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ARS Home Northeast Area Ithaca, New York Robert W. Holley Center for Agriculture & Health Emerging Pests and Pathogens Research Research





- Filiatrault, Melanie
- Stodghill, Paul
- Swingle, Bryan
- 2023
- 2022
- Publications
- Plant Diseases (303)

Research Project: Advancing Knowledge of the Biology and Etiology of Bacterial Plant Pathogens Towards Management Strategies **Location:
                            Emerging Pests and Pathogens Research** 2023 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. We continued to sequence and assemble the genomes of strains of bacteria pathogenic to onions, potatoes and nursery crops. Genome assemblies were performed for bacterial isolates associated with ornamental host plants (21 assemblies) and onions (22 assemblies). One of these is a closed, high-quality genome assembly for the type strain of Pantoea allii LMG 24248. High-quality genomes of type strains are valuable because these are used for the identification of novel strains and to organize strains of bacteria genera. Several manuscripts describing the Pantoea agglomerans and P. allii complete genome assemblies are in progress. These genomes are being used to assemble a comprehensive database of the genomic potential of pathogenic and non-pathogenic strains of Pantoea spp. All assemblies will be made publicly available deposited in National Center Biotech Info. (NCBI)’s GenBank repository. For some bacteria isolates we performed phenotypic assays and tested host range. We provided knowledge about the bacterial pathogens, increasing our understanding of bacterial pathogens of ornamental crops and potatoes. The new complete genome sequences are serving as important resources for researchers undertaking comparative genome studies, developing diagnostic tools, and discovering specific bacterial traits like those used to cause disease. During this period, we deployed pipelines for constructing pangenome/comparative-genomics databases. We have pipelines based on the packages, PyParanoid, Roary, and PIRATE. Our implementations allow pipelines for additional packages to be quickly deployed. We constructed databases for the genera Pantoea, Rahnella, Dickeya/Pectobacterium (as a single supergenus), and Xanthomonas. We modified previous pipelines and used these newer pipelines and databases of orthologous genes to identify orthologs and core genomes to produce phylogenetic trees which has aided in the identification of bacterial species. In addition, we developed a pipeline for identifying “classes” of plasmids across bacterial genomes and extended our pipelines to include plasmid “classes”, allowing us to study the movement of clusters of genes between organisms. With collaborators, we are preparing a manuscript that describes the movement of plasmids within P. agglomerans and how plasmids contribute to the distribution of virulence genes associated with bacterial strains that cause Center Rot in onions. We investigated the diversity of soft rot Pectobacteriaceae in collaboration with university scientists to obtain bacterial isolates from potato disease outbreaks in most of the major potato production areas of the United States. This year we sequenced and analyzed the genomes of 119 soft rot Pectobacteriaceae. These data were used to identify the species and compare the relationships of the bacteria reported in Accomplishment 2. 

Objective 2: Characterize biology and virulence factors of bacterial plant pathogens and identify their targets in host plants. A comprehensive investigation of genes required for the fitness of bacterial soft rot pathogens, like Dickeya was lacking. 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. To identify if common genes are required for fitness of Dickeya sp. in other parts of the potato plant, we set up experiments to inoculate the transposon libraries into potato stems. We found 381 genes that are important for fitness of Dickeya dadantii in stems tissue and 253 genes important for fitness of Dickeya dianthicola in stem tissue. Genes related to motility and chemotaxis are exclusively important for Dickeya to grow in potato stems and not necessary for growth of Dickeya in tubers. We discovered a cluster of virulence related genes (srfABC) to be exclusively required for growth in stems. We are now constructing mutants in genes of interest and testing for impact of growth and virulence in host plants. Most of the genes with strong impact on fitness are important for both D. dadantii and D.  dianthicola. We selected some of the genes we found to be important for fitness of Dickeya in potato tubers and/or stems and constructed mutants and evaluated if the mutants display altered phenotypes that impact the ability of bacteria to cause disease. The discoveries allow for a better understanding of the strategies bacteria use to cause disease and provide foundational knowledge that can be used to guide development of new strategies to prevent plant diseases caused by bacterial soft rot pathogens. The valuable libraries are being used to discover genes required for Dickeya’s broad host-range lifestyle, identify common virulence strategies used by related phytopathogenic Dickeya strains, and the role of diverse genes required for necrotrophic colonization of host plants and post-harvest diseases.

In the field, Dickeya and Pectobacterium spp are commonly co-isolated from diseased plants, suggesting a potential synergistic interaction. However, it is unclear if these co-infections are opportunistic or the result of active cooperation. Furthermore, mechanisms that would allow cooperation or synergy between these bacterial species are not known. To address these questions, we co-inoculated barcoded transposon libraries in the strains, D. dianthicola ME23, and D. dianthicola 67-19 with either wild-type P. carotovorum WPP14 or P. parmentieri WPP163 in potato tubers (cv. “Atlantic”). Measuring changes in the relative abundance of each Dickeya mutant during infection allowed us to quantify per-gene contributions to fitness during this interaction. Building upon our previous datasets, we aimed to identify genes specifically important for in planta growth of Dickeya in the presence of Pectobacterium spp. Bottlenecks and maintaining population growth was complicated by uneven strain growth with P. carotovorum. Interestingly, Pectobacterium gene fitness values were unchanged by the addition of Dickeya, supporting the hypothesis that these soft rot pathogens may grow synergistically in planta. Barcoded TnSeq is particularly useful for analyzing mixed cultures that are more representative of typical field conditions. Understanding the interactions of Dickeya and Pectobacterium will help in efforts to find innovative solutions for management of these pathogens.

We had initially proposed to develop TnSeq libraries for Pantoea to identify genes important for fitness and genes involved in the regulation of an important virulence cluster called HiVir. However, as an alternative, we pursued a new functional genomics approach called SorTnSeq. SorTnSeq uses transposon mutagenesis with fluorescence-activated cell sorting and allows for the identification of factors that affect expression of a gene of interest. Although we encountered difficulties performing the SorTn-Seq experiment, we obtained some mutants that have altered expression of HiVir. We continue to investigate these mutants in the hopes that they provide targets for further study.   

Some bacteria, like Pseudomonas syringae, change their behavior depending on their proximity and relationship to neighboring cells. To determine the mechanistic basis of these behaviors we first determined what type of bacterial functions are responsible. We found that flagellar motility has a large role in competitive expansion into areas occupied by unrelated bacteria. Next, we determined all genes necessary for flagellar motility and the set of genes differentially expressed in cells engaged in competitive expansion. Additional work is needed to determine a causal relationship between individual genes and this competitive behavior. Understanding this behavior may help explain why pathogens are able to outcompete other bacteria in pathogenic interactions. 

The transcription regulator, AlgU, is necessary for P. syringae to cause disease in plants. We pursued two lines of investigation to further our understanding of how AlgU controls the functions required for virulence. First, we identified a gene necessary for AlgU to down regulate flagellar expression, which helps the bacteria avoid activating the plant immune systems. Additional work is needed to determine the function of this gene. Second, we determined that AlgU is activated by low pH and that plants raise the pH at the site of infection, which suppresses AlgU activation. We also determined the complete set of genes induced in low pH and suppressed in high pH, and which of those genes require AlgU. 

Objective 3: Investigate sustainable strategies for control of bacterial plant diseases. We purchased purified peptides that we hypothesized would exhibit bactericidal or bacteriostatic activity against bacterial soft rot pathogens. Assay were performed with various concentrations of the peptides. We found one peptide that inhibited growth of several plant soft rot bacteria. We are currently determining if the peptide has antimicrobial activity against a broad range of plant pathogens and exploring the underlying mechanism of action of this peptide. This peptide has potential application for combating plant diseases caused by bacteria. Surprisingly, another peptide (a putative plant host defense peptide) increased growth of the plant pathogenic bacteria in vitro. Studies are in progress to determine if the peptide is inducing specific behavior of the bacteria and/or modifying bacterial physiology and able to stimulate growth of other plant pathogens. This year we surpassed our milestone to “Screen 200 individuals from 3 families of hybrid progeny for soft rot tolerance trait segregation”, by completing that goal early and then used that information to guide experiments to map chromosomal regions responsible for soft rot tolerance. **Accomplishments**
                                

1. One step closer to soft rot resistant potato. Scientists mapped the genetic basis of potato soft rot resistance from wild potato relatives. USDA scientists in Ithaca, New York, and Sturgeon Bay, Wisconsin, have identified the parts of three potato chromosomes that help protect potato tubers from soft rot disease caused by bacteria. Mapping was done by cross-pollinating a resistant wild potato plant with a domesticated potato to produce several families of progeny plants. We tested each of the progeny plants to determine their soft rot sensitivity and then correlated that with genetic features from either or both parents. This allowed us to find the parts of the chromosomes that came from the resistant parent and carry the resistance trait. Knowing the parts of the chromosome that carry soft rot disease resistance is a needed step to transferring resistance to protect cultivated potato. 2. Soft rot pathogen census. There are many species of bacteria that cause soft rot disease on potato and other crops (21 Pectobacterium and 12 Dickeya species). In cooperation with university colleagues, scientists in Ithaca, New York, surveyed bacteria responsible for soft rot diseases from all the major potato growing regions of the United States between 2016-2021. The species of each soft rot bacteria was determined by analyzing the full genome sequence. The results of the survey show that there are at least 11 soft rot bacterial species present and causing disease of potato in the United States. Pectobacterium carotovorum is the most widespread, being present in 9 of the 14 states tested. In contrast, other pathogens are restricted to certain regions, like Dickeya dianthicola, which was only found in the eastern United States. This information is essential to understand the ecology and epidemiology of these pathogens and to inform potato seed certification programs and federal regulatory programs helping to protect food security and livelihood of American farmers. **Review Publications**
                            *Helmann, T.C., Filiatrault, M.J., Stodghill, P. 2022. Genome-wide identification of genes important for growth of dickeya dadantii and dickeya dianthicola in potato (solanum tuberosum) tubers. Frontiers in Microbiology. 13:778927. https://doi.org/10.3389/fmicb.2022.778927.*
                                *Djami-Tchatchou, A., Li, Z., Stodghill, P., Filiatrault, M.J., Kunkel, B. 2021. Identification of IAA-regulated genes in Pseudomonas syringae pv. tomato strain DC3000. Journal of Bacteriology. https://doi.org/10.1128/JB.00380-21.*
                                *Canada-Bautista, M., Reyes-Proano, E., Cornejo-Franco, F., Alvarez-Quinto, R., Mollov, D.S., Quito-Avila, D. 2022. Genomic characterization of a new potyvirus infecting thevetia ahouai. Archives of Virology. https://doi.org/10.1007/s00705-022-05520-9.*
                                *Ma, X., Perry, K., Swingle, B.M. 2023. Complete genome sequence resource for potato ring rot pathogen, Clavibacter sepedonicus K496. Plant Disease. 107(4):1202-1206. https://doi.org/10.1094/pdis-06-22-1404-a.*
                                *Ma, X., Lofton, L., Bamberg, J.B., Swingle, B.M. 2022. Identification of resistance to Dickeya dianthicola soft rot in Solanum microdontum. American Journal of Potato Research. https://doi.org/10.1007/s12230-021-09859-8.*
                                *Zhang, X., Ge, T., Fan, X., Ma, X., Swingle, B.M., Leiby, R., Johnson, S., Larkin, R.P., Chim, B., Hao, J. 2023. First report of pectobacterium brasiliense causing bacterial blackleg and soft rot on potato in Pennsylvania. Plant Disease. 36774584. https://doi.org/10.1094/PDIS-09-22-2085-PDN.*
                                *Ma, X., Brazil, J., Rivedal, H.M., Perry, K.L., Frost, K., Swingle, B.M. 2022. First report of Pectobacterium versatile causing potato soft rot in Oregon and Washington. Plant Disease. https://apsjournals.apsnet.org/doi/epdf/10.1094/PDIS-08-21-1635-PDN.*


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