Browsing by Author "Charkowski, Amy O., committee member"

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Open Access
Biofilm dynamics and the response to N-oxides in Burkholderia pseudomallei
(Colorado State University. Libraries, 2019) Mangalea, Mihnea R., author; Borlee, Bradley R., advisor; Slayden, Richard A., committee member; Bowen, Richard A., committee member; Stenglein, Mark D., committee member; Charkowski, Amy O., committee member
Burkholderia pseudomallei is a saprophytic bacterium inhabiting wet soils in tropical regions and is the causative agent of melioidosis, an emerging infectious disease of high mortality. Although the incidence of melioidosis is more prevalent in the monsoonal wet season in Southeast Asia and Northern Australia, gardens and farms also serve as a reservoir for B. pseudomallei infection in the dry season, due to anthropogenic disturbances including irrigation and application of nitrogen (N)-based fertilizer use. Melioidosis is historically associated with rice farming in rural regions of the tropics where rain-fed lowland environments predominate and planting fields are often managed by the addition of N-based fertilizers to keep up with the demand for global rice consumption. In these oxygen-limiting environments, B. pseudomallei is a facultative anaerobic organism capable of growth in anoxic conditions by substituting nitrate (NO3-) as a terminal electron acceptor. B. pseudomallei is capable of complete denitrification, a step-wise enzymatic reaction that is carried out by four individual enzyme complexes or reductases, that reduce NO3- to N2. Denitrification among proteobacteria is regulated by sensing systems that depend on both the presence of substrate and hypoxic conditions, however little is known about this ecological and physiological phenomenon in B. pseudomallei. In hosts infected with B. pseudomallei, similar oxygen tensions are experienced by the organisms in abscesses, lesions, and during intracellular growth; however, little is known regarding the extent of anaerobic metabolism and defense from host-associated reactive nitrogen intermediates in B. pseudomallei. This study examines the predicted nitrate sensing and metabolism genes in a clinical isolate, B. pseudomallei 1026b, and specifically their role in regulating biofilm dynamics. We hypothesized that nitrate sensing and metabolism negatively regulate biofilm formation and aimed to describe the genetic and metabolic determinants of this phenotype in B. pseudomallei. In Aim I of this study, we characterized a dose-dependent biofilm inhibition model that responds to increasing concentrations of sodium nitrate and sodium nitrite, donors of the inorganic anions NO3- and NO2-, respectively. Based on in silico analyses of predicted nitrate sensing and metabolism loci, we screened transposon insertional mutants to identify candidates involved in the biofilm inhibitory response. We identified five mutants that no longer respond to nitrate-mediated biofilm inhibition in genes predicted to comprise key components of the denitrification pathway: the alpha and beta subunits of the dissimilatory nitrate reductase narGHJI-1, the narX-narL two-component regulatory system, and the nitrate/nitrite extrusion gene narK-1. Using LC-MS/MS, we quantified the intracellular concentration of the secondary metabolite cyclic-di-GMP, and observed a significant decrease of this key biofilm-associated molecule in response to sodium nitrate treatment. Furthermore, we evaluated the expression of cyclic-di-GMP regulatory enzymes to propose a mechanism for the nitrate-dependent biofilm inhibition phenotype in B. pseudomallei. In Aim II, we examined the functions of NarX and NarL in response to exogenous sodium nitrate and sodium nitrite and the biofilm inhibition model using separate in-frame deletion mutants. We characterized a disparity in biofilm inhibition that is dependent on nitrate but not nitrite in this two-component sensing system, before analyzing the global transcriptome of these mutants relative to the wild type in growth conditions supplemented with either N-oxide. Differential expression analysis of RNA sequencing reads revealed significant transcriptomic shifts in several gene clusters associated with biofilm formation, nitrate metabolism, general metabolism, antibiotic resistance, virulence, and secondary metabolite biosynthesis that responded similarly to both NO3- and NO2- supplementation. Additionally, we demonstrated that narX and narL mutants are deficient in intracellular survival in murine macrophages, providing a link between nitrate sensing and metabolism and B. pseudomallei host-pathogen interactions. These data suggest that denitrification is an important mechanism for biofilm dynamics and is also relevant to survival and pathogenicity in animal hosts during B. pseudomallei infection.
Open Access
Characterizing host plant-virus-vector interactions of the potato virus Y and aphid pathosystem
(Colorado State University. Libraries, 2023) Pitt, William Jacob, author; Nachappa, Punya, advisor; Charkowski, Amy O., committee member; MacRae, Ian V., committee member; Peairs, Frank B., committee member; Smith, Melinda D., committee member
Aphid-transmitted potato virus Y (PVY) is one of the most damaging pathogens of potato worldwide. Plant virus prevalence is influenced by landscape composition, host-use patterns of vectors, and the range of capable vector species. Regarding these influences, there are important knowledge gaps that remain within the PVY-aphid pathosystem. The overall goal of this research was to better understand host-virus-vector interactions within the PVY-aphid pathosystem at multiple levels of ecological organization. There is limited information on the effect of landscape-scale crop diversity on prevalence of insect-vectored viruses. In my dissertation, I investigate how landscape composition of crops (Shannon diversity of crops, percent crop cover) affects aphid vector communities and prevalence of aphid-transmitted PVY. I conducted a two-year field study in the San Luis Valley in Colorado where I sampled aphid communities with pan traps, quantified PVY incidence in potato crops with ELISA, and determined the association with landscape variables (Shannon diversity index of crops and percent crop cover) surrounding sampling sites. Crop diversity negatively influenced aphid species richness, but positively influenced PVY incidence. The negative association of crop diversity with aphid species richness could have been due to differences in management between crops and/or increased predation/parasitization of aphids. The positive association between crop diversity and PVY is likely because PVY has a wide host range and increasing crop diversity may positively influence PVY incidence due to increased inoculum in the landscape. Additionally, there was a positive association of potato (virus host) and a negative association of barley (virus non-host) with PVY incidence. In summary, I found that crop species diversity influenced both PVY prevalence and aphid communities, and that the virus host/non-host status of crops likely modulates this effect. Aphid vectors frequently probe upon various plants within a landscape, but the host use patterns of aphid vectors has not been adequately described. It would be useful to identify plants that aphid vectors are probing upon within a landscape in order to identify 1) vector movement within a landscape and 2) potential sources of aphid-transmitted virus inoculum. I used high throughput molecular gut content analysis (GCA) to characterize plant-aphid vector associations within a major potato producing region, the San Luis Valley, CO, where aphid-transmitted potato virus Y (PVY) is a major issue for potato production. Aphids were trapped weekly with suction traps during the growing seasons of 2020 and 2021. Plant-specific DNA in 200 individual aphids representing 9 vector species of PVY (Acyrthosiphon kondoi, A. pisum, Capitophorus elaeagni, Diuraphis noxia, Hayhurstia atriplicis, Myzus persicae, Phorodon cannabis, Protaphis middletonii, and Rhopalosiphum padi) were amplified by PCR, sequenced with the PacBio sequencing platform, and sequences were identified to genus using NCBI BLASTn. I found that all species of aphid vectors probed on plant genera that are outside of their reported host ranges, with Solanum, Medicago, Populus, Brassica, and Glycine as the most frequently detected plant genera. Moreover, aphids probed on many plant genera likely to be PVY host plants. These findings provide information that is essential to develop sustainable and effective management strategies to reduce PVY spread. With the increasing acreage of hemp (Cannabis sativa L.) (Rosales: Cannabaceae) in the United States, I was interested to know whether the cannabis aphid (Phorodon cannabis Passerini) (Hemiptera: Aphididae) is a potential vector of PVY. I conducted transmission assays and used the electrical penetration graph (EPG) technique to determine whether cannabis aphids can transmit PVY to hemp (host) and potato (non-host) (Solanum tuberosum L.) (Solanales: Solanaceace). I showed, for the first time, that the cannabis aphid is an efficient vector of PVY to both hemp (96% transmission rate) and potato (91%) using cohorts of cannabis aphids. In contrast, individual aphids transmitted the virus more efficiently to hemp (63%) compared to potato (19%). During the initial 15 min of EPG recordings, aphids performed fewer intracellular punctures and spent less time performing intracellular punctures on potato compared to hemp, which may in part explain low virus transmission to potato using individual aphids. During the entire 8-hour recording, viruliferous aphids spent less time ingesting phloem compared to non-viruliferous aphids on hemp. This reduced host acceptance could potentially cause viruliferous aphids to disperse thereby increasing virus transmission. Overall, my study shows that cannabis aphid is an efficient vector of PVY and that virus infection and host plant suitability affect feeding behaviors of the cannabis aphid in ways which may increase virus transmission.
Open Access
Fire-associated shifts in the soil microbiome in western conifer forests: implications for Armillaria root disease biocontrol and management
(Colorado State University. Libraries, 2024) Fitz Axen, Ada J., author; Stewart, Jane E., advisor; Kim, Mee-Sook, committee member; Charkowski, Amy O., committee member; Abdo, Zaid, committee member
The research presented in this thesis integrates the current understanding of environmental disturbances, plant associated microbiomes, and microbial biological control of fungal forest pathogens to contribute to improved disease management. In Chapter 2, I examined how fire disturbances affect soil microbial communities in areas where Armillaria root disease, caused by the pathogen Armillaria solidipes, is prevalent by documenting changes to bacterial and fungal community diversity and composition following three distinct levels of burn severity (high, low, and unburned) in a conifer forest in northern Idaho, United States. Expected reductions in bacterial community alpha diversity were observed when comparing burned communities with unburned communities; however, fungal communities showed a lack of significant change in alpha diversity in response to burn severity at the sampling time of 15-months post-fire. However, in both bacterial and fungal soil communities, compositional changes corresponding to burn severity levels were observed. Further examinations characterized similarities and differences between burn severity-associated communities and Armillaria species-associated communities to determine how these microbial changes might influence Armillaria root disease. At high-severity burn sites, colonization by A. solidipes and the associated microbial community was prevalent when compared with low-severity burn and unburned sites. In contrast, the presence and abundance of the weakly pathogenic species A. altimontana and its associated microbial community, including beneficial ectomycorrhizal fungi, appeared to be negatively impacted by high-severity burns. Further research is needed to determine which microbial taxa are critical for promoting or suppressing A. solidipes activity, yet the results from this study suggest that high-severity burns may create environments hospitable to this pathogen and thus monitoring for increased disease pressure following severe burns may be warranted. Chapter 3 focuses specifically on beneficial members of the native soil microbial community that exhibit antagonistic activity against A. solidipes. Because these native species are adapted to the environmental conditions and community interactions, they are more likely than foreign microbial species to successfully establish a stable population required for effective biological control. I isolated putative native biological control agents from soil samples collected under different burn severity conditions and tested their in vitro capabilities to inhibit the growth of A. solidipes with dual culture confrontation tests. Effective in vitro pathogen inhibition was observed with 10 microbial isolates, including five bacterial isolates from the genera Bacillus and Caballeronia and five fungal isolates from the genera Trichoderma and Mortierella. Further examination of the sites these microbes and communities originated from and their compositional changes documented in Chapter 2 revealed that the presence or abundance of our effective biological control organisms did not differ based on burn severity. Importantly, this suggests that fire disturbances may not directly influence the use of these species in management methods for Armillaria root disease in similar conifer forests. However, considering the increased presence of A. solidipes observed following a high-severity burn, there may be additional biotic or abiotic influences apart from biological control agents that are influencing the activity of A. solidipes after fire. These studies enhance our understanding of how abiotic and biotic influences interact to affect the presence of virulent soilborne forest pathogens and associated soil microbes. Considering the effects of these interactions is critical for the development of sustainable long-term management strategies that will help to preserve these ecosystems facing increasing environmental and pathogen-related stressors. The overall goals of this research are to build upon the growing body of research examining how the soil microbiome contributes to disease development and to provide tangible results that can be incorporated by forest managers to help reduce damage caused by Armillaria root disease in fire-prone conifer forests.