
Objective 1: Identify genomic resources for the development of diagnostics and detection tools for emerging and re-emerging bacterial plant pathogens. Using our bioinformatic pipelines, high quality genome assemblies were produced for 45 bacterial isolates. Studies this year continued to compare genomes of these bacterial strains with reported genomes in our collection and publicly available databases. The generation of whole genome sequences of bacteria provide useful resources for evaluating genomic relatedness and mechanisms bacteria used to cause disease in various hosts.
This year we continued research to track the sources of disease-causing bacteria in commercial potato production across the U.S. In FY25, we isolated and determined that a novel strain of Dickeya dianthicola was responsible for potato disease in Oregon. D. dianthicola has been a major problem for potato farming in the Eastern U.S., but this is the first time that this species was found in Oregon. Its emergence in the Northwest highlights the need for increased surveillance and management strategies in this production region.
With collaborators we developed a Whatman Plantsaver FTA® card-based pathogen detection protocol based on dormant tubers for detection of four potato viruses. Viruses tested included potato virus Y (PVY), potato virus S (PVS), potato mop top virus (PMTV), and tobacco rattle virus (TRV). Viruses were also detected from potato tubers, sprouts, and leaves. Bacterial pathogens such as Dickeya and Pectobacterium are a concern for the potato industry. These pathogens are poorly understood and express foliar symptoms differentially based on environmental factors and potato cultivar. Additionally, currently detection of Dickeya and Pectobacterium from potato is a labor-intensive process. Therefore, a goal was to expand the diagnostic assay developed for potato viruses, to detect other organisms, without adjustments that would alter labor, costs or automation throughput. Testing was performed to adapt our dormant tuber testing workflow to detect Dickeya sp and Pectobacterium sp. Direct tuber testing of asymptomatic tubers from plants showing signs of aerial stem rot detected Pectobacterium. Having a single workflow that can be used to detect multiple pathogens from potato is more efficient and cost effective for growers.
Objective 2: Strong progress was made with regard to Objective 2, Characterize biology and virulence factors of bacterial plant pathogens and identify their targets in host plants. Many aspects of Pantoea—including its interactions with plant hosts such as onions remain poorly understood. Pantoea ananatis OC5a (OC5a) causes center rot in onion bulbs and lesions on onion leaves. Previously we assembled and produced a high-quality annotated genome for OC5a. This strain contains an eleven (11) gene cluster (HiVir) responsible for the production of a phosphonate toxin, called pantaphos. Mutating a single gene pepM, completely disrupts the pantaphos biosynthesis pathway and eliminates disease systems. To identify genes important for P. ananatis growth, we performed large-scale transposon mutagenesis (Tn) screens in diseased and healthy onions. The screening involves randomly inserting transposons into the genome of an organism. By tracking where the transposons are inserted, genes critical for the organism’s survival in plants can be identified. Randomly-barcoded transposon mutagenesis, or RBTn, is a variation on the transposon sequencing (Tn-Seq) method in which each transposon sequence has an embedded, unique barcode that enables rapid and cheap library sequencing. We developed RBTn libraries for two strains of P. ananatis, wildtype OC5a (virulent on onion) and OC5a delta pepM (avirulent strain on onion). RBTn libraries were inoculated into onions to identify genes contributing to growth in diseased bulbs and those supporting growth of the avirulent strain in healthy bulbs, aiming to distinguish condition-specific and shared genes. Preliminary results indicate that genes in the HiVir cluster do not significantly provide a growth benefit in onion bulbs. Additionally, genes in the “alt” cluster, which confer resistance to toxic thiosulfinate defense compounds, did not show strong fitness effects in either condition. This suggests that “alt”-mediated tolerance likely works by altering the environment outside the bacterial cell, allowing even “alt” mutants to benefit from neighboring cells with functional “alt” genes. Results also showed that genes that encode proteins responsible for gene regulation have marginal fitness effects when disrupted. For example, mutants of rpoN, which are involved with stress response in other bacteria, have a slight fitness defect, suggesting rpoN might be involved with P. ananatis’s ability to survive the hostile onion environment. Also, mutants of rpoS, which regulates the transition from growth to stationary phase, have a slight fitness benefit. Additional experiments have been planned to test the P. ananatis RBTn libraries in more in-vitro and in-planta conditions.
Pectobacterium species are common pathogens of many crops, vegetables, and ornamentals, exhibiting extensive genetic variation across 19 recognized species. We constructed RB-Tn mutant libraries in five Pectobacterium species. Using fitness profiling, we identified genes essential for survival across species, distinguishing conserved functions from species-specific adaptations. Ortholog analysis further highlighted conserved and strain-specific functions. Coupled with in planta fitness profiling, shared and unique putative virulence determinants were identified. This work advances our understanding of the relationship between genomic variation, functional annotation, and niche specialization in Pectobacterium.
Dickeya spp. are adaptable bacterial plant pathogens and infect diverse tissue types across a broad range of host plants. However, the mechanisms Dickeya uses to modulate growth are largely unexplored. Tn-Seq was performed in three strains of Dickeya (encompassing two species) identified an ortholog group of putative transcription factors (TFs) important for growth in planta. Targeted mutants were constructed. In vitro and in planta growth assays revealed differences between mutant and wild-type in some strains, but not others. Phenotypes differed between species, and strains of the same species. To determine the specific regions of DNA that the putative TFs bind, fusion proteins were constructed and constitutively expressed in strains lacking TFs. Chromatin immunoprecipitation of the proteins was performed. Preliminary results indicate that only a small set of genes are directly regulated by the TFs across all three strains. In contrast, a larger number of strain-specific genes appear to be directly regulated by the TF, potentially accounting for the observed phenotypic differences. Additional research is required to confirm these findings. This work advances our understanding of how soft rot bacterial pathogens adapt to environmental stresses during infection.
Key questions in pathogenesis and plant health research include how pathogenic bacteria colonize host tissue and how multi-species communities form and function. Progress was made on determining the mechanistic basis of socially affected behaviors in bacteria. We found that plant pathogenic bacteria become hyper-motile when they sense the presence of unrelated bacteria. To help understand this process the sets of genes needed for motility were determined. This led to the discovery of genes that are specifically required to employ a competitive motility/colonization behavior in response to the presence of unrelated bacteria. These results provide new insights into the factors shaping plant pathogenic bacteria behavior and their colonization of environments and hosts.
Experiments were performed to determine the contribution of a widely conserved bacterial transcription factor named AlgU, to virulence and those environmental factors affecting AlgU activity. The contribution of AlgU to soft rot and blackleg disease of potato was quantified. This research showed that AlgU has a significant, positive role in soft rot and blackleg disease and without these genes the bacteria are impaired in their ability to cause disease. These results suggest that additional research aimed at translating this discovery into useful control methods is warranted.
Objective 3: Investigate sustainable strategies for control of bacterial plant diseases. Management options for bacterial plant pathogens are limited, highlighting the need for environmentally friendly disease control methods. Antimicrobial peptides have gained significant attention as potential alternatives to antibiotics and chemical pesticides, offering a promising strategy to combat bacterial plant diseases. Previously we discovered a synthetic peptide that showed broad spectrum activity to suppress in vitro growth of several bacterial plant pathogens, including Dickeya, Erwinia, Pseudomonas, and Pectobacterium sp. Studies this year focused on investigating the mode of action of the antimicrobial peptide. Using fitness profiling, we identified genes that when disrupted impact survival in the presence of the antimicrobial peptide. Genes that are implicated in the resistance to the antimicrobial peptide include those that encode for proteins involved in the biosynthesis of key components of the outer membrane of the bacteria, such as lipopolysaccharide (LPS), proteins whose predicted functions are to provide proper folding for maintaining structural integrity of the bacterial cell envelope, and proteins that act as pumps to export and prevent accumulation of antimicrobial peptides. These experiments identified vulnerabilities of the bacteria and represent potential targets for new treatments.
Accomplishments
1. Developed an assay to detect viruses in dormant potato tubers. Potatoes are vegetatively propagated, which increases risk to numerous diseases causing significant losses in quality and tuber yield. To monitor potato viruses, seed potato certification programs visually inspect plants during the growing season and perform time-consuming post-harvest grow-outs in the winter to eliminate seed potato lots with unacceptably high incidences of key tuber- borne diseases. Although these approaches can reduce crop losses, new pathogen detection strategies that are more rapid, sensitive, and accurate are needed to monitor the potato crop for existing and emerging pathogens. ARS scientists in Ithaca, New York, and collaborators developed a workflow for high throughput molecular testing of dormant seed potatoes to detect viruses and disease incidence in dormant tubers. Results showed that direct tuber testing provides reliable results for detecting four potato viruses (potato virus Y (PVY), potato virus S (PVS)), potato mop top virus (PMTV), and tobacco rattle virus (TRV). PVY detection results were comparable to the current seed potato certification tests. The protocol eliminates the need to ship tubers to seed certification agencies and winter grow out locations and provides earlier detection of pathogens of potatoes, thus limiting the spread of pathogens.
2. Mobilization of pathogenicity genes through plasmids of Pantoea agglomerans. Center rot of onions is caused by several bacterial species in the genus Pantoea and can result in significant pre- and post-harvest losses. Disease management is difficult because there are no known commercially available cultivars of onion with resistance to these pathogens. In order to help develop better management strategies, ARS scientists from Ithaca, New York, collected isolates of Pantoea agglomerans from onions from across the U.S. and tested the strains for onion pathogenicity and resistance to copper bactericides. Combining this information with whole genome sequencing analysis, we discovered that several important gene clusters (onion virulence, survival in onion tissue, and copper tolerance) are located on a small extra-chromosomal piece of DNA called a plasmid. Results showed that P. agglomerans can transfer these genes to other strains via movement of plasmids between strains. This work demonstrates that there is an association between possession of plasmids containing specific genes associated with copper resistance, P. agglomerans ability to survive in onion tissue, and cause disease in onions. This knowledge will help onion growers more quickly and precisely identify emerging threats and develop effective management practices.
Review Publications
Gonzalez-Tobon, J., Helmann, T., Daughtry, M., Stodghill, P., Filiatrault, M.J. 2024. Complete genome sequence resource for Xanthomonas hortorum isolated from Greek oregano. Phytopathology. 107:3259-3263. https://doi.org/10.1094/PDIS-10-22-2399-A.
Shing, G.Y., Asselin, J.E., Smith, A., Aegerter, B., Coutinho, T., Zhao, M., Dutta, B., Mazzone, J., Neupane, R., Gugino, B., Hoepting, C., Khanal, M., Malla, S., Nischwitz, C., Sidhu, J., Burke, A.M., Davey, J., Uchanski, M., Derie, M.L., Du Toit, L.J., Stresow, S., Bonasera, J.M., Stodghill, P., Kvitko, B. 2025. Plasmids encode and can mobilize onion pathogenicity in Pantoea agglomerans. The ISME Journal: Multidisciplinary Journal of Microbial Ecology. 19(1). Article wraf019. https://doi.org/10.1093/ismejo/wraf019.
Gonzales-Tobon, J., Daughtrey, M., Karp, M.A., Stodghill, P., Filiatrault, M.J. 2025. First report of Pseudomonas amygdali causing leaf spot on Hibiscus rosa-sinensis in New York State, U.S.A. Plant Disease. 109(3). https://doi.org/10.1094/PDIS-07-24-1556-PDN.
Yang, Z., Helmann, T.C., Baudin, M., Schreiber, K.J., Bao, Z.N., Stodghill, P., Deutschbauer, A., Lewis, J.D., Swingle, B.M. 2025. Genome-wide identification of novel flagellar motility genes in Pseudomonas syringae pv. tomato DC3000. Frontiers in Microbiology. 16. https://doi.org/10.3389/fmicb.2025.1535114.
Ingram, J.T., Mudrack, E., Tran, L., Curtis, M., Mattupalli, C., Gudmestad, N., Charkowski, A., Groves, R., Babler, B., Whitworth, J.L., Frost, K., Brown, C., Karasev, A., Gray, S., Filiatrault, M.J. 2025. A robust potato tuber tissue collection method to investigate potato virus Y, potato mop-top virus, and tobacco rattle virus localization patterns. Plant Disease. https://doi.org/10.1094/PDIS-11-24-2453-RE.
Ingram, J.T., Mattupalli, C., Mudrak, E., Curtis, M., O'Neill, P., Davenport, B., Gudmestad, N., Charkowski, A., Groves, R., Babler, B., Frost, K., Karasev, A., Gray, S., Whitworth, J.L., Filiatrault, M.J. 2025. Multi-year evaluations of an FTA card-based detection protocol for four vector-borne viruses affecting potato. Plant Disease. https://doi.org/10.1094/PDIS-11-24-2453-RE.
Yang, Z., Swingle, B.M. 2025. Pseudomonas syringae socially-induced swimming motility requires the molybdenum cofactor. Molecular Microbiology. https://doi.org/10.1111/mmi.15378.
Ma, X., Frost, K., Zhang, X., Hao, J., Swingle, B.M. 2025. First Report of Dickeya dianthicola Causing Potato blackleg in Oregon. Plant Disease. https://doi.org/10.1094/PDIS-01-25-0154-PDN.