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Title: A microbiological study of Erwinia amylovora exopolysaccharide ooze
Creator: Slack, Suzanne Marie
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Fire blight, caused by the pathogen Erwinia amylovora (Burrill) Winslow et al, is the most devastating bacterial disease of pome fruits around the world. The primary dispersal of E. amylovora is through ooze, a mass of exopolysaccharides and bacterial cells that is exuded from infected host tissue. Over the 2013 and 2014 field seasons, 631 ooze droplets (201 in 2013 and 435 in 2014) were collected from field inoculated trees. Populations of E. amylovora in ooze droplets range from 108 to 1011...
Show moreFire blight, caused by the pathogen Erwinia amylovora (Burrill) Winslow et al, is the most devastating bacterial disease of pome fruits around the world. The primary dispersal of E. amylovora is through ooze, a mass of exopolysaccharides and bacterial cells that is exuded from infected host tissue. Over the 2013 and 2014 field seasons, 631 ooze droplets (201 in 2013 and 435 in 2014) were collected from field inoculated trees. Populations of E. amylovora in ooze droplets range from 108 to 1011 colony forming units per micro liter (cfu/μl). In the host tissue surrounding the droplets even larger populations of E. amylovora reside in the surrounding 1 cm of tissue. Three apple cultivars with varying levels of resistance were also infected with four Michigan E. amylovora strains. Using scanning electron microscopy, host tissue was examined for the origin of the ooze droplets and erumpent mounds and small (10 μm) tears were the only bacterial sources observed. Genetic expression analysis indicated that E. amylovora cells in stem sections located above ooze drops and in ooze drops were actively expressing virulence genes. If disseminated to susceptible host tissue, these cells would be primed for infection. The current study suggests the following: high populations of E. amylovora are present in ooze droplets which larger populations found in darker pigmented, smaller volume droplets. These droplets are rupturing out from the parenchyma and epidermis of the host, with evidence of immense pressure being involved from SEM observations. Ooze droplet volume and population can vary between host cultivar and the virulence of a specific E. amylovora strain. Genetic expression analysis of virulent factors in E. amylovora indicated that the bacteria in ooze were primed and ready to infect a new susceptible host.
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Title: Cloning of the polysaccharide depolymerase gene of bacteriophage pea1 (h) and its expression in Erwinia amylovora
Creator: Hartung, John Stephen
Date: 1985
Collection: Electronic Theses & Dissertations

Title: Cyclic di-GMP dependent regulation in the plant pathogenic bacterium Erwinia amylovora
Creator: Castiblanco Mosos, Luisa F.
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Bacterial pathogenesis generally depends on the ability to effectively coordinate the synthesis and expression of pathogenicity and virulence factors in order to successfully colonize a host. In some cases, this involves a need to rapidly reprogram cellular behaviors in response to host defenses or based on spatial location in the host. This genetic regulation often involves small signaling molecules such as cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger synthesized...
Show moreBacterial pathogenesis generally depends on the ability to effectively coordinate the synthesis and expression of pathogenicity and virulence factors in order to successfully colonize a host. In some cases, this involves a need to rapidly reprogram cellular behaviors in response to host defenses or based on spatial location in the host. This genetic regulation often involves small signaling molecules such as cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger synthesized intracellularly and degraded by diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. This nucleotide signaling molecule is involved in the regulation of many cellular processes in numerous bacterial species, including the transition from motile to sessile lifestyle, virulence, biosynthesis of exopolysaccharides and adhesion structures, and cell differentiation. Signal transduction and phenotypic modulation is determined by binding of c-di-GMP to specific downstream receptor molecules.Erwinia amylovora uses motility and the hypertensive response and pathogenicity- (Hrp-) type III secretion system (T3SS) during initial phases of plant infection. Once inside the xylem, this pathogen aggregates due to the production of exopolysaccharides (EPS) and attachment structures forming a biofilm that ultimately plugs the xylem vessels. Although c-di-GMP has been shown to be an important intracellular signal in several plant pathogenic bacteria, the importance of this molecule in Erwinia amylovora has not been previously investigated. This doctoral research explores the role of c-di-GMP in the genetic regulation of key cellular processes associated with pathogenesis in E. amylovora. Five active DGC enzymes (EdcA, EdcB, EdcC, EdcD and EdcE) were identified in this bacterial pathogen. Phenotypic analyses demonstrated that c-di-GMP positively regulates the biosynthesis of both cellulose and amylovoran, positively regulates biofilm formation, and represses motility. Disease assays demonstrated that c-di-GMP negatively regulates virulence in these infection models.The exopolysaccharides amylovoran and levan and attachment structures such as fimbriae, type IV pili and curli, have been demonstrated to be critical for the formation of a mature biofilm in E. amylovora. The results presented in this work demonstrated that c-di-GMP binds to the receptor protein BcsA, the cellulose synthase subunit, and activates the biosynthesis of cellulose in E. amylovora at a post-translational level. In addition, using SEM and confocal microscopy, it was demonstrated that cellulose is a main component of the biofilms formed by E. amylovora in vitro and in the host. Gene overexpression and site-directed mutagenesis analyses, suggest that BcsZ, an endoglucanase, is also required for cyclic di-GMP activation of cellulose biosynthesis and biofilm formation.Further investigation into the mechanisms of c-di-GMP dependent regulation of virulence, with a special emphasis on the regulation of hrp-T3SS gene expression, revealed that high intracellular levels of c-di-GMP via DGC overexpression lead to a significant reduction in gene expression for hrpL, the hrp alternative sigma factor, and dspE. C-di-GMP binding assays suggest that HrpS, the σ54 dependent transcriptional regulator of hrpL, is a candidate receptor of c-di-GMP. Moreover, the results presented in this work indicate that HrpS can bind to other guanosine-containing signaling molecules including the c-di-GMP degradation product pGpG. This work provides an overview of some of the molecular mechanisms for c-di-GMP-dependent regulation on the main pathogenicity determinants in E. amylovora, in order to orchestrate pathogenesis and successfully cause disease.
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Title: Cyclic di-GMP regulates EPS secretion, biofilm formation, and motility in the plant pathogen Erwinia amylovora
Creator: Edmunds, Adam Christopher
Date: 2011
Collection: Electronic Theses & Dissertations
Description: The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes including biofilm formation, motility, and virulence. This intracellular concentration of this molecule is inversely governed by GGDEF-containing proteins (GCPs) and EAL-containing proteins (ECPs), which synthesize and degrade c-di-GMP, respectively. The role of this regulatory molecule in the plant pathogen and causal agent of fire blight disease, ...
Show moreThe second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes including biofilm formation, motility, and virulence. This intracellular concentration of this molecule is inversely governed by GGDEF-containing proteins (GCPs) and EAL-containing proteins (ECPs), which synthesize and degrade c-di-GMP, respectively. The role of this regulatory molecule in the plant pathogen and causal agent of fire blight disease, Erwinia amylovora was explored. In this study, it is demonstrated that E. amylovora contains three functional diguanylate cyclases (DGCs) that synthesize c-di-GMP: gcp1, gcp3, and gcp5; and two functional c-di-GMP specific phosophodiesterases (PDEs) that degrade c-di-GMP: ecp1 and ecp3. C-di-GMP was not detected in the wild type strain. C-di-GMP was shown to positively regulate biofilm formation and secretion of the main expolysaccharide amylovoran and to inversely regulate swimming motility and virulence. A deletion of ecp3 resulted in a hyper-biofilm forming phenotype. Interestingly, over-expression of dgc1, which contains both an EAL and a GGDEF domain, resulted in no observable phenotypic differences from the wild type strain, even though over-expression of dgc1 increased the levels of c-di-GMP.
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Title: Ecological and genetic analysis of streptomycin-resistant Erwinia amylovora in Michigan and epidemiology of fire blight in an apple nursery
Creator: McManus, Patricia S.
Date: 1994
Collection: Electronic Theses & Dissertations

Title: Fungicide resistance analysis of major tree fruit pathogens in Michigan
Creator: Lesniak, Kimberley E.
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Chemical and/or antibiotic resistance management is critical in order to reduce the progression or complete loss of a product while effectively controlling plant pathogen populations. This dissertation focuses on the observation for control and resistance management of four major worldwide fruit tree pathogens: Venturia inaequalis (apple scab), Monilinia fructicola (American brown rot), Erwinia amylovora (Ea; fire blight) and Pseudomonas syringae pv. syringae (Pss; bacterial canker). Chemical...
Show moreChemical and/or antibiotic resistance management is critical in order to reduce the progression or complete loss of a product while effectively controlling plant pathogen populations. This dissertation focuses on the observation for control and resistance management of four major worldwide fruit tree pathogens: Venturia inaequalis (apple scab), Monilinia fructicola (American brown rot), Erwinia amylovora (Ea; fire blight) and Pseudomonas syringae pv. syringae (Pss; bacterial canker). Chemical fungicides have been and are still heavily relied on for management of the fungal plant pathogens V. inaequalis and M. fructicola. Single-site fungicides such as the quinone-outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) have shown to provide excellent control but can also select for resistant pathogen populations in a relatively short time period, as in the case for both V. inaequalis and M. fructicola. Adequate control of bacterial pathogens, such as Ea and Pss has been a challenge for pome and stone fruit growers for decades. Antibiotics have been intensively used for the management of Ea while only copper formulations are registered for Pss. The current research implicates that orchards in Michigan and in other states cannot further rely on QoI fungicides for apple scab control. Further investigation of peach and cherry orchards indicates that SDHI applications have shifted Michigan populations toward resistance. Results from studies performed with Ea and Pss indicate that non-chemical applications such as acibenzolar-S-methyl (ASM) and phosphate salts have a potential to reduce the use of antibiotics and copper for control in seasons when disease pressure is low to moderate.
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Title: Identification and analysis of induced genes from Erwinia amylovora and Malus x domestica during fire blight infection
Creator: Blumer-Schuette, Sara E.
Date: 2006
Collection: Electronic Theses & Dissertations

Title: Investigating carbohydrate utilization and virulence in Erwinia amylovora
Creator: Sweeney, Emma M.
Date: 2018
Collection: Electronic Theses & Dissertations
Description: Fire blight, caused by the gram-negative bacterium Erwinia amylovora, is a destructive disease of apple and pear trees worldwide. A unique aspect of apple and pear physiology is the presence of sorbitol in the leaves and shoots, glucose on the flower stigma surface and sucrose in the flower nectary. Erwinia amylovora cells encounter all three carbohydrates at different stages of infection, and it is unknown how the carbohydrate utilization genes are regulated between these changing nutrient...
Show moreFire blight, caused by the gram-negative bacterium Erwinia amylovora, is a destructive disease of apple and pear trees worldwide. A unique aspect of apple and pear physiology is the presence of sorbitol in the leaves and shoots, glucose on the flower stigma surface and sucrose in the flower nectary. Erwinia amylovora cells encounter all three carbohydrates at different stages of infection, and it is unknown how the carbohydrate utilization genes are regulated between these changing nutrient environments. This thesis explores carbohydrate utilization by E. amylovora in relation to virulence, regulatory small RNAs (sRNAs), other virulence factors and host specificity. The findings presented here indicate that sorbitol utilization (srl) gene mutants of E. amylovora are amylovoran-deficient, and they are unable to obtain the energy base needed to infect apple shoots and immature pear fruits. Additionally, the sRNA Spot 42 does not regulate sorbitol as it does in Escherichia coli, and we hypothesize that E. amylovora has evolved to evade Spot 42 regulation in order to adapt to the high-sorbitol content of apple and pear hosts.
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Title: Resistance against Erwinia amylovora induced in apple trees by acibenzolar-s-methyl
Creator: Maxson, Kimberly Lynn
Date: 2000
Collection: Electronic Theses & Dissertations

Title: Role of waaL and umuDC in Erwinia amylovora EA1189 in oxidative stress and ultraviolet radiation survival
Creator: Berry, Matthew
Date: 2009
Collection: Electronic Theses & Dissertations

Title: Roles of hfq-dependent srnas in e. amylovora regulation of virulence
Creator: Schachterle, Jeffrey Kent
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Erwinia amylovora is the causative agent of fire blight disease of apple and pear trees, causing annual losses of over 100 million USD in the USA. E. amylovora cells are disseminated to new hosts by insects, wind, and rain, and then invade susceptible tissues and migrate systemically throughout the host, requiring coordinate regulation of several virulence factors, including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III...
Show moreErwinia amylovora is the causative agent of fire blight disease of apple and pear trees, causing annual losses of over 100 million USD in the USA. E. amylovora cells are disseminated to new hosts by insects, wind, and rain, and then invade susceptible tissues and migrate systemically throughout the host, requiring coordinate regulation of several virulence factors, including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III secretion. Complex regulatory mechanisms have evolved in E. amylovora that occur at the transcriptional, post-transcriptional, and post-translational levels to control these virulence factors. In my work, I analyze the role of small RNAs (sRNAs) as post-transcriptional regulators of virulence-associated traits in E. amylovora.The Hfq chaperone protein stabilizes sRNAs in the cell, allowing them to interact with and regulate mRNA targets. An hfq mutant differs from wild-type cells in several virulence-associated phenotypes including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III secretion. E. amylovora encodes at least 40 Hfq-dependent sRNAs; in my work, I have systematically made deletion mutants of each sRNA singly, as well as constructed inducible expression vectors for each sRNA. Screening of this sRNA library has shown that several sRNAs contribute to regulation of each virulence phenotype, indicating complex regulation of the traits assessed. Of particular interest, the ArcZ sRNA regulates several of the virulence-associated traits we have assessed, and an arcZ deletion mutant loses virulence in both immature pear and apple shoot infection models.Flagellar motility, which enables E. amylovora cells to swim through flower nectar to invade natural openings in host flowers, is regulated by ArcZ. We have shown that ArcZ regulates motility by regulating the flagellar transcription factor FlhD at both the transcriptional and post-transcriptional levels. Because the ArcZ regulation of FlhD at the transcriptional and post-transcriptional levels has a contradiction in sign, we searched for additional layers of regulation between ArcZ and FlhD. We did so by conducting a transposon screen in the arcZ mutant background for suppressor mutants that restored flagellar motility. This screen yielded as the most common suppressor mutation the leucine responsive regulator protein (Lrp), a global transcription factor known for regulation of amino acid metabolism. We have found that Lrp not only acts as a regulator of flagellar motility between ArcZ and FlhD, but that it also reverses the regulatory effects of arcZ deletion on amylovoran and levan production, as well as biofilm formation. Our work shows that Lrp is a novel virulence regulator that plays an important role in regulating several virulence-associated traits in conjunction with the sRNA ArcZ.Transcriptomic comparison between the arcZ mutant and wild-type cells confirmed that ArcZ regulates several genes known to also be regulated by Lrp, and also indicated that ArcZ regulates several genes involved in mitigating the threat of reactive oxygen species, including genes encoding a catalase, a thiol-peroxidase, and a peroxiredoxin. We found that catalase makes the greatest contribution to diminishing the threat of exogenous hydrogen peroxide. Additional analysis suggests that ArcZ participates in regulation with an oxidative sensing transcription factor network that includes the transcription factors ArcA, Fnr, and Fur.This work shows that several sRNAs make small contributions to virulence trait regulation, and that a few sRNAs, like ArcZ, make major contributions to E. amylovora virulence. ArcZ regulates several virulence-associated traits through the global transcription factor Lrp, which we have found to be a novel virulence regulator. ArcZ also regulates genes involved in mitigating the threat of reactive oxygen species, which can protect E. amylovora cells from host defenses during infection. Thus, ArcZ plays an integral role in modulating phenotypic expression during fire blight disease progression that enables E. amylovora to successfully colonize and infect host plants. Mechanistic understanding of E. amylovora gene regulation moves us closer to understanding weaknesses that can be exploited for development of novel disease control strategies.
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Title: Secretion signals and chaperone function in Erwinia amylovora
Creator: Triplett, LIndsay R.
Date: 2009
Collection: Electronic Theses & Dissertations

Title: Studies on the nutrition of Bartlett pear trees (Pyrus dommunis L) and fire blight (Erwinia amylovora, Burr.)
Creator: Lewis, Lowell Nelson
Date: 1960
Collection: Electronic Theses & Dissertations

Title: The mechanisms of streptomycin resistance in Erwinia amylovora
Creator: Chiou, Chien-Shun
Date: 1994
Collection: Electronic Theses & Dissertations

Title: The role of biofilm formation in systemic movement of Erwinia amylovora in apple
Creator: Koczan, Jessica
Date: 2011
Collection: Electronic Theses & Dissertations
Description: Erwinia amylovora is a highly virulent, necrogenic plant pathogen that causes fire blight disease on apple, pear, and other rosaceous plants. The fire blight pathogen is highly invasive and capable of rapid systemic movement through plants. Current methods of control focus on chemical and antibiotic treatments. The popularity of highly susceptible cultivars and the emergence of antibiotic resistance have driven the need to find other methods to control fire...
Show moreErwinia amylovora is a highly virulent, necrogenic plant pathogen that causes fire blight disease on apple, pear, and other rosaceous plants. The fire blight pathogen is highly invasive and capable of rapid systemic movement through plants. Current methods of control focus on chemical and antibiotic treatments. The popularity of highly susceptible cultivars and the emergence of antibiotic resistance have driven the need to find other methods to control fire blight. The study of pathogen virulence factors has the potential to identify novel control methods. Several bacterial virulence factors have been shown to be critical for biofilm formation, the production in a complex aggregated network of bacterial cells, exopolysaccharides, and other macromolecules. In addition, vascular plant pathogens commonly use the ability of biofilm formation to aid in the systemic movement of the pathogen. Research presented here used in silico analysis to identify several virulence factors. Biofilm formation and virulence assays determined that virulence factors that contribute to the systemic movement of E. amylovora in vascular tissue of apple. The production of exopolysaccharides amylovoran and levan was determined to be needed for the formation of a mature biofilm. Though cells are motile, amylovoran deficient mutants are unable to move past the site of inoculation. Levan deficient mutants display a delayed, reduced virulence phenotype. Several putative bacterial surface proteins, or attachment structures, assist in the initial attachment (both reversible and irreversible) of the pathogen to host tissue. Deletions in genes encoding for the production of attachment structures drastically reduce the biofilm capability of E. amylovora, and the ability to get into the xylem tissue. In addition, mutation of functional flagellar motor stators demonstrated that motility was important in mediating contact of bacterial cells to surfaces. Motility of the fire blight pathogen is shown to be important in the movement of bacterial cells within host tissue. Additionally, motility seems to be important in the expansion of biofilms. In total, the contribution of the virulence factors to biofilm formation and the localization of the pathogen in host tissue imply that biofilm formation assists in the systemic movement of E. amylovora in apple.
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