Abstracts from the College of Agriculture
Undergraduate Summer Research Symposium August 8, 2002

Ordered alphabetically by student's last name

The Effects of Microbial Inoculants on Nitrate Reduction During the Ensiling Process

Jennifer Carr and Limin Kung Jr., Department of Animal and Food Sciences

The condition known as "nitrate toxicity" is especially problematic for farmers with high levels of nitrates in their water or feed. Upon ingestion, nitrate is converted into nitrite in the rumen of cattle. Nitrite is then absorbed into the blood stream where it takes the place of oxygen molecules on hemoglobin, converting it to methemoglobin. Animals can die of asphyxiation if not treated immediately.  Fermenting forages prior to their feeding to ruminants is a common practice. This process, also known as ensiling, reduces nitrate levels in forages by 30 to 70 %, but further reductions are warranted. Thus, the objective of this study was to evaluate the effect of microbial inoculants (Propionibacteria and Pediococcus) on their ability to reduce nitrates in silage. Our hypothesis was that these inoculants would metabolize nitrates and thus further reduce their concentrations in silages.  Silages will be analyzed for volatile fatty acids, lactic acid, nitrates, nitrites and otherfermentation end products. Preliminary data collected has shown that microbial inoculation increased the rate of acid production when compared to untreated silage during ensiling.  Microbial inoculation also affected the populations of enterobacteria and lactic acid bacteria in silage. Collection of data and analyses are ongoing.

Fatty Acid Analysis of Commercially Important Fungi in the Genus Pleurotus

Jamie Ellis1, Caroline Golt1, and Cliff Keil2
Departments of 1Plant & Soil Sciences and 2Entomology & Applied Ecology

Traditionally used for edible and medicinal purposes, the fungi in the genus Pleurotus, are currently being used for bioremediation, and are commercially cultivated on a variety of agricultural wastes and several lignocellulosic products.  The taxonomy of this group is difficult and controversial.  Additionally, many commercially important strains are unstable when cultured on artificial media.  Thirty six different isolates taken from ten different species and strains of Pleurotus, from all over the world were analyzed using fatty acid analysis.  The fatty acid composition of the isolates was determined by gas chromatography.  Fatty acid analyses is widely used not only to characterize different species of bacteria and yeasts, but also has been recently extended to characterize different species and races of fungi.  Our results suggest that fatty acid analyses of these isolates presents a characterization tool with sufficient resolving power that can be used to separate different species and strains based on their variable fatty acids.  Some species that are used widely commercially present great variability between their different strains.  This provides the basis to investigate these particular species and discover where this variation is occurring.

Potential Biological Control 
of the Alien Invasive Weed, Polygonum perfoliatum L.

Kevin R. Harkins, Keith Colpetzer, Douglas W. Tallamy, and Judith Hough-Goldstein
Department of Entomology and Applied Ecology 

Dr. Jeremy Nathans with Kevin Harkins

Mile-a-Minute weed or Polygonum perfoliatum L. is an annual vine from southeastern Asia that has become established in the eastern United States since its introduction in the early 1940’s. Mile-a-minute grows so quickly that it is beginning to replace native vegetation in some areas. Because of its invasive nature and limited control methods, an experiment was designed to simulate the feeding behavior of a possible biological control, a weevil, on P. perfoliatum. The weevil, Homorosoma chinensis, was imitated by cutting back nodes of the plant with scissors to simulate the damage caused by the egg-laying adults. The experiment simulated beetle damage at four different levels, from one to four beetles per plant, and plants were trimmed beginning when they were small (15 leaves), medium (60 to 90 leaves), or large (240 leaves).  Mortality was highest in the treatments that simulated beetle damage with the highest density of beetles per plant, where trimming was started when plants were small. In addition, a preliminary study was conducted on potential seed predation on mile-a-minute seeds in comparison to common buckwheat (Fagopyrum esculentum) and tall fescue (Festuca arundinacea). The treatments that excluded only invertebrates resulted in more seeds predated than excluding only vertebrates. Overall, there was no dramatic difference in predation among seed types. In the laboratory, three common carabid species prefer imbibed mile-a-minute seeds to dry seeds. 

Inter-year Distance Between Nest Sites of Wood Thrush  with Respect to 
Age, Sex, Previous Year's Success and Mate's Age 

Isis Johnson, Courtney Howland, Dan Nelson, Jared Judy, and Roland Roth
Department of Entomology and Applied Ecology

The successful fledging of avian young depends, in part, on territory location and placement of nests within the territory. Thus, natural selection should favor adults that return to sites where they were successful in the previous year yet avoid previously used but less successful sites. Age and sex may also influence this site faithfulness. We are examining these hypotheses with the Wood Thrush (Hylocichla mustelina) population in the University of Delaware Woods by testing pertinent independent variables as predictors of the distance between the first nest of a returning bird in Year X +1 and its first nest in the previous year X, our measure of site fidelity. During Summer 2002 we located nests, monitored them to determine the uniquely color-banded owners (and returnees) and their success, and mapped their locations. Similarly collected data from 2001 gave us the age, sex, mate age, and success (2 measures) of each returnee and allowed us to determine between-year inter-nest distances (BYD) for their first nests.  Based on studies that used broader definitions of site fidelity, we predict that males, older birds, birds mated to older birds, and birds with more success in Year X will have smaller BYDs. After the initial test with the 9 male and 9 female returnees from 2001, we will use Roth’s data from 10 other, non-overlapping pairs of years to enlarge our test.

Genetic Characterization of HR Despite NOS (hdn) Arabidopsis Mutants

Bevan B. Kirley, Barb A. Farnworth, JoAnne Z. Lynch, Allan D. Shapiro, Hajime Sakai
Department of Plant and Soil Science

Activation tagging is a mutagenesis procedure by which a strong enhancer is inserted randomly throughout the genome.  The result is usually the overexpression of the gene closest to the insertion site.  Activation tagged mutants were selected that showed a hypersensitive response (HR) despite the presence of a nitric oxide synthase inhibitor (hdn mutants) that has been previously shown to block the HR.  This indicates that these  mutants are bypassing the hypothetical pathway leading to the HR.  The HR is a programmed cell death (PCD) response resulting in the controlled death of the affected area.  In one mutant, hdn-101, a likely candidate for the overexpressed gene has been identified and experiments are underway to confirm this.  Phenotypically, the HR is associated with irreverisble damage to the plant plasma membrane resulting in ion leakage from the cells.  This leakage can be measured macroscopically by measuring the conductivity of leaf discs in distilled water.  Wild-type plants display a characteristic sigmoid increase in conductivity with time after an initial ~3.5 h lag.  By contrast, mutant hdn-101 displays a much shorter lag phase (~2 h) and a much more rapid initial increase in ion leakage.  These results indicate that the rate-limiting step for committment to PCD has been overcome in this mutant.  However, the final level of PCD is similar to wild type.  This result highlights the function of negative feedback control in limiting the limiting the extent of programmed cell death.

Use of a Dilution Method to Estimate Viral Production Rates 
in the Delaware Bay and West Florida Shelf

Robin White and K. Eric Wommack, Department of Plant and Soil Science

Aquatic viruses outnumber the bacterial population by at least a factor of ten.  With bacteria being ubiquitous, viruses have an immense impact quantitatively and ecologically on microbial environments.  In this study, the objectives were to test a dilution method, and to measure viral production rates in different aquatic systems.  A few revisions were made to the dilution technique.  One change was adding a peristaltic pump to keep raw water and ultra filtrate diluent continually mixing, thus preventing excessive loss of bacteria. In the dilution method, viruses within a seawater sample are diluted to at least 10% of original concentration.  During the twelve-hour incubation triplicate sub samples were collected at three-hour intervals and fixed to a final concentration of 1% formaldehyde. Bacteria and viruses in sub samples were vacuum filtered onto a 0.02 µm Anodisc filter, stained with SYBR Gold, and examined using an epifluorescence microscope.  Virus-like particles (VLP) and bacteria on micrographs were enumerated using Adobe Photoshop and Fovea Pro software.  VLP and bacteria abundance were plotted and first order linear regression lines calculated for each experiment.  Slope of the regression lines were used to estimate viral production rate. Viral production rates were higher in the Delaware Bay, an estuarine environment (2.25 x 105 VLP/mL/hr), compared to the West Florida Shelf, an oligotrophic environment (1.17 x 105 VLP/mL/hr). The dilution method was a success, and can be used as a reliable technique for estimating viral production.  Viral production estimates indicate that daily between 0.5% and 5% of bacteria are killed by viral lysis.

Pollinator Preference of Native Versus Introduced Flowering Plants 
in the Northern Delaware Piedmont Region

Susanna Wingard, Douglas Tallamy, and Dewey M. Caron
Department of Entomology and Applied Ecology

Learning the effects of introduced flora and fauna species on their new ecosystems is necessary in order to preserve the biodiversity of our native habitats. Determining pollinator preference for native versus introduced species of plants can provide information as to how introduced species can overtake natives as well as to how much biodiversity their respective flowers comparatively support.  Presumably native insect pollinators will be better adapted to utilizing pollen/nectar from a native flower source than from an exotic. Thus introduced species with a large number of visitors would most probably be polylectic, or generalist foragers, such as flower flies and honey bees. Floral visitor biodiversity was measured via visitor counts of 26 species including seven pairings from four Northern Delaware meadow sites which contained similar native and exotic flower species. Insect pollinator preference for native species was most noticeable in the pairs where shape was similiar.  In spite of the many variables affecting pollinator activity, this study should better determine pollinator preference for native or alien species in a meadow environment.

A Macroscopic Quantification of Cell Death in HR Despite NOS Inhibitor (hdn) Mutants

Alleen Yu, Barbara A. Farnworth, Bevan B. Kirley, Chu Zhang, Allan D. Shapiro
Department of Plant and Soil Science

Arabidopsis mutants showing a hypersensitive response (HR) despite a nitric oxide synthase inhibitor, which previous studies had shown to block the HR, were selected for genetic characterization. The HR is a programmed cell death (PCD) response, resulting in a regulated cell death. On the microscopic level, this means irreversible damage to the plasma membrane, causing ions to ‘leak out’ of dying cells.  The progression of cell death can be quantified by measuring the amount of ion leakage at pre-determined, post-inoculation times by using a conductivity meter. In this assay (optimized by Zhang), plants were inoculated with Pseudomonas syringae PV. tomato DC3000 bacteria carrying the avirulence (resistance – eliciting) gene avrRpt2. After a pre-determined amount of time, sample disks were taken from the leaves, washed, and placed in distilled water in order to measure ion leakage. Two mutants were identified as having clear and significantly different responses from the Columbia wild type control: hdn2-1 and hdn1-1D. Data analysis reveals that the typical response in a wild type Columbia plant is a sigmoidal increase in conductivity with time after an initial 3.5-hour lag. However, these mutants exhibit deviations from the control response including differences in initial response and in the amount of cell death.

Links: Summer 2002 Undergraduate Research Symposium, Symposium Abstracts from other Colleges and Departments,
Undergraduate Research Summer Enrichment ProgramUnversity of Delaware Undergraduate Research Program, Howard Hughes Undergraduate Program.
Created 3 August 2002. Last up dated 9 August 2002 by Hal White
Copyright 2002, University of Delaware