Location: Emerging Pests and Pathogens Research
2024 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 development of diagnostics and detection tools for emerging and re-emerging bacterial plant pathogens. Bacterial soft-rot pathogens cause disease on a wide range of crops and ornamental plants. A better understanding of bacterial species causing diseases on ornamental plants may help in the development of tools to combat soft rot pathogens. We sequenced and assembled additional genomes of strains of bacteria pathogenic to onions, potatoes and nursery crops. We isolated 24 bacterial strains from ornamental host plants. Genome assemblies were performed for bacterial strains associated with ornamental host plants (9 assemblies). Studies this year focused on comparing genomes of these newly discovered bacterial strains with reported genomes in our collection and publicly available databases. For one bacterial genome, we discovered some unique genomic characteristics and new effectors molecules that may play a role in causing disease. The generation of whole genome sequences for soft rot bacteria provide useful resources for evaluating genomic relatedness and mechanisms used to avoid the plant defense response. Bacteria of the genus Pantoea are a diverse group of bacteria that are considered emerging pathogens able to cause disease in many economically important crops worldwide. To help improve and develop appropriate control strategies a better understanding of the taxonomy and factors these pathogens use to cause disease is needed. Bacterial genomes of Pantoea species that were sequenced in previous reporting cycles were further analyzed. Refinements of the pangenome and the plasmid clustering pipelines were made to identify and characterize plasmids found in onion-associated strains of Pantoea agglomerans. Consistent with the literature, all strains we evaluated contained a large plasmid, termed Large Pantoea (or LPP-1). Encoded on this plasmid were genes for biosynthesis of yellowish pigments, called carotenoids. We identified and characterized a second plasmid with unique distribution among the bacterial strains. This plasmid, named “pAggl, was found in every sequenced strain of P. agglomerans, and only in strains of P. agglomerans,”. Plasmid pAggl contains genes encoding for environment and signaling proteins, transcriptional regulators, iron transport proteins, lipopolysaccharide modification enzymes, acetoin biosynthesis proteins, and proteins involved in sugar transport and metabolism and appears to be vertically transmitted (passaged to daughter cells upon bacterial replication). Thus, we hypothesize that the presence of pAggl benefits strains of P. agglomerans. Plasmid analyses also identified a set of three plasmid classes (A, B, and C) with an overlapping, but identical sets of conserved genes. We found that plasmids A and C shared a number of genes, and B and C number of genes, but A and B shared no genes. Our data and statistical analysis of the plasmids sequences favors our hypothesis that in the distant past, instances of plasmids A and B combined to make a C plasmid. In general, these plasmids were found in onion-associated P. agglomerans strains. Hence, we named this class of plasmids “pOnion”. Many pOnion plasmids were found to contain genes that provide resistance to copper, a common bactericide for onions and other crops. A manuscript describing these results is in preparation. Efforts this year were also spent tracking the sources of disease-causing bacteria in commercial potato production across the U.S. In FY24 bacteria were isolated from diseased potato samples that originated from major potato growing regions of the U.S. and their genomes were analyzed to determine if the strains in different areas were related. Two sources of pathogens in the U.S. were found: those that are randomly distributed and those moving between farms in contaminated seed material. From this study we found one bacterial species (Pectobacterium parmentieri) that appears to be spreading between the Northeast and Midwest production regions and should continue to be monitored closely to see if this problem worsens.
Objective 2. Characterize biology and virulence factors of bacterial plant pathogens and identify their targets in host plants. Dickeya is a genus of plant pathogenic bacteria that affects economically important crops and ornamentals worldwide. There are no effective options to manage such pathogens and there is still much that we do not know about their interaction with the plant and the environment. Previously we constructed a high-density barcoded transposon library in several Dickeya species and used the libraries to identify genes important for fitness of Dickeya in potato tubers and potato stems. We selected some of the genes, such as those that encode for putative transcriptional regulators, constructed mutants then evaluated the ability of the mutants to colonize and cause symptoms in potato stems and tubers. Additionally, we expressed the novel transcriptional regulators with tags and will use these constructs to identify the sets of genes that are regulated by these proteins and impact disease progression. Our results are providing insights into the mechanisms used by Dickeya when interacting with and colonizing plants, and thus might provide targets for management. We are using the transposon libraries to discover genes required for Dickeya’s broad host-range lifestyle, identify common virulence strategies used by related phytopathogenic bacteria, and define the role of diverse genes required for necrotrophic colonization of host plants and post-harvest diseases. Furthermore, we developed transposon libraries in several Pantoea sp to identify genes important for fitness and involved in the regulation of an important virulence cluster called HiVir. Experiments are underway to inoculate these libraries into onions. Using results from a previous experiment where we inoculated transposon libraries of the soft rot pathogens P. carotovorum or P. parmentieri into potato tubers (cv. “Atlantic”), we are in the process of determining shared fitness determinants with other pathogens such as Dickeya sp, as well as unique fitness determinants required for these pathogens to survive in plant hosts. This work advances our understanding of how soft rot bacterial pathogens adapt to environmental stresses during infection.
Members of the Dickeya genus present notoriously more proteins that serve as receptors to mediate movement towards compounds and nutrients than other closely related bacteria. However, the functions and signals of many of these proteins (known as MCPs) remain unknown. Interestingly, long untranslated regions exist upstream of the coding regions of these MCPs in Dickeya. Using molecular and biocomputational methods we identified potential gene regulatory mechanisms that control expression of these putative ncRNAs and MCPs. Mutants lacking the putative ncRNAs or the MCP genes were constructed and tested alterations in movement towards compounds and their ability to cause symptoms in potato stems and tubers. Differences in motility and symptom development were found between some mutants and the wild-type strain. Our results provide new insight into the sensing and signaling mechanisms used by Dickeya.
For Objective 3A: Investigate the role of antimicrobials in tolerance to bacterial soft rot pathogens. The development of environmentally friendly methods of disease control are desired. Antimicrobial peptides have received considerable interest as promising alternatives to antibiotics and chemical pesticides and provide a promising strategy to combat bacterial plant pathogens. We tested the antibacterial activity of a synthetic peptide against Dickeya, Erwinia, Pseudomonas, and Pectobacterium sp. Minimum inhibitory concentrations (MICs) for the peptide were determined by broth dilution method. Of the strains we tested, the MIC for Dickeya, Pectobacterium, and Erwinia ranged from 40-60 ug/ml. Pseudomonas syringae was more sensitive to the peptide and had a MIC of 10 ug/ml. After 72 hrs., bacterial growth was observed for some of the species. To investigate if the population of bacteria that grew after 72 hrs. were resistant to the antimicrobial peptide, bacteria were retested. Retesting showed the bacteria were still sensitive to the peptide suggesting the action of the peptide is bacteriostatic. Our study revealed broad-spectrum efficacy of a peptide in suppressing in vitro growth of several plant pathogens. Co-inoculation experiments with bacteria and the peptide appear to inhibit symptom development in plants. Studies are underway to investigate the mode of action of this antimicrobial peptide. Research continued to identify the genetic basis of soft rot resistance by screening inbred lines for bacterial soft rot tolerance trait segregation. We screened a family of 161 potato plant progenies that were the product of crossing a highly resistant wild potato relative with a sensitive domesticated potato variety and then self-pollinating (inbreeding) one of the progeny plants. We then used genomic methods to identify markers associated with the soft rot resistance trait. This work was published and can be used by potato breeders to begin preparing to introduce soft rot resistance to cultivated varieties of potato. This will help advance our goals of food security in the United States.
Accomplishments
1. Metal containing enzymes control bacterial social interactions. Bacteria are single-celled organism that often form colonies of millions or even billions of identical copies of themselves. Bacterial colonies will sometimes coordinate their behavior and organize themselves as a group, such as when they try to protect themselves from physical or chemical stresses or acquire resources from unrelated bacteria. The mechanisms involved in formation of multicellular bacterial communities and coordination of bacterial behavior are complex and information is lacking. USDA-ARS scientists in Ithaca, New York, discovered that some plant pathogenic bacteria can detect unrelated bacteria when they are nearby. The pathogenic bacteria respond by swimming towards the unrelated bacteria, presumably to prevent potential competitors from using up the resources. Using molecular approaches and genomic analysis, they found that the bacteria use genes involved in making a metal binding molecule for coordinated the swimming behavior. This research is helping us to understand why plant pathogenic bacteria can invade plant tissues and displace the other bacteria. Furthermore, our results can be used identify and assemble bacterial communities that cannot be displaced by pathogens, thereby preventing plants from being infected by aggressive pathogens.
2. Influence of pH on plant pathogenic bacteria. Microbes such as bacteria are exposed to a variety of stresses when colonizing plants. One of the first responses during the plant defense response is a change in pH. How plant pathogenic bacteria adapt to this change is not thoroughly understood. ARS scientists in Ithaca, New York, discovered a new molecular strategy that the plant pathogenic bacterium Pseudomonas syringae uses to sense the change in pH inside plants. Upon sensing an increase in pH, (alkalization) the bacteria suppress expression of effector molecules, iron uptake systems, and production of toxins. In contrast, a decrease in pH (acidification) promotes production of bacterial components critical for disease development. Understanding the phenotypic changes that take place in microbes during exposure to different pH levels will help in designing management strategies that block the development of bacterial diseases.
3. Vulnerabilities of soft rot pathogens revealed. Soft rot bacteria such as Dickeya are devastating plant pathogens that have significant impacts on agriculture globally. Recently there has been a rapid emergence and spread of some Dickeya species with increased aggressiveness. Understanding the traits and molecular mechanisms that allow different species to have diverse lifestyles, different phenotypes, and colonize plants is key to developing new control methods. With university collaborators, ARS scientists in Ithaca, New York, used a high throughput screening approach to identify genes that contribute to bacterial fitness during the development of potato stem rot. The approach identified genes not previously described as having roles in host colonization and/or survival in potato stems. By comparing the results of two different Dickeya species, we identified shared genes important for fitness of the bacteria in potato stems. This research provides insight into the mechanisms soft rot pathogens use to colonize plants and cause disease and provides novel targets for disease management.
Review Publications
Gonzalez-Tobon, J., Helmann, T., Stodghill, P., Filiatrault, M.J. 2024. Surviving the potato stems: differences in genes required for fitness by Dickeya dadantii and Dickeya dianthicola. Phytopathology. 114:1106-1117. https://doi.org/10.1094/PHYTO-09-23-0351-KC.
Zhang, N., Gan, J., Carneal, L., Tobon, J., Filiatrault, M.J., Martin, G. 2023. Helper NLRs Nrc2 and Nrc3 act codependently with Prf/Pto and activate MAPK signaling to induce immunity in tomato. The Plant Journal. 117:7-22. https://doi.org/10.1111/tpj.16502.
Rodriguez-Herrera, K.D., Ma, X., Swingle, B.M., Pethybridge, S.J., Gonzalez-Giron, J.L., Herrmann, T.Q., Damann, K., Smart, C.D. 2023. First report of serratia marcescens causing cucurbit yellow vine disease in New York. Plant Disease. https://doi.org/10.1094/PDIS-06-23-1051-PDN.
Fenstemaker, S.M., Ma, X., Bamberg, J.B., Swingle, B.M. 2023. Reproducible Quantitative Trait Loci for Resistance to Soft Rot Caused byDickeya dianthicola Derived from the Wild Potato Solanum microdontum(PI 458355) Are Located on Chromosomes 1, 3, and 5. American Phytopathological Society. 114:580-589. https://doi.org/10.1094/PHYTO-05-23-0158-R.