Plant Science & Landscape Architecture Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2797
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Item POSITIONAL CLONING OF BROAD-SPECTRUM LEAF RUST RESISTANCE GENE, LR57, FROM AEGILOPS GENICULATA, A TERTIARY GENE POOL MEMBER OF WHEAT(2023) Schulden, Taylor Francis; Rawat, Nidhi; Erwin, John; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The tertiary gene pool of wheat includes wild relatives like Aegilops geniculata (UUMM, 2n=4x=28) that are valuable genetic reservoirs for novel abiotic and biotic resistance genes. However, modern wheat varieties share limited genomic commonality with these gene pool members presenting barriers to recombination and genetic mapping of desirable genes. We mapped a broad-spectrum leaf rust resistance gene Lr57 located on chromosome 5Mg of Ae. geniculata using a simple but powerful methodology for high-resolution genetic mapping in tertiary gene pool members of wheat. Five gene candidates were revealed all with possible defense related functions. Strategic application of differential expression analysis, Virus-Induced Gene Silencing, and mutagenesis analysis reduced the candidate gene of interest to a singular and novel ID-NLR resistance gene containg a protein kinase, NB-Arc, and LRR domain. Using multiple strategies, validation of Lr57 candidate was completed. Gene complementation by transformation of Lr57 candidate is currently being conducted.Item Investigating the hyperdiversity of fungal endophytes in wild Rubiaceae tropical plants and coffee plantations.(2022) Castillo Gonzalez, Humberto; Yarwood, Stephanie A; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal endophytes are an essential component of a plant’s microbiome, their effect spreads to fitness, disease dynamics, stress tolerance, water acquisition and nutrient uptake. Plant ecosystems, from natural forest to plantations bear the indelible signature of its presence. The current investigation was designed to understand the diversity of endophytes in the Rubiaceae family, in plants associated to natural and managed ecosystems. The effect of location, leaf developmental stage, tissue type, host genotype, and anthropogenic interference was evaluated through amplicon sequencing. Costa Rica served as base for the sample collection. Leaves and sapwood from a variety of tropical plant species were collected in old-growth natural forests and foliar tissue from domesticated coffee plants were sampled in two plantations under different management. Fungal diversity was assessed by metabarcoding using the ITS2 nrDNA region fITS7 – ITS4, and library sequencing was completed by Ion Torrent. We identified a hyperdiversity of endophytes inhabiting these plants and were able to isolate a total of 659 fungi from coffee leaves. This investigation provides relevant information about overall community composition, the ecological drivers of community assemblage and the characteristics of the fungal endophytic communities, including potential interactions among the identified taxa. Endophytes may harness the potential to transform agriculture and conservation science, however we currently lack the knowledge to engineer microbial communities through breeding or management. It is essential to continue the efforts on understanding community functions and dynamics, and how host, endophyte interactions, and other ecological and human- related mechanisms influence their diversity in both forest species and agronomically important crops.Item TEMPORAL DYNAMICS, ANTIMICROBIAL RESISTANCE AND PHYLOGENETIC RELATIONSHIPS OF BACTERIAL TAXA IN IRRIGATION WATER SOURCES AND RELEVANCE TO FOOD SAFETY(2021) Solaiman, Sultana; Micallef, Shirley SAM; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. Unfortunately, open water sources are often contaminated with many known human pathogens such as E. coli, Salmonella and Listeria and unknown/understudied pathogens such as Aeromonas that are associated with foodborne outbreaks. To facilitate the adoption of microbiologically safe irrigation water sources, a comprehensive study on the prevalence and virulence potential of human pathogens and their transferability to fruit and fresh produce vegetables was conducted. The effect of irrigation water types on crop surface microbial community structure, presence of virulence factors and antimicrobial resistance were investigated to evaluate the potential of transfer of pathogenic and antimicrobial resistant bacteria in humans. Initially, the prevalence of indicator bacteria was determined using culture methods and then microbiological water quality profiles (MWQP) was created to identify water sources that complied with the U.S Food Safety Modernization Act water standards. Next, using culture and molecular methods, investigation of the antimicrobial resistance profile of one known foodborne pathogen Escherichia coli retrieved over a two-year period was done. E. coli resistance against widely prescribed antibiotics, extended spectrum β-lactams, was determined phenotypically and genetically. The diversity, distribution and potential for pathogenesis of one understudied pathogen, Aeromonas, prevalent in a variety of typical or potential irrigation water sources and collected over a one-year period was investigated. The study revealed spatial and temporal patterns in species richness, evenness, virulence gene carriage and attachment behaviors on both biotic and abiotic surfaces, of this bacterial taxon. Finally, the effect of using highly treated reclaimed water and pond water on lettuce surface microbiomes was investigated. The study provided an integrated assessment of the shifts in microbial community that result from using different irrigation water sources for irrigation of lettuce. Understanding the ecology of lettuce associated microbiota can be useful to infer risks of transfer and establishment potential of possible pathogenic strains from irrigation water sources to minimally processed raw consumed fresh produce crops.Item SCLEROTINIA SCLEROTIORUM DIVERSITY AND MANAGEMENT OF WHITE MOLD ON LIMA BEAN IN MID-ATLANTIC REGION, USA(2020) Demissie, Habtamu Bekele; Everts, Kathryne L.; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lima bean (Phaseolus lunatus L.) is one of the most important vegetables grown in the mid-Atlantic region of the US. Delaware has more acreage of land per year allocated for lima bean production, primarily used for processing, than any other state. The yield of lima bean is severely affected by white mold caused by Sclerotinia sclerotiorum. Currently, there is limited information on the population diversity of S. sclerotiorum in the mid-Atlantic region compared to other production regions, such as New York. Due to lack of research conducted in mid-Atlantic region, there are no specific fungicide application guidelines for lima beans. Improved understanding of the diversity within or among S. sclerotiorum isolates obtained from different geographical regions and various hosts will assist selecting representative isolates for use in developing improved disease management strategies including development of host resistance and effective fungicide guidelines. The main objectives of this research includes 1) studying the diversity of S. sclerotiorum isolates from lima bean and other crops in the mid-Atlantic and other regions and 2) improving disease management guidelines for white mold. Forty-two S. sclerotiorum isolates were collected from ten crops within eight different states in the US. The diversity of the collected isolates was evaluated for, a) lesion length and oxalic acid production on nine cultivars (five lima bean, two soybean, and two common bean), b) mycelial compatibility groupings (MCGs) and molecular characterization, and c) fungicide sensitivity (in-vitro) to two concentrations of boscalid, cyprodinil, fludioxonil, fluazinam, prothioconazole, and thiophanate-methyl . A field study also evaluated six application timings of boscalid, at 20% flowering, 100% flowering, two weeks, and three weeks after 20% flowering, a double applications, and non-treated control for management of white mold in lima bean. The collected isolates produced different lesion lengths, which were dependent on the crops and cultivars tested. Isolate 13, which was obtained from soybean, NJ, was the most aggressive in causing the longest lesions. Isolate 6, which was obtained from snap bean, DE, was the least aggressive isolate in causing the shortest lesion. Isolates were also significantly differed in oxalic acid production. Isolate 13 and isolate 4 were the highest oxalic acid producers. Seventy-five percent of the MCGs interactions were incompatible. The Shannon index (Ho) values of the MCGs were between 0 - 0.35 indicate that there is high diversity among the S. sclerotiorum isolates tested and that the isolates may reproduce sexually rather than via vegetative reproduction. The molecular characterization of the sequences examined at the ITS region and β-tubulin gene provided high sequence similarities among our isolates. The low variability did not allow us to evaluate differences among isolates. The molecular/genetic variability within the population was 1 - 2%. To evaluate the fungicide sensitivity of isolates, the percent reduction in mycelial growth (PRMG) of each isolate in presence of Dimethyl Sulfoxide (DMSO) and the fungicide was compared to the control (the isolate grown in the presence of DMSO). The collected isolates varied in PRMG to all six fungicides. The PRMG of the isolates differed at the two concentrations, except for cyprodinil and fludioxonil. There was a significant interaction between the concentrations and isolates sensitivity to all fungicides except boscalid and thiophanate-methyl. Correlations were conducted to identify associations between fungicide sensitivities, lesion length, and oxalic acid production. Isolates’ sensitivity to boscalid was negatively correlated to lesion length (r=-0.28397; P=0.0004) and oxalic acid production (r=-0.23370; P=0.0040). In addition, fungicide sensitivity to fluazinam was positively correlated to fungicide sensitivity to prothioconazole (r=0.35695; P<.0001) and thiophanate methyl (r=0.46247; P=<.0001). Likewise, fungicide sensitivity to fludioxonil was positively correlated to fungicide sensitivity to boscalid (r=0.19309; P=0.0179) and thiophanate methyl (0.28760; P=0.0004). However, fluazinam sensitivity was negatively correlated to boscalid sensitivity (r= -0.20119; P=0.0136). In the fungicide timing evaluation, the disease incidence was reduced by 6.4%, 5.4%, 3.9%, and 7.6% compared to no treatment when fungicides were applied at 20% flowering (P<0.0001), 100% flowering (P<0.0001), one week after 100% flowering (P<0.0128), or at 20% and 100% flowering (P<0.0001), respectively. These application timings also reduced the disease severity by 5.7%, 8.0%, 6.0%, and 7.0% compared to no treatment, respectively. Earlier, within 2 weeks of 20% flowering and double fungicide treatment reduced disease incidence and disease severity and improved yield of lima bean. This research improves our understanding of the diversity of the mid-Atlantic Sclerotinia sclerotiorum population and suggests that, during selection of resistant lima bean cultivars, plants should be challenged by an array of S. sclerotiorum isolates, not just one putatively aggressive or susceptible isolate. My research also establishes guidelines for timing of fungicide management of white mold and developed baseline data on isolate sensitivity to fungicides.Item FUSARIUM SPECIES OF CUCUMIS MELO IN THE MID-ATLANTIC REGION OF THE US AND THEIR IMPACT ON SALMONELLA ENTERICA NEWPORT SURVIVAL AND INTERNALIZATION ON VARIOUS MELON CULTIVARS.(2019) Korir, Robert Cheruiyot; Everts, Dr. Kathryne L; Micallef, Dr. Shirley A; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fruit rots caused by Fusarium spp. can lead to economic yield losses on melon (Cucumis melo). However, which Fusarium spp. are the most prevalent in Maryland and Delaware has not been documented. Several Salmonella enterica subsp. serovar Newport (S. Newport) outbreaks on melon have occurred over the past 25 years. Fusarium spp. infestation on melon have potential impact on survival and colonization of Salmonella. Our objectives were to identify Fusarium spp. infestations on melons within the Delmarva region, and evaluate their impact on survival and internalization of S. Newport on various melon cultivars. Fifty-six isolates were molecularly identified, according to Fusarium-ID online database, as Fusarium spp. (Fusarium fujikuroi-20, Fusarium proliferatum-18, Fusarium oxysporum-15, Fusarium graminearium-2, Fusarium verticilloides-1). Our findings revealed that most of the Fusarium isolates we collected were not pathogenic to melon fruit. We evaluated the impact of four Fusarium spp. (F. armeniacum, F. oxysporum, F. fujikuroi, and F. proliferatum) on S. Newport survival in five melon cultivars; ‘Arava’ (C. melo var. reticulatus, Galia), ‘Athena’ (var. reticulatus, muskmelon), ‘Dulce Nectar’ (var. inodorus, honeydew), ‘Jaune de Canaries’ (var. inodorus, Canary), and ‘Sivan’ (var. cantalupensis, Charentais). Impact of F. proliferatum on survival and interlization of S. Newport was evaluated on honeydew (smooth) and cantaloupe (netted) melons. Generally, Fusarium did not impact the survival of S. Newport, however greater survival of S. Newport was observed on the netted cultivars compared to the smooth surface melons. Fusarium fujikuroi significantly enhanced survival of Salmonella when inoculated on riper ‘Jaune de Canaries’ melons (above ¾ slip). However, when the experiments were replicated with less ripe (about ¾ slip) melon, F. fujikuroi did not significantly influence the growth of S. Newport. Salmonella Newport internalized in all treatments and the cantaloupe and honeydew melons, but variation in population levels were observed across the treatments. Overall, Fusarium proliferatum did not impact internalization of S. Newport on either melon type. This may be attributed to that Fusarium species used during this study were non-pathogenic. Salmonella Newport recovered gradually decreased with time. Fusarium species on melon, influence S. Newport colonization differently. Also, melon rind type affects the ability of S. Newport to survive and colonize differently.Item SALMONELLA ENTERICA STRATEGIES FOR PERSISTENCE ON TOMATO (SOLANUM LYCOPERSICUM) AND SEROVAR DYNAMICS IN SURFACE AND RECLAIMED WATER(2019) Ferelli, Angela Marie Cecelia; Micallef, Shirley A.; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While select aspects of Salmonella enterica subspecies enterica persistence in agricultural matrices have been illustrated, serovar specific survival strategies in surface water, transmission, and persistence on plants are multifaceted and remain only partially examined. In the present work, we utilized an interdisciplinary approach to illustrate novel mechanisms by which S. enterica may adapt to plants as an alternative host. Furthermore, we leveraged the wealth of diversity in S. enterica serovars to investigate specific dynamics and drivers of persistence in water and transfer onto produce crops. Through biochemical, gene expression, and plant challenge assays of both tomato (Solanum lycopersicum) vegetative and fruit organs, we found that plant-derived NO was generated in response to S. Newport recognition. Furthermore, bacterial gene expression on both leaves and fruit was indicative of adaptation to a novel environment including upregulation in NO detoxification machinery, indicating plant-derived NO as a novel bacterial stress. NO tolerance of various S. enterica was then evaluated to investigate drivers of “produce associated’ S. enterica adaptation to the plant niche. We identified that plant derived NO can negatively affect titers of all S. enterica serovars tested and that serovar specific tolerance to NO in vitro was apparent in a concentration and exposure time dependent manner. Finally, the survival of various S. enterica in surface and reclaimed water was investigated while evaluating the potential for transition to viable but non-culturable (VBNC) organisms. Furthermore, surface water used for irrigation, a common water environment for S. enterica, was investigated as a priming reservoir for various S. enterica serovars for enhanced transmission onto tomato crops. Persistence in water included VBNC subpopulations and was driven by water type. Transfer success onto tomato was driven by serovar, and prolonged incubation in water increased the transfer ability of serovars that initially transferred poorly onto tomato. Finally, attachment to polystyrene and water oxidation-reduction potential were identified as possible indicators of foodborne pathogen transfer success onto tomato. Moving forward, a greater understanding of the environmental queues used by S. enterica subspecies enterica responding to the agricultural environment will aid researchers in developing S. enterica targeted on-farm management techniques to ensure safe yet sustainable fresh produce cultivation practices.Item Bacterial communities of the specialty crop phyllosphere: response to biological soil amendment use, rainfall, and insect visitation(2016) Allard, Sarah Michelle; Micallef, Shirley A; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microorganisms in the plant rhizosphere, the zone under the influence of roots, and phyllosphere, the aboveground plant habitat, exert a strong influence on plant growth, health, and protection. Tomatoes and cucumbers are important players in produce safety, and the microbial life on their surfaces may contribute to their fitness as hosts for foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. External factors such as agricultural inputs and environmental conditions likely also play a major role. However, the relative contributions of the various factors at play concerning the plant surface microbiome remain obscure, although this knowledge could be applied to crop protection from plant and human pathogens. Recent advances in genomic technology have made investigations into the diversity and structure of microbial communities possible in many systems and at multiple scales. Using Illumina sequencing to profile particular regions of the 16S rRNA gene, this study investigates the influences of climate and crop management practices on the field-grown tomato and cucumber microbiome. The first research chapter (Chapter 3) involved application of 4 different soil amendments to a tomato field and profiling of harvest-time phyllosphere and rhizosphere microbial communities. Factors such as water activity, soil texture, and field location influenced microbial community structure more than soil amendment use, indicating that field conditions may exert more influence on the tomato microbiome than certain agricultural inputs. In Chapter 4, the impact of rain on tomato and cucumber-associated microbial community structures was evaluated. Shifts in bacterial community composition and structure were recorded immediately following rain events, an effect which was partially reversed after 4 days and was strongest on cucumber fruit surfaces. Chapter 5 focused on the contribution of insect visitors to the tomato microbiota, finding that insects introduced diverse bacterial taxa to the blossom and green tomato fruit microbiome. This study advances our understanding of the factors that influence the microbiomes of tomato and cucumber. Farms are complex environments, and untangling the interactions between farming practices, the environment, and microbial diversity will help us develop a comprehensive understanding of how microbial life, including foodborne pathogens, may be influenced by agricultural conditions.Item SALMONELLA ENTERICA INTERACTIONS WITH TOMATO: PLANT GENOTYPE EFFECTS AND SALMONELLA GENETIC RESPONSES(2015) Han, Sanghyun; Micallef, Shirley A.; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Several outbreaks of Salmonella enterica infections have been linked to tomatoes. One cost-effective way to complement on-farm preventive Good Agricultural Practices would be to identify cultivars with inherent decreased susceptibility to Salmonella colonization. Various tomato cultivars with distinct phenotypes were screened to evaluate their susceptibility to Salmonella epiphytic colonization. The potential role of plant exudates, collected from the same cultivars, on the growth kinetics of Salmonella was examined. These investigations were supplemented with Salmonella genome-wide transcriptomics that showed bacterial responses to colonization of tomato shoots and roots. Epiphytic colonization of fruit by S. enterica was cultivar-dependent and serotype-specific, but did not correlate with leaf colonization. Fruit and leaves of the same cultivar differed in their ability to support Salmonella growth. Quantitative and qualitative analysis of tomato exudates provided a possible explanation for the differential susceptibility to bacterial colonization among tomato cultivars. Tomato exudates alone were capable of supporting Salmonella growth, and the growth kinetics of Salmonella in tomato exudates differed by cultivar. Characterization of the chemical composition of primary and secondary metabolites in tomato exudates pointed to potential causes for the differential growth of Salmonella observed in the exudates of various tomato cultivars. Key transcriptomic signals that were down- and up-regulated in Salmonella upon interacting with tomato were identified, enabling us to elucidate the molecular mechanisms underlying this enteric pathogen-plant interaction. Overall, the identified signals lead to a proposed model that depicts the cellular processes needed to preserve cell viability when multiple abiotic stresses in conjunction with low nutrient availability are encountered, while simultaneously repressing unnecessary energy demands or maintaining them at a level equivalent to growth in a nutritious medium. These findings strongly support the hypothesis that plant-regulated mechanisms influence enteric pathogen colonization. It is clear that Salmonella can sense subtle environmental cues brought about by the genotype or physiological state of plants and can respond with distinct patterns of gene expression. Future work should focus on whether this bacterial behavior on plants results from an evolutionary adaptation to use plants as a vector to re-enter animal hosts.Item SALMONELLA-INDUCED SYSTEMIC ACQUIRED RESISTANCE IN TOMATO AND ITS IMPACT ON SALMONELLA COLONIZATION OF TOMATO LEAVES(2015) Phannareth, Tommy; Micallef, Shirley A; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Salmonella enterica is an enteric human pathogen that lives in gastrointestinal tract; however, Salmonella are able to survive in plants. Thus, vegetables such as tomato are vectors for Salmonella. Evidence suggests that Salmonella induces PAMP-triggered immunity (PTI) in plants, however, plant systemic acquired resistance (SAR), which may act to suppress Salmonella populations, has not been explored. This research investigates whether Salmonella triggers SAR in tomato, and whether SAR activation restricts epiphytic Salmonella populations. Inoculation of tomato leaves with Salmonella increased SAR marker gene expression in distal tomato leaves, but did not reduce populations of the phytopathogen Pseudomonas syringae or Salmonella on distal leaves, even following treatment with chemical SAR activators. NahG plants, which are deficient in SAR signaling, supported higher Salmonella populations, and nitric oxide depletion on leaf surfaces favored Salmonella growth, suggesting that SAR is involved. SAR alone is insufficient to restrict Salmonella growth on tomato, despite being triggered.Item MOLECULAR PHYLOGENY AND TAXONOMIC REVISION OF FUNGI IN THE GENUS Thelonectria AND RELATED SPECIES WITH Cylindrocarpon-LIKE ANAMORPHS(2014) Salgado-Salazar, Catalina; Chaverri, Priscila; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genus Thelonectria and related species with Cylindrocarpon-like anamorphs are a group of perithecial ascomycetes in the family Nectriaceae that occur as saprobes and in few cases as pathogens of hardwood trees, shrubs or other plant substrates. Despite of being a key component of forest ecosystems around the world, species relationships and distribution are largely unknown. The objectives of this study were to: 1) infer species level phylogenetic relationships of the genus Thelonectria and related species with Cylindrocarpon-like anamorphs with uncertain classification, testing monophyly of each one of the groups studied; 2) delimit taxa, establishing taxon circumscriptions and providing brief descriptions; 3) resolve nomenclatural issues by identifying redundantly used names and synonyms; 4) provide identification tools, specifically, diagnostic keys and molecular data that can be used further as molecular barcodes; 4) provide distribution data and to take the first steps into the identification of speciation patterns observed in these fungi. To achieve these goals, herbarium materials, as well as freshly collected material obtained from the field or from fungal repositories were compared using phylogenetic analyses of multiple loci, morphology and geographic distribution. This research resulted in the narrower circumscription of the genus Thelonectria, not to contain one of the most common species in the group, T. jungneri. According to the results of the phylogenetic analyses it was found T. jungneri is a segregating clade that needs to be recognized as a different genus. For the genus Thelonectria, a total of 31 new species were described, and three new genera, closely related to Thelonectria were created to accommodate the diversity of other species with Cylindrocarpon-like anamorphs: Cinnamonectria gen nov. with C. cinnamomea as type taxon, Macronectria gen. nov. with M. jungneri as type taxon, and Tumenectria gen. nov. with T. laetidisca as type taxon. Species in this group of fungi present extensive morphological conservationism, representing a challenge for species identification without the use of molecular techniques, however offering a great opportunity to explore mechanisms of speciation and evolutionary diversification.