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Title:Discovery, surveillance, and management of herbicide-resistant amaranthus spp
Author(s):Murphy, Brent Peter
Advisor(s):Tranel, Patrick J.
Contributor(s):Riggins, Chance; Clough, Steven; Jamann, Tiffany
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Amaranthus tuberculatus
Amaranthus palmeri
Amaranthus retroflexus
Conservation Reserve Program
Abstract:Amaranthus species are considered key driver weed species within Midwestern agricultural production systems. Herbicides are often relied upon for the control of Amaranthus spp. In recent years, herbicide resistance at multiple sites of action, such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and protoporphyrinogen oxidase (PPO), have become widespread within a narrow subset of the Amaranthus genus, such as A. tuberculatus and A. palmeri. Species within the genus possesses a distinct geographic distribution. Maintenance of these geographical boundaries can prevent the spread of more competitive species into new regions. The concept of geographical boundaries can be further applied to herbicide-resistant and susceptible biotypes of a given species. By maintaining isolation of herbicide-resistant populations from herbicide-sensitive populations, the spread of resistance may be mitigated. The objectives of this Master’s research were the i) identification of single nucleotide polymorphisms (SNPs) which delimit common weedy and cultivated Amaranthus spp., ii) development of quantitative assays to detect key Amaranthus spp., A. tuberculatus and A. palmeri, in mixed seed samples, iii) investigation of herbicide resistance in soybean fields of Ohio, iv) characterization of herbicide resistance within A. retroflexus accessions from Ohio, v) quantification of gene flow between A, tuberculatus and A. albus, and vi) development of a full plant regeneration procedure for A. tuberculatus through tissue culture. The research objectives were addressed utilizing a wide range of resources, including in-house and National Plant Germplasm System (NPGS) germplasm collections, population collections from Ohio and interspecific crosses. Amaranthus spp. delimiting barcodes in the nuclear ribosomal internal transcribed spacer (ITS) region were identified utilizing 75 sequences from the National Center for Biotechnology Information (NCBI) and 92 accessions from in-house collections and the NPGS. Nine species-specific barcodes were identified, and five species were delimited with single SNPs within the ITS region. SNPs specific to key weedy species A. tuberculatus and A. palmeri were utilized to develop quantitative polymerase chain reaction (qPCR) assays for species detection in mixed seed samples. A DNA extraction procedure from 100 seed samples was optimized to complement the qPCR assays, allowing high-throughput screening of seedlot scale samples. Amaranthus tuberculatus populations were collected in the 2016 growing season in Ohio as part of a long-term herbicide resistance surveillance initiative. A total of 24 populations were investigated for herbicide resistance to the photosystem II inhibitor atrazine, PPO-inhibitor lactofen, and EPSPS-inhibitor glyphosate. Resistance to atrazine and lactofen were sporadically observed, while high levels of resistance to EPSPS inhibitor glyphosate were consistently observed throughout the state. Continued surveillance of herbicide-resistant population is critical for the maintenance of sensitive populations to common herbicides. Accessions of A. retroflexus from Ohio were identified as potentially resistant to PPO-inhibitor fomesafen and glyphosate in greenhouse screening. Herbicide resistance was characterized through a ten-step dose response to both herbicides. Four-parameter log-logistic models were constructed for resistant and sensitive accessions to each herbicide. A significant difference between the accession in the dose required for 50% inhibition (ED50 value) and lower limit were observed for response to fomesafen (P-value<0.01). No significantly different parameter relationships were observed for response to glyphosate. Sequence comparison of the PPO gene between resistant and sensitive accessions revealed no known resistance causing mutations, however two unreported SNPs which result in amino acid substitutions (D414N and C277S) were observed. This is the first report of PPO resistance in A. retroflexus from the United States. Early and rapid identification of developing herbicide-resistant populations is essential for control and eradication. Weedy Amaranthus spp. each possess unique morphological and genetic features beneficial for proliferation. Hybridization potential between A. tuberculatus and A. albus was determined through reciprocal crossing in a controlled environment. Progeny of each cross was collected separately and analyzed for hybrid frequency. A unidirectional hybridization frequency of 0.0046% was observed from A. tuberculatus to A. albus, as confirmed through molecular markers. No putative hybrids were observed from A. tuberculatus females. Interestingly, a significantly skewed male:female ratio from 1:1 was observed (87% female, P-value < 0.001). DNA content analysis of the first back cross population indicated polyploidization may be required for hybridization. Gene flow between two species allows highly beneficial traits, such as herbicide resistance, to spread outside a given species, influencing control options. Quantification of gene flow is necessary for predictive modelling of the spread of herbicide resistance within the Amaranthus genus. Full plant regeneration is a necessary protocol for many aspects of basic science, including the analysis of gene function. The development of plant regeneration procedures relies on variation of plant hormones and media conditions. Regeneration of callus from leaf discs was consistently achieved utilizing a Murashige and Skoog media (MS) supplemented with the auxin indole-3-acetic acid (IAA) at 0.9 mg L-1 and the cytokinin trans-zeatin-riboside (zeatin) at 2.8 mg L-1. Root regeneration was consistently achieved from callus utilizing a MS media supplemented with 4.2 mg L-1 zeatin in the absence of IAA. Utilizing a linear dilution series, zeatin was determined to be unable to elicit shoot organogenesis under tested conditions. Continued utilization of dilution series of alternative cytokinins, such as 6-benzylaminopurine and thidiazuron, is expected to increase the probability of identifying optimal conditions for shoot organogenesis. As weeds evolve within existing management systems, both surveillance and new advances are required to protect producers. An understanding of how weed species and herbicide-resistance traits move geographically can advise management practices to limit their spread. Quantifying gene flow between species can assist in predicting the next species to develop resistance to a given herbicide, facilitating surveillance and early detection. Characterizing new herbicide-resistance traits is required for rapid and high-throughput diagnosis of resistance in the absence of phenotyping. Finally, the development of new technologies, both to study key driver weeds and to control them, provide a future where, while existing management practices may break, new practices are available to take their place.
Issue Date:2018-04-09
Rights Information:Copyright 2018 Brent Murphy
Date Available in IDEALS:2018-09-04
Date Deposited:2018-05

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