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Browsing by Author "Leach, Jan, advisor"

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    Detecting durable resistance to rice bacterial blight
    (Colorado State University. Libraries, 2016) Delorean, Emily Elizabeth, author; Leach, Jan, advisor; Verdier, Valerie, committee member; Argueso, Cris, committee member; Brick, Mark, committee member
    The productivity of rice, a staple crop worldwide, is limited by pathogens such as Xanthomonas oryzae pv oryzae (Xoo). Controlling yield loss to the resulting disease, bacterial blight, is most effective through growing genetically pathogen resistant rice varieties. However, widespread deployment of varieties containing single gene resistance to bacterial blight places an immense selection pressure on Xoo to evolve virulence. The major virulence factors employed by Xoo to drive infection are transcription activator like (TAL) effectors. TAL effectors are secreted into the host cells where they target the transcription of particular host susceptibility genes to favor infection. Previous TAL effector research indicates that not all TALs are created equal and some are crucial to the virulence of Xoo. By breeding for resistance genes targeting necessary TAL effectors we may find more durable resistance as selection pressure on the pathogen will result in loss of the TAL effector function and therefore a decrease in virulence and pathogen fitness. In the present study, we characterized a novel and widespread TAL effector through quantitative trait loci (QTL) mapping. We used the indica rice Multi-Parent Advanced Generation Inter-Cross (MAGIC) population to screen for resistance to the cloned TAL effector, TAL7b, and the Philippine race 6 Xoo strain PXO99A. Our results confirm that TAL7b is a virulence enhancing factor and that the MAGIC population contains six loci targeting resistance to TAL7b.We also identified another seven resistance QTL to the highly virulent Xoo strain, PXO99A.
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    Molecular genetics of glyphosate resistance in Palmer amaranth (Amaranthus palmeri L.)
    (Colorado State University. Libraries, 2009) Gaines, Todd A., author; Westra, Philip, advisor; Leach, Jan, advisor
    Glyphosate resistant Palmer amaranth populations were identified in Georgia in 2004. Studies were undertaken to characterize inheritance, the molecular basis of resistance, and the potential for gene transfer to related Amaranthus species. Dose response results support rejecting a monogenic inheritance hypothesis in favor of an alternative polygenic, additive inheritance model. Apomixis in genetic populations used for inheritance studies is probably occurring and makes interpretation of inheritance difficult. Glyphosate resistance in Palmer amaranth appears to be incompletely dominant and may be polygenic. No target site mutations known to confer resistance were identified in resistant alleles of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the target of glyphosate. Estimation of gene copy numbers of EPSPS relative to acetolactate synthase (ALS) in gDNA by quantitative PCR (qPCR) suggested that resistant plant genomes contain 64 to 128 times more copies of EPSPS than susceptible plants. qPCR on cDNA revealed that EPSPS was expressed approximately 35 times higher in resistant plants. Elevated EPSPS copy number is heritable and correlates with expression level and resistance in F2 populations. The molecular basis of resistance is likely due to increased production of EPSPS due to gene amplification. This is the first documented occurrence of EPSPS gene amplification in a weed population under glyphosate selection pressure. The risk of resistance gene transfer was measured with field studies and hand crosses with A. hybridus, A. retroflexus, A. powellii, A. spinosus, and A. tuberculatus. Glyphosate application (0.4 kg ha-1) was used to screen for resistant progeny from the crosses. Hybridization with A. spinosus occurred in both years of the field study and in hand crosses, with average frequency ranging from <0.01% to 1.4%. Hybrids with A. spinosus were either monoecious or dioecious. Monoecious plants produced seed through self-pollination, and the F2 progeny were segregating for resistance. Hybridization occurred in the 2007 field study with A. hybridus (<0.01%) and A. tuberculatus (0.08% and 0.19% for two accessions), all of the hybrid plants were dioecious, and none produced seed. The highest risk for glyphosate resistance gene transfer from A. palmeri is to A. spinosus.
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    Phylogenetic analysis of Xanthomonas translucens & genome-enabled diagnostic tools for pathogens of cereals and non-cereals
    (Colorado State University. Libraries, 2016) Langlois, Paul, author; Leach, Jan, advisor; Jahn, Courtney, committee member; Steingraeber, David, committee member
    Prevalence of Xanthomonas translucens, which causes bacterial leaf streak (BLS) in cereal crops and bacterial wilt in forage and turfgrass species, has increased in many regions in recent years. Because the pathogen is seedborne in economically important cereals, it is a concern for international and interstate germplasm exchange, and thus, reliable and robust protocols for its detection in seed are needed. However, historical confusion in the taxonomy within the species has complicated the development of accurate and reliable diagnostic tools for X. translucens. The goal of this study was to clarify the genetic relationships of X. translucens pathovars, and to use that information to develop useful and robust diagnostic tools. We sequenced genomes of 15 X. translucens isolates representing six different pathovars. Based on Multilocus Sequence Typing (MLST), wheat isolates designated as X. translucens pv. undulosa are in the same phylogenetic clade as barley isolates identified as X. translucens pv. translucens. The wheat and barley pathovars, undulosa and translucens, are genetically distinct from the cerealis pathovar isolated from either cereals or non-cereals, as well as pathovars isolated from other non-cereals, including arrhenatheri, graminis, and poae. Using unique genomic regions, Loop Mediated Isothermal Amplification (LAMP) primer sets were designed that selectively amplified X. translucens (species-specific), or that selectively amplified strains belonging to cerealis and poae pathovars. In addition, LAMP PCR assays were developed that distinguished X. translucens strains associated with cereal leaf streak (CLS), such as undulosa, translucens, hordei, and secalis, from the other cereal or non-cereal pathovars.
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    Regional whole plant and molecular response of Kochia scoparia to glyphosate
    (Colorado State University. Libraries, 2012) Wiersma, Andrew, author; Westra, Philip, advisor; Leach, Jan, advisor; Reddy, Anireddy, committee member; Holtzer, Thomas, committee member
    Globally, glyphosate (Roundup®) resistant weeds pose a serious challenge to modern agricultural practices that utilize glyphosate for weed control, including Roundup Ready® cropping regimes. Locally, glyphosate resistant Kochia scoparia have been identified throughout the central Great Plains, and the infested range is expanding rapidly. Glyphosate and Roundup Ready® crops form the foundation of no-till technology, which has considerably reduced water use and soil loss in arid to semi-arid regions of North America. Unfortunately, the continued spread of glyphosate-resistant K. scoparia will jeopardize the utility of glyphosate and the sustainability of no-till agricultural practices. In an effort to suppress glyphosate-resistant K. scoparia, more needs to be known about 1) the spread of resistance, 2) the level of resistance, and 3) the mechanism responsible for glyphosate resistance in K. scoparia. Suspected glyphosate-resistant K. scoparia accessions were collected from Kansas, Colorado, North Dakota, South Dakota, and Alberta. Whole plant glyphosate dose response and shikimate assays were used to confirm resistance and assess the level of resistance. Then PCR, quantitative PCR, sequencing, and immunoblotting techniques were used to determine the mechanism responsible for glyphosate resistance. Sequence of the EPSPS binding site proline confirmed that amino acid substitution at that residue was not responsible for resistance in K. scoparia. However, quantitative PCR estimates of EPSPS copy number revealed increased copy number in all glyphosate-resistant individuals —ranging from 3 to 9 EPSPS copies relative to the reference ALS gene. Furthermore, increased EPSPS copy number was correlated to increased transcript and protein abundance. Based on these finding, I confirm resistance for all tested accessions throughout the North American central Great Plains, and conclude that increased glyphosate rates will have little effect in controlling glyphosate-resistant K. scoparia. Furthermore, I suggest that EPSPS gene amplification may be the mechanism responsible for glyphosate resistance in K. scoparia, and that lower level increases in EPSPS expression (as compared to A. palmeri) are sufficient for glyphosate resistance. Moreover, this research, again, demonstrates the adaptability of plants and foreshadows the need for diversifying weed management practices.
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    Role of Oryza sativa chitinases in disease resistance
    (Colorado State University. Libraries, 2010) Snelling, Jacob Wayne, author; Leach, Jan, advisor; Lapitan, Nora Lyssa V., committee member; Chisholm, Stephen Thomas, 1972-, committee member
    Plant chitinases have long been implicated in defense responses against invading pests and pathogens. In Oryza sativa cv. Nipponbare (rice), chitinases have been shown to co-localize with disease resistance QTL, and are thought to contribute to a multigenic basal defense response. Previous work has shown that overexpression of several O. sativa class I chitinases resulted in heightened resistance to the fungal pathogens Rhizoctonia solani and Magnaporthe oryzae pv. oryzae. Here, I examined the responses of several O. sativa chitinase classes to the RNAi silencing of two transcriptionally active rice chitinase genes LOC_Os02g39330 and LOC_Os04g41620. Silencing of these genes results in a reduction of expression in several additional rice chitinases, and also shows an increased susceptibility phenotype to fungal rice pathogens R. solani, M. oryzae pv. oryzae, and bacterial rice pathogen Xanthomonas oryzae pv oryzae. The relative amounts of silencing of the individual genes LOC_Os02g39330 or LOC_Os04g41620 were not significantly correlated with disease phenotype. Thus, we conclude that silencing of these target genes altered the expression of other chitinases, and perhaps other defense response genes that were not assayed, and that the combination of altered expression profiles contributed to increased susceptibility to the various pathogens.
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    Understanding the disease ecology of the corn bacterial leaf streak pathogen Xanthomonas vasicola pv. vasculorum
    (Colorado State University. Libraries, 2019) Ortiz Castro, Mary Carmen, author; Leach, Jan, advisor; Broders, Kirk, advisor; Charkowski, Amy, committee member; Schipanski, Meagan, committee member
    Bacterial leaf streak, caused by Xanthomonas vasicola pv. vasculorum (Xvv), is an emerging disease of corn in North and South America. Based on the combined $52.4 billion value of the corn industry, early reports of Xvv disease severity, and lack of management methods, this emerging pathogen represents an economic threat to corn production in the United States. The primary goal of this research is to provide a basic understanding of the infection ecology and survival of the corn bacterial leaf streak pathogen. Through genetic transformations of the bacteria with fluorescent proteins and confocal microscopy, we were able to show the localization of the bacteria within plant leaves. In addition, we found that there is a significant interaction between Xvv isolates and two corn varieties. By evaluating the bacterial fitness across representative isolates of Xvv, we showed that 22°C is the optimal temperature for bacterial growth in culture. We also evaluated the interaction of Xvv with the endophyte Pantoea ananatis and found that the presence of the endophyte significantly decreases Xvv's disease response. Finally, through litter studies at multiple locations, we demonstrated that infected residue left on the surface of the soil harbored significantly greater quantities of Xvv than infected residue buried 10 cm below the surface. These findings will be useful to understand the bacterial leaf streak disease cycle and aid in the development of management strategies that may limit the distribution of Xvv within corn fields and prevent its spread to other corn producing regions.