Theses and Dissertations at Montana State University (MSU)
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Item Identification of economic wireworms using traditional and molecular methods(Montana State University - Bozeman, College of Agriculture, 2013) Etzler, Frank Eric; Chairperson, Graduate Committee: Michael A. Ivie; Kevin W. Wanner, Anuar Morales-Rodriquez and Michael A. Ivie were co-authors of the article, 'DNA barcoding to improve the species level management of wireworms' submitted to the journal 'Journal of economic entomology' which is contained within this thesis.; Michael A. Ivie was a co-author of the article, 'Review of the Limonius canus LeConte, 1853 (Coleoptera: Elateridae)' submitted to the journal 'The coleopterists bulletin' which is contained within this thesis.Interest in wireworms has grown in the past decade due to their increasing pest status, largely due to the removal of effective seed treatments from the market. Currently, there is no effective Integrated Pest Management (IPM) strategy to control for wireworms, due to the diverse number of species that make up complexes in cropland. The purpose of this study was to determine what wireworm species are present in Montana's croplands and develop tools to make species concepts accessible to non-specialists. This was done using DNA barcoding to associate wireworms with adults. DNA barcoding was done by amplifying the Cytochrome-Oxidase I (COI) region of the mitochondrial genome. Twenty-nine (29) species were successfully sequenced and 13 species had adult and larval associations made, including three new associations. In addition, a LUCID pictorial key was also created to help identify species occurring in Montana. A LUCID key is a computer-based key where a user identifies a specimen with the help of pictures of each character. During the wireworm study, one species-group in the genus Limonius was found to include many economic species, including two that are important in Montana. This group needed to be reevaluated due to controversies raised in a recent revision, many of which dealt with economic species. With the combined use of morphological characters and DNA data, eight species are now recognized as belonging to the group. All of these subprojects show the combined use of DNA and morphology as essential to fully understanding wireworm species. With a more precise knowledge of the species that make up the complexes in Montana's croplands, we can focus on developing IPM stratetgies for efficient control.Item Selection and characterization of genomic DNA clones of Pyrenophora teres and their application for disease diagnosis via the polymerase chain reaction (PCR)(Montana State University - Bozeman, College of Agriculture, 1990) Baltazar, Baltazar MontesPolymerase Chain Reaction (PCR) protocols were developed for the diagnosis of net and spot forms of Pvrenophora teres. Low copy number sequences selected from a P. teres f. sp. maculata random genomic library were used as a source of probes. Emphasis was placed on those sequences identifying DNA polymorphisms between net and spot isolates and with little or no sequence similarity with barley, wheat, or triticale genomes. Sequences identifying a large deletion in genomic DNAs of net and spot isolates were preferred over sequences detecting small DNA changes. Sequence data of two informative clones, pPtm-290, and pPtm-60, were used to construct primer sets to amplify the corresponding sequence in genomic DNAs of net and spot isolates present in barley plants infected with these pathogens. PCR results demonstrated the potential of the PCR as a diagnostic tool for P. teres. All the PCR experiments conducted with primers designated as Pt-1 and Pt-2 constructed using the sequence data from pPtm-290, showed a strict correlation between the presence of a 430 bp band and the presence of the pathogen in genomic DNAs of barley infected with the net form, spot form or both pathogens. PCR experiments with primers Pt-3 and Pt-4 constructed using sequence data from pPtm-60, indicated that it is possible to detect polymorphic bands between net and spot isolates as evidenced by the PCR products analyzed in an ethidium bromide agarose gel. PCR analysis offers a sensitive, rapid, inexpensive, and non-radioactive technique for the diagnosis of P. teres infection in field-grown barley plants. Future experiments should focus on the ability of the PCR to detect P. teres and P. graminea in infected barley seeds. Additionally, PCR-based protocols for P. teres diagnosis could possibly be incorporated in seed certification programs to avoid the distribution of infected seed in farmer fields.