UMD Theses and Dissertations
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Item INVESTIGATING MECHANISMS UNDERLYING MLO’S ROLE AS A HOST FACTOR ESSENTIAL FOR PATHOGENESIS OF POWDERY MILDEW FUNGI(2024) Bloodgood, David; Xiao, Shunyuan; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Loss-of-function mutations in Mildew Locus O (MLO) family genes confer broad-spectrum resistance to powdery mildew (PM) fungi in various plant species. mlo-mediated resistance is invariably coupled with increased defense responses and early leaf senescence; hence the molecular basis of mlo-mediated resistance remains unresolved. A saturated genetic screen in the background of an Arabidopsis triple mutant where three essential immune components, EDS1, PAD4 and SID2 are mutated, led to the identification of five allelic mutations in MLO2, each of which results in compromised immunity yet poor infection (cipi) to PM. Further CRISPR-targeted mutagenesis of two functional homologs, MLO6 and MLO12 in a cipi mutant background result in complete lack of infection from PM fungi. The sextuple mutant, eds1pad4sid2mlo2mlo6mlo12 (epsm3) showed no early leaf senescence, ROS accumulation or expression of defense genes, indicating that MLO2, MLO6 and MLO12 are bona fide host susceptibility factors for PM. Expression of MLO2-GFP as a transgene in epsm3 restores susceptibility to PM and MLO2-GFP focally accumulates at the fungal penetration site. Thus, restoration of susceptibility to PM in the epsm3 background can be used as a sensitive reporter to assess whether other MLO family members share a conserved molecular function when expressed in leaf epidermal cells. The Barley MLO and Arabidopsis MLO7 enabled PM pathogenesis whereas MLO1, MLO3 and MLO4 could not, suggesting the existence of two distinct classes of MLO family members. Sequence alignment identified three conserved amino acid residues in the C terminal calmodulin-binding domain of MLO2, and MLO7, which are absent in MLO1, MLO3 and MLO4. This observation suggests that the C-terminal domain of MLO proteins could contribute to their functional divergence. Creation and functional assays of chimeric MLO2/MLO1 proteins by swapping their C terminal domains revealed that the C terminus determines the localization pattern of MLO proteins. The Feronia (FER) receptor-like kinase is required for localization of MLO7 in synergid cells; however, CRISPR-targeted mutagenesis of FER did not disrupt the localization of MLO2 to the fungal penetration site. Based on the results described above, it can be inferred that MLO2 localization to and possible stabilization of the plasma membrane at the fungal penetration site is essential for allowing PM fungi to penetrate the host cell and subsequently differentiate the haustorium. Further multiplexed CRISPR mutagenesis of other gene families suggests that SYP121 and SYP122, two closely related SNARE genes play essential roles in focal accumulation of MLO2 at the fungal penetration site.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 SPRAY STRATEGIES AND SELECTION FOR FUNGICIDE RESISTANCE: FENHEXAMID RESISTANCE IN BOTRYTIS CINEREA AS A CASE STUDY(2023) Boushell, Stephen Carl; Hu, Mengjun; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungicide resistance is a limiting factor in sustainable crop production. Despite the wide adoption of general resistance management strategies by growers, the recent rate of resistance development in important fungal pathogens is concerning. In this study, Botrytis cinerea and the high-risk fungicide fenhexamid were used to determine the effects of fungicide dose, tank mixture, and application timing on resistance selection across varied frequencies of resistance via both detached fruit assays and greenhouse trials. The results showed that application of doses lower than the fungicide label dose, mixture with the low-risk fungicide captan, and application post-infection seem to be the most effective management strategies in our experimental settings. In addition, even a small resistant B. cinerea population can lead to a dramatic reduction of disease control efficacy. Our findings were largely consistent with the recent modeling studies which favored the use of the lowest possible fungicide dose for improved resistance management.Item MULTIFACETED APPROACHES TO CONTROL FUSARIUM HEAD BLIGHT IN WHEAT: GENETIC MAPPING, MECHANISTIC STUDIES, AND FUNGICIDE EFFICACY ANALYSES(2022) Singh, Lovepreet; Rawat, Nidhi; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fusarium Head Blight (FHB), caused by the fungal pathogen Fusarium graminearum, is the second most important disease of wheat globally. This doctoral research contributes toward improving the two most widely used FHB management strategies: development of resistant cultivars and the use of fungicides. Given the quantitative nature of FHB resistance, pyramiding of multiple quantitative trait loci (QTLs) is required to develop resistant cultivars. Marker-assisted selection using diagnostic Kompetitive Allele-Specific PCR (KASP) assays is a cost and time-efficient approach. Toward this end, a previously identified resistance QTL from a moderately resistant soft red winter wheat cultivar Jamestown located on chromosome 1B was genetically mapped and diagnostic KASP assay was developed for it. Furthermore, a robust gene specific KASP marker was developed and validated for Fhb1, the most widely used QTL for resistance against FHB. Previously, a Pore-forming toxin-Like (PFT) gene was identified as the major underlying gene for Fhb1-mediated resistance in wheat. In the present research, functional characterization of PFT was done toward improving the understanding of the molecular mechanisms of FHB resistance. Ectopic expression of PFT in Arabidopsis conferred a broad-spectrum resistance against multiple fungal pathogens. PFT protein was purified from heterologous expression in Nicotiana benthamiana and used for various bioassays to elucidate the biological function of its individual domains. The experimental evidence supported our working hypothesis of PFT’s atypical mechanism of resistance. Working on the chemical control options for FHB, the efficacy and appropriate timing of application was investigated of a newly recommended fungicide for FHB-‘Miravis Ace.’ Overall, the knowledge and resources developed in this research would contribute towards improved integrated disease management of FHB in wheat.Item Epidemiology and Fungicide Sensitivity of Grape Late Season Bunch Rots in the Mid-Atlantic(2022) Cosseboom, Scott David; Hu, Mengjun; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This project aims to improve the management of late season bunch rots of grape (LSBR) whichcan be caused by a wide range of fungal pathogens. LSBR collectively have been an increasing issue in Mid-Atlantic vineyards, severely affecting grape yield and quality. Despite intensive fungicide spray programs and cultural practices, severe LSBR epidemics threaten the budding Mid-Atlantic wine industry. The basic plant pathological variables of host, pathogen, and environment were investigated to improve knowledge of the diseases involved, and therefore improve management strategies. The most common causal agents of LSBR in the Mid-Atlantic were found to be Botrytis cinerea and Colletotrichum spp. and the species identity of less common fungi was also investigated. The next most prevalent fungi associated with LSBR, Alternaria alternata, Aspergillus uvarum, and Neopestalotiopsis rosae were evaluated for pathogenicity in field experiments through the artificial inoculation of grape clusters. Second, the sensitivity of A. uvarum, B. cinerea, and N. rosae to commonly used chemical classes of fungicides was tested. Lastly, the optimal infection conditions and timing for Colletotrichum spp. were evaluated in laboratory, field, and greenhouse experiments, resulting in a quantitative inoculum tracking technique and a disease prediction model. These experiments were focused on solving practical and important disease management issues experienced by local grape growers, while conducting novel research that was applicable to the broader science community. Beyond the increased knowledge of the etiology and epidemiology of LSBR, the conclusions of this research could lead to reformed LSBR management strategies with the elimination of unnecessary and ineffective fungicide applications, increased accuracy and timing of management efforts, and increased marketable grape yield.Item TRANSLATION AND MOVEMENT OF AN INFECTIOUS UMBRAVIRUS-LIKE RNA CITRUS YELLOW VEIN ASSOCIATED VIRUS(2021) Liu, Jingyuan; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While the citrus yellow vein disease was first reported more than 60 years ago, its causal agent, citrus yellow vein associated virus (CYVaV), was only sequenced in 2013. CYVaV was recently structurally characterized and phylogenetically classified as a Class 2 umbravirus-like associated RNA (ulaRNA), a new category of coat-protein dependent subviral RNA replicons. There is a dearth of structural and biological characterizations of ulaRNAs as well as studies on their translation regulation. CYVaV has a limited genome size (2.7 kb), and contains only two ORFs that encode replicase proteins p21 and p81. Here I show that CYVaV transcripts are infectious in Arabidopsis protoplasts and Nicotiana benthamiana plants, and CYVaV can systemically infect the latter using agro-infiltration, despite the absence of encoded movement proteins or silencing suppressors. Fluorescent in situ hybridization (FISH) revealed that CYVaV is phloem-limited, and restricted to sieve elements, companion cells, and phloem parenchyma cells. In this work, the secondary structures of the CYVaV 5ʹ end and 3ʹ UTR were determined using SHAPE structure probing and phylogenic comparisons, and were used to infer the putative structures of other Class 2 ulaRNAs, revealing a number of distinctive structural features. Here I report the identification of a novel 3ʹCITE in the 3ʹUTR of CYVaV that is strongly conserved in Class 2 ulaRNAs and structurally resembles an I-shaped structure (ISS) 3ʹCITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5ʹ end. We also report that the ~30 nt 5ʹ terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3ʹCITE, or the 3ʹCITEs of umbravirus PEMV2, or even independent of a 3ʹCITE. These findings introduce a new type of 3ʹCITE and provide the first information on translation of ulaRNAs.Item Investigating Cultural Practices to Improve the Efficacy and Reliability of Biological Control Organisms in Turfgrass Systems(2021) Doherty, Joseph; Murphy, Angus; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biological control, or suppressing plant pathogens through natural predators or competitors, has been an area of scientific intrigue for many decades. However, inconsistent efficacy remains the chief reason for a lack of widespread adoption by growers. This dissertation was developed to address the inconsistencies of biological control through three practices. First, reducing competition from resident foliar microbial communities, or niche clearing, was explored for brown patch suppression and biological control organism establishment. Second, biological control organisms were applied to the rhizosphere and evaluated for suppression of root infecting pathogens. Finally, combinations of monthly biochar topdressing and biweekly or weekly biological control organism applications were evaluated for foliar pathogen suppression and biological control organism establishment. In each study, biological control organisms were observed to suppress pathogens and reduce disease severity. However, neither niche clearing nor biochar topdressing increased disease control compared to the biological control organism applied alone. While biochar applications did not improve the efficacy of biological control organisms, they did reduce the severity of disease unaffected by biological control organisms and increased turfgrass quality. Furthermore, biological control organisms were most successful under low to moderate levels of disease pressure, as control was lost when disease pressure peaked each year. Once again, neither niche clearing nor biochar topdressing increased populations of biological control organisms compared to the organisms applied alone. Weekly applications of biological control organisms did result in higher population levels compared to biweekly applications, and rhizosphere targeted applications appeared to have resulted in an establishment of the biological control organism into the rhizosphere community. Findings from these research projects suggest that biological control organisms should be applied weekly to increase odds of successful disease reduction. Given the lack of control under high disease pressure in all studies, successful implementation of biological controls should be targeted to the shoulders of the season when disease pressure is lower, or biological control organisms should be implemented into a season long program to supplement a traditional fungicide program.Item CUCURBIT DISEASE MANAGEMENT WITH REDUCED CHLOROTHALONIL AND IMPROVED UNDERSTANDING OF PSEUDOPERONOSPORA CUBENSIS POPULATION DYNAMICS(2020) Jones, Jake Gardner; Everts, Kathryne L; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Research has linked chlorothalonil exposure to declines in pollinator health due to an increased likelihood of Nosema ceranae infection, altered gut microbial community, and a reduction in colony fitness and survival of honey bees (Apis mellifera). Therefore, a reduction in use of chlorothalonil, a large component of cucurbit disease management, may be needed. Without chlorothalonil, a widely used, broad-spectrum fungicide, the fungal and oomycete pathogens in cucurbit cropping systems can more quickly evolve resistance to targeted fungicides due to a limited number of efficacious modes of action and frequent sprays. Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew, for example, has a short life cycle, experiences repeated applications of fungicides, and has a wide host range making it a high risk for fungicide resistance development. Our research focused on the development of an alternative fungicide spray program in melons to reduce the use of chlorothalonil, identifying the fungicide insensitivities of local P. cubensis populations and determining the efficacy of fungicides used to manage cucurbit downy mildew, and investigating the clade-host relationship and formation of oospores in regional P. cubensis samples. Efficacy on two important diseases in melon, powdery mildew and gummy stem blight, can be largely maintained without chlorothalonil but anthracnose control was not adequate without the inclusion of chlorothalonil. Currently, there are a number of highly effective targeted fungicides available to growers for management of cucurbit downy mildew including oxathiapiprolin, zoxamide + chlorothalonil, chlorothalonil, and cyazofamid. Our research shows evidence of P. cubensis clade-host associations, with clade 1 preferentially infecting acorn and summer squash (Cucurbita pepo), butternut squash (Cucurbita moschata), and watermelon (Citrullus lanatus), while clade 2 preferentially infects cucumber (Cucumis sativus). Melons (Cucumis melo) and pumpkin (Cucurbita maxima) are hosts to both clade 1 and clade 2 P. cubensis. Using these findings, producers can choose the fungicide that most appropriately targets the more virulent clade 2 or less virulent clade 1 infections.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.