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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Emerging Pests and Pathogens Research » Research » Research Project #432528

Research Project: Characterization of Molecular Networks in Diseases Caused by Emerging and Persistent Bacterial Plant Pathogens

Location: Emerging Pests and Pathogens Research

2019 Annual Report

Objective 1: Characterize the genomes of emerging and persistent bacterial plant pathogens, including Pectobacterium and Dickeya species, to identify pathogenicity and virulence factors. Objective 2: Functionally characterize key metabolic and virulence pathways that contribute to pathogenesis in emerging and persistent bacterial pathogens of potato and tomato. Sub-Objective 2.1: Characterize bacterial regulators that contribute to virulence. Sub-Objective 2.2: Characterize the roles of bacterial genes involved in calcium precipitation. Sub-Objective 2.3: Identify genes involved in host-pathogen interactions. Objective 3: Develop and test strategies that target pathogen biology or host interactions for control of emerging and persistent bacterial plant pathogens. Sub-Objective 3.1: Test anti-virulence (AV) approaches for inhibiting bacterial virulence and plant disease. Sub-Objective 3.2: Identify novel inhibitors that target bacterial genes involved in calcium precipitation. Sub-Objective 3.3: Identify and characterize antisense RNA molecules that target metabolic or virulence factors of bacterial pathogens.

Bacterial plant pathogens cause significant economic losses by reducing crop yields and value or by degrading post harvest handling and storage qualities. High value, vegetable, fruit and nursery crops, are particularly vulnerable because diseases reduce productivity and value by diminishing appearance. The threat of newly emerging plant pathogens has increased due to the combined influence of globalization and climate change, which serve to introduce and alter pathogen range and disease dynamics. As such, research is needed to develop novel control strategies that enable growers to quickly and effectively respond to emerging and persistent bacterial plant pathogens. Our proposed studies will use state of the art high-throughput genomic and molecular methods to understand how bacteria infect and cause plant disease and how this information can be directed toward the development of novel methods to manage bacterial plant pathogens of agricultural importance. Specifically, we will focus our efforts on bacterial pathogens of solanaceous crops, such as bacterial speck of tomato caused by Pseudomonas syringae pv. tomato and blackleg disease of potatoes caused by a disease complex that includes Pectobacterium spp. and Dickeya spp. We expected to discover novel conceptual information regarding microbial adaptations that facilitate plant associations and disease. This information will guide new and environmentally sound management strategies that target features of the pathogen's biology or host interactions, specifically virulence factors. Our proposed studies are expected to result in new and innovative approaches for managing plant pathogens and ultimately increase plant health and production.

Progress Report
Objective 1: Genomic analysis of bacteria causing potato blackleg disease was conducted. Scientists in Ithaca, New York fully sequenced the genomes of 50 pathogenic bacterial strains isolated from diseased potato plants collected from New York State farms. Genome sequences are currently being analyzed to determine the genetic relationships among these bacteria. This information will help the researchers understand how these bacteria spread in the United States potato production system. The genetic relationship information will be used to determine whether the current American blackleg disease outbreak resulted from a recent introduction of a single, highly virulent strain from a foreign source or resulted from the emergence a population already present (endemic) in the United States. Understanding how this disease emerged is important because this information can inform policy regulating import of plant material from foreign sources. Examination of genome sequences also revealed that some of the pathogenic bacteria isolated belong to a new species of bacteria. ARS researchers in Ithaca, New York are working with scientists to characterize this new species of bacteria, which will aid in understanding the range of bacterial species existing in the United States that are capable of causing Blackleg and soft rot disease symptoms in potato and other crops. Researchers are continuing to analyze and determine the cause of blackleg and soft rot diseases in plants collected on farms, mainly in New York State. This work has led to the discovery that the bacteria causing the American blackleg outbreak is also responsible for an outbreak of a rotting disease in Dahlia plants growing on a fresh cut flower farm in New York State. This is an important discovery because this pathogen has been a costly and destructive problem in the Australia and Europe fresh cut flower production. This is the first time this pathogen has been found in United States commercial fresh cut flower production and researchers will continue to monitor the impact of this disease in the United States. Sub-Objective 2.1: Characterize bacterial regulators that contribute to virulence. This led to the discovery of a new hypothesis about the timing and coordination of gene expression that occurs as bacteria enter plant tissue. Failure to correctly coordinate the timing of gene expression can cause bacteria to lose their ability to successfully infect and cause disease in plants. Researchers are continuing this work to understand the molecular basis of this process. This is an important discovery because it demonstrates that plant pathogenic bacteria have evolved mechanisms to control the timing of changes in the pathogen physiology and structures. Additionally, this discovery suggests that anti-virulence drugs/chemicals targeting the bacterial proteins that control timing and expression of virulence factors may be an effective method for limiting disease and supports the need for further research to identify chemicals that effectively alter the timing of bacterial virulence gene expression. Objective 2.3: Characterizing the roles of bacterial genes involved in calcium precipitation. Calcium signaling is important in the recognition of microbes in plants and the plant defense response. Despite reports showing that bacteria sequester calcium on the surface and this influences disease outcome, it was unknown if plant pathogenic bacteria contain systems for sensing calcium. For this objective, studies continued on further characterization of a calcium-responsive bacterial signaling system in the plant-pathogenic bacterium P. syringae previously shown to regulate expression of factors that contribute to disease and control the ability of the bacterium to produce calcium precipitates on the bacterial cell surface. Further functional assays of the calcium-responsive bacterial signaling system determined that it impacts ability of the bacterium to move by controlling formation and dissolution of calcium precipitates on the bacterial cell surface. The results provide a better understanding of how bacteria interpret signals from the host and coordinate systems necessary for eliciting disease. Importantly, results revealed a previously undescribed mechanism used by bacteria in plant pathogenesis. The results opened up new avenues of research pertaining to the role of calcium precipitation in pathogenesis of bacterial pathogens. The discoveries may lead to new technological and environmental applications. Studies this year were also aimed at investigation of bacterial carbonic anhydrases (CAs). CAs are enzymes that impact the bacterium’s ability to precipitate calcium. They have described roles in survival, invasion and pathogenicity of microorganisms. Information regarding the role CAs play in bacterial plant pathogens is lacking. ARS researchers in Ithaca, New York previously identified a CA produced by the plant pathogenic bacterium P. syringae, described it’s expression by calcium and showed that deletion of a specific CA in P. syringae impacted the ability of the bacterium to cause disease in plants. Further characterization determined the CA in P. syringae influences gene expression of a select number of factors known to be involved in virulence in the bacterium. Studies are underway to evaluate global impacts on virulence gene expression and in vivo inhibition of the CA to investigate effects on pathogenicity and growth P. syringae in planta. Preliminary studies investigating in vivo inhibition of the P. syringae CA did not result in successful inhibition of symptoms of disease or growth. Therefore the experimental approach was redesigned for future experiments. Global transcriptional and proteomic studies to determine the mechanism of bacterial calcium dissolution are in progress. CAs are widely distributed in many organisms. Researchers have identified putative CAs in other plant pathogenic bacteria and constructed deletion mutants to determine if similar phenotypes and mechanisms of calcium precipitation/dissolution exist in other bacteria. The research provides critical information regarding mechanisms used by bacterial plant pathogens to sense and interpret signals and nutritional cues from the host to coordinate virulence and metabolism. It is anticipated that further understanding the process of calcium perception and precipitation will aid in the development of new management strategies for bacterial plant pathogens. Objective 2.3: Identification of genes involved in host pathogen interactions. Focus was on experiments to identify plant defense genes involved in the Dickeya-potato interaction. Dickeya spp. are necrotrophic bacterial pathogens that can cause blackleg disease on potatoes. The blackleg disease has resulted in significant economic losses in the United States and continues to devastate the potato industry. Breeding for resistance to blackleg in commercial potato cultivars is difficult due to the limited knowledge of the host-microbe interactions in this pathosystem. Previously, researchers conducted global transcriptomic analysis to understand the molecular interactions between Dickeya and susceptible or tolerant diploid potatoes. Data mining revealed several enriched terms and gene expression patterns when comparing the expression profiles between tolerant and susceptible potatoes, indicating possible roles in disease resistance. One class of genes that represent promising candidates for future functional investigations encode for putative, previously uncharacterized, antimicrobial peptides (AMPs). AMPs, also known as host defense peptides, are small naturally occurring microbicidal molecules produced by plant innate immune response that function as a first line of defense to kill pathogenic microorganisms. AMPs are recognized as a good source of plant host resistance. The results have led to the hypothesize that the unique AMPs discovered are involved in resistance to soft rot pathogens. Researchers in Ithaca, New York have also identified other genes of interest for breeding for blackleg tolerance in potatoes because their homologs are involved in resistance to other pathogens in other plant hosts. This study of potato global gene expression patterns provides critical information to facilitate the development of novel disease management strategies and accelerate disease-resistance potato breeding processes. Sub-objective 3.2: Identify novel inhibitors that target bacterial genes involved in calcium precipitation. Potato blackleg and soft rot diseases from Dickeya and Pectobacterium spp. are currently controlled using cultural practices, while suitable and effective chemical treatment options are being explored. Current disinfecting agents used for potato storage include hypochlorites, chlorine dioxide, copper quinolinolate, quaternary ammonium, hydrogen peroxide. Recently, the inorganic salt potassium tetraborate tetrahydrate (PTB), has been shown to alleviate soft rot disease caused by Pectobacterium spp. on tomato fruits. The ability of PTB to inhibit growth of Dickeya was investigated. The minimum inhibition concentration of PTB for the six reference Dickeya and Pectobacterium strains was determined. Other studies involved determining the mode of action of PTB against plant pathogens including evaluating mechanisms by which bacteria may become resistant to PTB. Our findings suggest that PTB represents a promising inexpensive antibacterial agent for controlling soft rot pathogens of potatoes in transit and/or storage. However, pathogens may become resistant.

1. Completion of a community resource to aid research progress on finding a solution to the American potato disease outbreak. In 2014 an especially destructive form of blackleg disease became a wide-spread and costly problem in U.S. potato production. ARS researchers in Ithaca, New York, determined and published the genome sequence of the bacterium believed to be responsible for this outbreak. This sequence is publicly available and is being used to facilitate progress on understanding the cause, spread and control of this disease.

Review Publications
Fishman, M., Filiatrault, M.J. 2019. Prevention of surface-associated calcium phosphate by the Pseudomonas syringae two-component system CvsSR. Journal of Bacteriology. 201(7).
Swingle, B.M., Perna, N.T., Glasner, J.D., Hao, J., Johnson, S., Charkowski, A., Perry, K.L., Stodghill, P. 2019. Complete genome sequence of the potato blackleg pathogen Dickeya dianthicola ME23. Microbiology Resource Announcements. 8(7).
Swingle, B.M., Cui, W.G., Zheng, H.L., Zhang, F.B., Zhu, H.T., Gao, M. 2019. First report of Rhizopus oryzae causing Potato Soft Rot in the Hebei Province of China. Plant Disease. 104.
Ma, X., Perry, K., Swingle, B.M., Perry, K. 2018. Pectobacterium and Dickeya responsible for potato blackleg disease in New York state in 2016. Plant Disease. 102(9):1834-1840.