Master of Science / Department of Plant Pathology / John P. Fellers / Harold N. Trick / Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV), are two of the major viruses in the Great Plains of the United States. Cultural practices and mite vector control are the primary methods of disease management; however, they are not fully effective. Resistant varieties are also deployed, although some of the lines present temperature sensitive resistance or negative agronomic properties are linked to resistance. Alternative approaches to viral resistance are needed. RNAi has been shown to play a role in viral defense response and has been successfully used as a biotechnological tool to preprogram viral resistance in transgenic plants. RNAi reduces the expression of specific genes by targeting the accumulation of mRNA. The mechanism is activated by the presence of dsRNA, which is processed into small non-coding sequence that serves as a guide for degrading RNA in a sequence specific manner. In this work, the RNAi approach was used to elicit resistance against WSMV and TriMV. Immature embryos of the wheat cv "Bobwhite" were independently co-transformed with pAHC20, containing the bar gene for glufosinate selection, and either the hairpin construct targeting the coat protein of WSMV or TriMV. After tissue culture, PCR was used to determined the presence of the RNAi CP transgene in putative transformed plants. Eight WSMV and ten TriMV CP RNAi transgenic plants were obtained from the bombardment experiments. Transgenic T1 and T2 seeds were collected and transgene expression was established through RT-PCR. In order to determine viral resistance, T1 and T2 progeny was mechanically inoculated. ELISA results indicated a differential resistance response among the tillers evaluated in each line in T1 generation for both WSMV and TriMV constructs. In T1 generation resistance was seen in up to 60% of the plants evaluated for both constructs, although some events that showed transgene presence did not exhibited resistant phenotype. Analyses of transgene presence and expression in T2 generation evidenced events of transgene silencing and deletion. Regardless of these phenomena, consistent resistance response in two lines of WSMV CP construct and one TriMV CP transgenic line was found.

Many plant species, in response to stresses, accumulate low molecular weight secondary metabolites called phytoalexins. Pea (Pisum sativum ) makes a pterocarpanoid phytoalexin called pisatin which is relatively unique among pterocarpans because its stereochemical configuration is different at two adjacent carbons from the corresponding carbons in pterocarpan phytoalexins synthesized by alfalfa, soybean, clover and other legumes. Previous research demonstrated that an (-) isoflavanone-synthesizing isoflavone reductase (EFR) is induced during (+) pisatin biosynthesis and the final step in the biosynthesis is the methylation of (+) cis-6a-hydroxymaackiain (HMK) by 6a-hydroxymaackiainmethyltransferase (HMM). And, contrary to a predominant model of (+) pisatin biosynthesis, the 6a-OH of pisatin was shown to involve oxygen from H₂O rather than O₂. This work describes the role of (-) isoflavanone (sophorol) in (+) pisatin biosynthesis. Radioactive tracer techniques were used both in vivo and in vitro to analyze metabolism of (-) sophorol and related isoflavonoids. I have found that, in vivo, the incorporation of (-) sophorol into (+) pisatin is more efficient than the incorporation of (+) sophorol and (+) maackiain, suggesting that the normal biosynthetic route to (+) pisatin utilizes (-) and not (+) sophorol and does not use maackiain. (+) Sophorol is not metabolized in vitro by pea protein extracts, although isoflavene, 7,2 '-Dihydroxy-4',5'-methylenedioxyisoflavanol (DMDI) and a novel diastereomer of HMK, trans-HMK, accumulate when (-) sophorol is used as substrate. A cDNA from pea, which encodes sophorol reductase (SOR), was cloned by homology to an alfalfa cDNA coding for isoflavanone reductase. The SOR cDNA was found to be transcribed in response to CuCl₂ treatment of pea seedlings, as was previously found for cDNAs of IFR and HMM, which are involved in pisatin biosynthesis. The SOR cDNA gene product specifically reduces (-) and not (+) sophorol in vitro. DMDI, the product formed by the activity from the recombinant protein, is incorporated in vivo into (+) pisatin. My current model of (+) HMK synthesis proposes that (-) sophorol and (3R) DMDI are normal in vivo pathway intermediates. However, trans-HMK is likely an artifact as it is a poor pisatin intermediate in vivo and is also a poor substrate in vitro for HMM.

I examine three aspects forest vegetation of northern Florida: the Land Office Survey records of the abundance and distribution of woody species across northern Florida in the early nineteenth century; the abundance and distribution of woody species across a sharp elevation gradient in the hardwood slope forests along the Apalachicola River; and the population decline of Torreya taxifolia. / Land Office Survey data was tabulated for all available bearing trees north of Gainesville. This study includes 131,227 trees sampled at 49,896 section corners and mid-point lines. This data tabulation shows that northern Florida was dominated by pines prior to U.S. settlement. Over 77 percent of bearing trees were pines. Pineland oaks are the next most abundant species after pine. Wetlands trees, such as bay, gum, and cypress were dominant species in the absence of pine. The mixed hardwood species are a diverse group, including beech, magnolia, and dogwood, with no clear dominant among them. / The second portion of this research examines the spatial relationships among the species of the hardwood slope forests along the Apalachicola River. Species distributions were sampled using 80 belt transects in four ravine systems. Species turnover across elevation is high, but measures of species diversity and forest structure do not change with elevation. Distribution patterns of the major species indicate that assemblages in the adjacent elevation samples in the middle and upper slope positions are more similar to one another than are assemblages from the lower elevations. The assemblages do not, however, appear to form sub-communities with respect to elevation. / Finally, I examine hypotheses for the decline of Torreya taxifolia, a narrowly endemic conifer native to ravines along the Apalachicola River. Included in this analysis of the torreya decline is a demographic summary of 108 torreya on preserve land. Most individual torreya are multiple stemmed and less than 14 years old. Further, these individuals are most frequently grown at low elevations and under moderate to dense canopy shade. Among the many hypotheses proposed to explain the decline, I present evidence that fire suppression may have allowed fungal populations to soar, contributing to the onset of disease. / Source: Dissertation Abstracts International, Volume: 51-12, Section: B, page: 5692. / Major Professor: Joseph Travis. / Thesis (Ph.D.)--The Florida State University, 1990.

Withholding irrigation to alfalfa (Medicago sativa L.) during summer, a management strategy referred to as summer irrigation termination (SIT), has been suggested as a way to conserve water in desert environments. SIT may decrease productivity of alfalfa stands, although such negative effects may be reduced if cultivars with improved tolerance to SIT could be developed. This research was undertaken to determine how improved tolerance to SIT could be achieved through plant breeding. Single spaced plants of an extremely nondormant alfalfa population were grown in a field trial in Tucson, AZ and exposed to SIT in 1994 and 1995. These plants were used to identify traits associated with tolerance to SIT and represented parental material in a selection experiment. Direct selection for minimal reduction of forage yield following SIT was conducted under two stress intensities (lengths of SIT) and compared to indirect selection for characteristics potentially associated with dehydration avoidance. None of these selection criteria improved post-SIT forage yield relative to a random sample of plants from the parental population. This lack of response from selection was attributed to stress intensities that were not sufficiently high to fully expose genetic variation for yield following SIT. Physiological studies showed that high concentrations of total nonstructural carbohydrates (TNC) in crown tissue are positively associated with tolerance to SIT. Using TNC concentrations as an indirect selection criterion may therefore represent a more promising approach in improving tolerance to SIT than direct selection for post-SIT yield. Crown tissue was shown to die if the tissue moisture content fell below about 42%. This threshold value was used to predict whole-plant mortality of alfalfa grown in solid-seeded plots comparable to commercial fields. Crown samples were taken at five locations within the field along a soil gradient that caused whole-plant mortality to vary from 0.5 ± 0.5 to 48.7 ± 4.1%. Predicted values closely followed this change in observed mortality rates (r² = 0.97*) but tended to overestimate actual mortality on average by 4.2%. Alfalfa growers may be able to minimize mortality using this simple method to predict mortality during SIT and to reschedule irrigation accordingly.

The effects of extracts from purple nutsedge tubers were determined on the germination, growth, root leakage, water status, and photosynthesis of an Upland cotton, DPL 5415, and a Pima cotton, Pima S-7. Tubers extracts inhibited secondary root growth of seedlings more than primary root growth. At 500 ppmw, primary root growth was inhibited 44 percent whereas inhibition of secondary root growth was 64 percent. Non-polar extracts were more inhibitory to growth than polar extracts. Cotton plants grown in soil treated with hexane extracts of tubers containing non-polar allelopathic substances also lost electrolytes from their roots indicating an effect on root function. The effects on root function resulted in perturbations to the capacity of the plants to maintain efficient water status. At 250 ppmw of the hexane extract, the plant water potential, the leaf water content, and the leaf osmotic potential decreased from -0.7 to -1.3 MPa, from 89 to 79 percent, and from -0.8 to -1.0 MPa, respectively. In addition, the photosynthetic capacity of cotton was decreased 50 percent in both cotton cultivars in the second and third day after transplanting to soil treated with 62 ppmw of the hexane extracts. Leaf dehydration to below 70 percent relative water content and a reduction of quantum yield was detected in DPL 5415 at 125 ppmw of the hexane extracts. However, Pima S-7 was capable of tolerating higher levels of dehydration and did not show the reduction of quantum yield. Leaf expansion and epicotyl growth were also inhibited by 30 and 37 percent, respectively, by the hexane extracts at 250 ppmw. Purple nutsedge tubers released volatile substances that inhibited growth when trapped and tested on cotton seedlings, and caused root leakage. GC analyses showed that both the hexane extracts of purple nutsedge tubers and the volatile compounds released from the tubers contained substances with retention times that are characteristic of sesquiterpenes. These results demonstrate that purple nutsedge tubers contain allelopathic substances capable of inhibiting the growth of cotton by interfering with membranes of root cells, disrupting water status, and affecting photosynthesis.

Saguaro cactus virus (SCV) is a single-stranded RNA virus which belongs to the carmovirus genus within the family Tombusviridae. A full-length infectious clone of SCV has been generated and in this study was used to: (1) elucidate the role of the capsid protein (CP) in cell-to-cell and long distance movement, and (2) to better understand the various function(s) of the p26 and p86 proteins in viral replication. Analysis of a series of frameshift mutants and a deletion mutant has demonstrated that the CP is required for cell-to-cell movement in both Chenopodium amaranticolor and C. capitatum. This analysis also revealed a requirement of the CP coding region for viral replication in protoplasts. This is the first report of a cis-element, required for tombusvirus replication, which extends beyond the 3'-untranslated region into the CP coding region. The p26 and p86 constitute the putative SCV RNA-dependent RNA polymerase (RdRp). To better understand the structure and function of the RdRp, 16 clustered charged-to-alanine mutants were generated in the p26 and p86. The infectivity as well as the ability of each of these mutants to replicate in protoplasts was analyzed and compared to the infectivity and replication level of the wild type (pSCV15). Of the 16 mutants, five of them were nearly as infectious as wild type and were also able to replicate at near wild type levels. Four of the mutants consistently displayed a lower replication rate as determined by Northern analysis with two of these four demonstrating a lower level of infectivity on indicator plants. Two other mutants demonstrated a level of replication which was only able to be detected by RT-PCR. These mutants were not able to elicit the formation of local lesions on C. amaranticolor or induce symptoms on either inoculated or systemic leaves of C. capitatum. The ability of these mutants to synthesize negative-strand RNA, was examined. It was determined that all of the mutants which were able to produce positive-strand RNA were also able to synthesize negative strand RNA as determined by RT-PCR. Five of the mutants were not able to replicate in protoplasts and were not infectious on either host. These remaining five uninfectious mutants were also unable to replicate either negative or positive-strand RNA.

The inability of a plant to run from danger or seek nutrients necessitates its capacity to change the environment of the surrounding soil for protection and sustenance. A unique plant process, the release of thousands of autonomous cells from the root cap, called root border cells, may play a role in the ability of the plant to regulate microbial populations and nutrient availability in the rhizosphere. In this study, evidence is presented showing that root border cells are a differentiated tissue, that the production of border cells is highly regulated and tied to cell turnover in the root cap and that products of border cells regulate cell division in the root cap meristem. In vivo labeling experiments demonstrate that 13% of the proteins that are abundant in preparations from border cells are undetectable in root tip cells. Differences between the two cell populations are apparent as soon as border cells separate from each other, even when they are still adhered to the root tip. Twenty-five percent of the proteins synthesized by border cells in a 1-hour period are rapidly excreted into the incubation medium. Border cells arise within the root cap meristem by cell division and their production is tightly regulated both developmentally and in response to border cell removal. Removal of border cells results in the induction of cell division in the transverse root cap meristem to 400% of the basal rate within 30 minutes. This elevated rate of mitosis is maintained for 1.5 h and falls to basal levels within 6 hours. During this time, mitosis in the root apical meristem remains constant. mRNA differential display analysis showed changes in gene expression in the root cap within 5 to 15 minutes of removal of border cells. Genes putatively involved in cell functions in three regions of the cap showed expected distribution patterns by in situ hybridization and RNA blot analysis revealed changes in their expression patterns were seen in response to border cell removal. The presence of border cells acts as an inhibitor to continued mitosis and border cell production in the root cap. Evidence from fractionation studies shows that a heat stable, protease insensitive molecule in the range of 25 to 80 kDa, produced by the border cells themselves, is responsible for this inhibition.

<p> Butternut (<i>Juglans cinerea</i> L.), or white walnut, has suffered large population declines in the past half-century due to poor regeneration and mortality caused by an introduced fungus, <i>Ophiognomonia clavigignenti-juglandacearum </i> (Nair, Kostichka &amp; Kuntz) Broders &amp; Boland. This fungus causes branch and trunk cankers that can coalesce to girdle adult trees. Chapter 1 provides background information on butternut and butternut canker. We used next-generation sequencing to identify new nuclear DNA markers for butternut and Japanese walnut, a congener with which butternut readily hybridizes. We also examined the alignment of SSR repeat sequences in butternut and Japanese walnut with similar sequences from other angiosperms in public sequence databases. The methods used and results obtained in this process are detailed in Chapter 2. Chapter 3 summarizes an investigation of the environmental and genetic factors contributing to canker disease incidence, severity, and mortality in a large (n=113) population of butternut in southern Wisconsin and two other populations of butternut, one near the main study site in southern Wisconsin and another in the Great Smoky Mountains National Park. We present evidence for weak correlations of genetic similarity and phenotypic similarity for several disease traits, parentage analysis of regeneration in the smaller Wisconsin population, and evidence for significant microsite influences on butternut mortality over an 11-year period in the large Slocum's Woods butternut population.</p>

<p> Developing maize kernels are vulnerable to colonization by microbes. When colonization allows proliferation of the microbe at the expense of the host, disease occurs. The ascomycete fungal pathogens <i>Aspergillus flavus</i> and <i>Fusarium verticillioides</i> are capable of colonizing maize kernels, causing ear rots and contamination of the kernel with mycotoxins. These diseases lead to significant losses of crop yield and quality, and constitute a threat to food safety and human health. Thus, the significance of these diseases has prompted extensive research efforts to understand these plant-parasite interactions. However, pathogenesis and resistance mechanisms remain poorly characterized, hampering the development of effective control strategies. No commercial maize lines are completely resistant to these fungi. We applied an integrated approach consisting of histology, in situ gene expression and transcriptional profiling to better understand the nature of the interactions that occur between maize kernels and these fungi. Maize inbred line B73 was hand pollinated and inoculated with either <i> A. flavus</i> or <i>F. verticillioides</i> by wounding the kernel with a needle bearing conidia. Histological staining of the kernel sections revealed fungal mycelium in kernels adjacent to the inoculation site by 48 hours post inoculation (hpi), and in all tissues at 96 hpi. Compared with <i>F. verticillioides, A. flavus</i> more aggressively colonized kernel tissue and formed a unique biofilm-like structure around the scutellum. Transcriptome profiling using RNA-sequencing (RNA-seq) coupled with pathway analysis showed that these fungi were recognized by the kernel tissues prior to visible colonization. Infection of the kernel by these fungi induced transcriptional changes in defense-related genes, hormone signaling networks, as well as primary and secondary metabolism pathways. To dissect tissue-specific responses of the kernel, RNA in situ hybridization and histological staining were carried out in adjacent serial sections. We found that two maize genes, <i>pathogenesis related protein, maize seeds (PRms)</i> and <i>shrunken-1 (Sh1) </i>, were expressed in the aleurone and scutellum during infection by these fungi. By staining the adjacent sections, we found that these genes were induced in the tissue before the establishment of fungal colonization. Integration of histology, in situ gene expression and transcriptional profiling to study pathogenesis of maize kernels by these two fungi revealed distinctive and common features between the two pathosystems, and provided information that will facilitate the development of resistance genotypes in maize.</p>

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7040. Adviser: Robert M. Skirvin. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.