Browsing by Author "Schmale, David G. III"

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    Accurate identification and grouping of Rhizoctonia isolates infecting turfgrasses in MD and VA and their sensitivity to selected fungicides in vitro
    Amaradasa, Bimal Sajeewa (Virginia Tech, 2011-07-19)
    Rhizoctonia blight (sensu lato) is a common and serious disease of many turfgrass species. The most widespread causal agent R. solani consists of several genetically different anastomosis groups (AGs) and subgroups. Though anastomosis or hyphal fusion reactions have been used to group Rhizoctonia species, they are time consuming and sometimes difficult to interpret. Anastomosis reactions are incapable of identifying isolates belonging to different AG subgroups within an AG. This study evaluated molecular techniques in comparison with traditional anastomosis grouping (AG) to identify and group isolates of Rhizoctonia. More than 400 Rhizoctonia isolates were collected from diseased turfgrass leaves from eight geographic areas in Virginia and Maryland. A random sample of 86 isolates was selected and initially characterized by colony morphology, nuclei staining and anastomosis grouping. Molecular identification was performed by analysis of rDNA-ITS region and DNA fingerprinting techniques universally primed PCR (UP-PCR) and amplified fragment length polymorphism (AFLP). The cladistic analysis of ITS sequences and UP-PCR fragments supported seven clusters. Isolates of R. solani AG 1-IB (n=18), AG 2-2IIIB (n=30) and AG 5 (n=1) clustered separately. Waitea circinata var. zeae (n=11), and var. circinata (n=4) grouped separately. A cluster of six isolates (UWC) did not fall into any known Waitea group. Most of the binucleate Rhizoctonia-like fungi (BNR) (n=16) grouped separately. AFLP grouping also largely agreed with the above results. However, UWC isolates clustered into two groups. Molecular analyses corresponded well with traditional anastomosis grouping by clustering isolates within an AG or AG subgroup together. UP-PCR cross-hybridization could distinguish closely related Rhizoctonia isolates to their infraspecies level. Genetically related isolates belonging to the same AG subgroups cross-hybridized strongly, while isolates of different AGs did not cross-hybridize or did so weakly. Sequence-characterized amplified region (SCAR) markers were generated from UP-PCR products to identify isolates of major pathogenic groups AG 1-IB and AG 2-2IIIB. Specific primer pairs successfully distinguished isolates of AG 1-IB and AG 2-2IIIB from isolates of other AGs. Sensitivity of Rhizoctonia species and AGs was tested in vitro to commercial formulations of iprodione, triticonazole and pyraclostrobin. W. circinata isolates were moderately sensitive to iprodione while isolates of R. solani and BNR were extremely sensitive. Isolates of AG 2-2IIIB showed less sensitivity to triticonazole than other Rhizoctonia isolates. W. circinata var. zeae isolates were moderately sensitive to pyraclostrobin while most of the other isolates were extremely sensitive.
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    Association of Campylobacter spp. Levels between Chicken Grow-Out Environmental Samples and Processed Carcasses
    Schroeder, Matthew William (Virginia Tech, 2012-04-20)
    Campylobacter spp. have been isolated from live poultry, production environment, processing facility, and raw poultry products. The detection of Campylobacter using both quantitative and qualitative techniques would provide a more accurate assessment of pre- or post harvest contamination. Environmental sampling in a poultry grow-out house, combined with carcass rinse sampling from the same flock may provide a relative assessment of Campylobacter contamination and transmission. Air samples, fecal/litter samples, and feed pan/drink line samples were collected from four commercial chicken grow-out houses. Birds from the sampled house were the first flock slaughtered the following day, and were sampled by post-chill carcass rinses. Quantitative (direct plating) and qualitative (direct plating after enrichment step) detection methods were used to determine Campylobacter contamination in each environmental sample and carcass rinse. Campylobacter, from post-enrichment samples, was detected from 27% (32/120) of house environmental samples and 37.5% (45/120) of carcass rinse samples. All sample types from each house included at least one positive sample except the house 2 air samples. Samples from house 1 and associated carcass rinses accounted for the highest total of Campylobacter positives (29/60). The fewest number of Campylobacter positives, based on both house environmental (4/30) and carcass rinse samples (8/30) were detected from flock B. Environmental sampling techniques provide a non-invasive and efficient way to test for foodborne pathogens. Correlating qualitative or quantitative Campylobacter levels from house and plant samples may enable the scheduled processing of flocks with lower pathogen incidence or concentrations, as a way to reduce post-slaughter pathogen transmission.
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    Atmospheric Lagrangian transport structures and their applications to aerobiology
    Bozorg Magham, Amir Ebrahim (Virginia Tech, 2014-02-21)
    Exploring the concepts of long range aerial transport of microorganisms is the main motivation of this study. For this purpose we use theories and concepts of dynamical systems in the context of geophysical fluid systems. We apply powerful notions such as finite-time Lyapunov exponent (FTLE) and the associated Lagrangian coherent structures (LCS) and we attempt to provide mathematical explanations and frameworks for some applied questions which are based on realistic concerns of atmospheric transport phenomena. Accordingly, we quantify the accuracy of prediction of FTLE-LCS features and we determine the sensitivity of such predictions to forecasting parameters. In addition, we consider the spatiotemporal resolution of the operational data sets and we propose the concept of probabilistic source and destination regions which leads to the definition of stochastic FTLE fields. Moreover, we put forward the idea of using ensemble forecasting to quantify the uncertainty of the forecast results. Finally, we investigate the statistical properties of localized measurements of atmospheric microbial structure and their connections to the concept of local FTLE time-series. Results of this study would pave the way for more efficient models and management strategies for the spread of infectious diseases affecting plants, domestic animals, and humans.
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    Biochemical, Molecular and Functional Analysis of Volatile Terpene Formation in Arabidopsis Roots
    Huh, Jung-Hyun (Virginia Tech, 2011-07-20)
    Plants produce secondary (or specialized) metabolites to respond to a variety of environmental changes and threats. Especially, volatile compounds released by plants facilitate short and long distance interaction with both beneficial and harmful organisms. Comparatively little is known about the organization and role of specialized metabolism in root tissues. In this study, we have investigated the root-specific formation and function of volatile terpenes in the model plant Arabidopsis. As one objective, we have characterized the two root-specific terpene synthases, TPS22 and TPS25. Both enzymes catalyze the formation of several volatile sesquiterpenes with (E)-β-farnesene as the major product. TPS22 and TPS25 are expressed in the root in distinct different cell type-specific patterns and both genes are induced by jasmonic acid. Unexpectedly, both TPS proteins are localized to mitochondria, demonstrating a subcellular localization of terpene specialized metabolism in compartments other than the cytosol and plastids. (E)-β-Farnesene is produced at low concentrations suggesting posttranslational modifications of the TPS proteins and/or limited substrate availability in mitochondria. We hypothesize that the mitochondrial localization of TPS22 and TPS25 reflects evolutionary plasticity in subcellular compartmentation of TPS proteins with emerging or declining activity. Since (E)-β-farnesene inhibits Arabidopsis root growth in vitro, mitochondrial targeting of both proteins may fine tune (E)-β-farnesene concentrations to prevent possible autotoxic or inhibitory effects of this terpene in vivo. We further investigated the role of volatile terpenes in Arabidopsis roots in interaction with the soil-borne oomycete, Pythium irregulare. Infection of roots with P. irregulare causes emission of the C11-homoterpene (or better called C4-norterpene) 4,8-dimethylnona-1,3,7-triene (DMNT), which is a common volatile induced by biotic stress in aerial parts of plants but was not previously known to be produced in plant roots. We demonstrate that DMNT is synthesized by a novel, root-specific pathway via oxidative degradation of the C30-triterpene, arabidiol. DMNT exhibits inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro. Moreover, arabidiol and DMNT biosynthetic mutants were found to be more susceptible to P. irregulare infection and showed higher rates of Pythium colonization in comparison to wild type plants. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues.
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    Birch leaves and branches as a source of ice-nucleating macromolecules
    Felgitsch, Laura; Baloh, Philipp; Burkart, Julia; Mayr, Maximilian; Momken, Mohammad E.; Seifried, Teresa M.; Winkler, Philipp L.; Schmale, David G. III; Grothe, Hinrich (European Geosciences Union, 2018-11-08)
    Birch pollen are known to release ice-nucleating macromolecules (INM), but little is known about the production and release of INM from other parts of the tree. We examined the ice nucleation activity of samples from 10 different birch trees (Betula spp.). Samples were taken from nine birch trees in Tyrol, Austria, and from one tree in a small urban park in Vienna, Austria. Filtered aqueous extracts of 30 samples of leaves, primary wood (new branch wood, green in colour, photosynthetically active), and secondary wood (older branch wood, brown in colour, with no photosynthetic activity) were analysed in terms of ice nucleation activity using VODCA (Vienna Optical Droplet Crystallization Analyser), a cryo microscope for emulsion samples. All samples contained ice-nucleating particles in the submicron size range. Concentrations of ice nuclei ranged from 6:7 x 10⁴ to 6:1 x 10⁹ mg⁻¹ sample. Mean freezing temperatures varied between -15:6 and -31:3 °C; the range of temperatures where washes of birch pollen and dilutions thereof typically freeze. The freezing behaviour of three concentrations of birch pollen washing water (initial wash, 1 : 100, and 1 : 10 000) were significantly associated with more than a quarter of our samples, including some of the samples with highest and lowest activity. This indicates a relationship between the INM of wood, leaves, and pollen. Extracts derived from secondary wood showed the highest concentrations of INM and the highest freezing temperatures. Extracts from the leaves exhibited the highest variation in INM and freezing temperatures. Infrared spectra of the extracts and tested birch samples show qualitative similarity, suggesting the chemical components may be broadly similar.
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    Caging the blob: using a slime mold to teach concepts about barriers that constrain the movement of organisms
    Bohland, Cynthia E.; Schmale, David G. III; Ross, Shane D. (University of California Press, 2011-11-01)
    Few laboratory exercises are designed to teach biology students about barriers that may constrain the movement of organisms. We describe a unique inquiry-based exercise involving Lego mazes (the barrier) and the plasmodial slime mold, Physarum polycephalum (the organism). During guided inquiry, students construct mazes using Lego brand building blocks and the slime mold is allowed to "navigate" through the maze and "respond" to the barrier. Students then generate and test hypotheses about the movement of the slime mold in response to different barriers in the open-inquiry phase of the investigation.
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    Campylobacter jejuni and Salmonella spp. Detection in Chicken Grow Out Houses by Environmental Sampling Methods
    Kuntz, Thomas James (Virginia Tech, 2009-04-24)
    Campylobacter and Salmonella are foodborne pathogens commonly associated with raw poultry. Although there has been much research done on isolating these pathogens from poultry production environments using cloacal swabs, fecal samples, intestinal tract contents and dissection, research involving environmental sampling has been limited. New and/or improved environmental sampling methods may provide an easy, convenient, and less time-consuming way to collect samples. Coupling these sampling methods with PCR may provide a relatively simple, rapid, and robust means of testing for foodborne pathogens in a chicken house or flock prior to slaughter. Air, boot and sponge samples were collected from three commercial chicken grow-out houses located in southwestern Virginia when flocks were three, four, and five weeks old. Air samples were collected onto gelatin filters. Fecal/litter samples were collected from disposable booties worn over investigator's protective shoe coverings. Pre-moistened sponges were used to sample house feed pans and water dispensers on drink lines. A PCR method was used to qualitatively detect Campylobacter jejuni and Salmonella spp. Campylobacter jejuni was detected at each farm (house), across all three ages (3, 4, and 5 weeks), and from each sample type. Salmonella was not detected in any of the environmental samples. For all 270 samples, 41% (110/270) were positive for Campylobacter. Collectively, 28% (25/90) of air, 44% (40/90) of sponge, and 50% (45/90) of bootie samples were positive for Campylobacter. The methods used in this study are non-invasive to live animals, relatively rapid and specific, and could enable poultry processing facilities to coordinate scheduled processing of flocks with lower pathogen incidence, as a way to reduce post-slaughter pathogen transmission.
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    Characterization and management of major fungal diseases and mycotoxin contamination of grain sorghum in the mid-Atlantic U.S.
    Acharya, Bhupendra (Virginia Tech, 2019-06-11)
    Industry demand for local sources of grain for animal feed has increased sorghum production in the mid-Atlantic region of the U.S. Sorghum anthracnose (causal agent Colletotrichum sublineola) and the grain mold complex, which includes mycotoxin-producing Fusarium spp., limit the yield and quality of grain sorghum in humid climates worldwide. A majority of U.S. grain sorghum production is in arid regions, and management strategies have not been developed for the mid-Atlantic U.S. where warm, wet conditions favor disease. The specific objectives of this research were to: (1) determine the effectiveness of fungicides and their application timing for the management of sorghum foliar anthracnose, (2) compare five grain sorghum hybrids for their susceptibility to foliar anthracnose, grain mold and mycotoxin contamination under field conditions, (3) integrate host resistance and fungicide application to manage anthracnose and grain mold, and (4) identify Fusarium spp. associated with grain mold and mycotoxin contamination of sorghum in the mid-Atlantic U.S. For Objective 1, it was determined that a single application of pyraclostrobin-containing fungicide no later than flowering reduced anthrancose, protected yield and maximized farm income. Objective 2 focused on sorghum hybrid selection as a disease management tactic, and it was determined that hybrids with high yield potential and moderate disease resistance should be selected for mid-Atlantic sorghum production in order to maximize grain yield and quality while minimizing the need for fungicide inputs. Objective 3 focused on integrated management and demonstrated that under moderate disease pressure, a high-yielding susceptible hybrid required a single application of pyraclostrobin-based fungicide to minimize fungal diseases and maintain acceptable yields, whereas under high disease pressure it was necessary to integrate hybrid resistance and judicous applications of fungicides. The aim of Objective 4 was to characterize potential causal agents of mycotoxin contamination in mid-Atlantic sorghum, and thirteen phylogenetically distinct Fusarium species (F. lacertarum, F. graminearum. F. armeniacum, F. proliferatum, F. fujikuroi, F. verticillioides, F. thapsinum and several in Fusarium incarnatum-equiseti species complex) were found to be associated with grain mold and fumonisin and/or deoxynivalenol contamination of sorghum grain. This work has provided insights into the impacts of fungal diseases on grain sorghum yield and quality in the mid-Atlantic and has aided in development of best management practices for the region.
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    Characterization of fungicide resistance in grape powdery and downy mildew using field trials, bioassays, genomic, and transcriptomic approaches: quinoxyfen, phosphite, and mandipropamid
    Feng, Xuewen (Virginia Tech, 2018-02-06)
    Development of fungicide resistance in fungal and oomycete pathogens is a serious problem in grape production. Quinoxyfen is a fungicide widely used against grape powdery mildew (Erysiphe necator). In 2013, E. necator isolates with reduced quinoxyfen sensitivity (designated as quinoxyfen lab resistance or QLR) were detected in Virginia. Field trials were conducted in 2014, 2015, and 2016 at the affected vineyard to determine to what extent quinoxyfen might still contribute to disease control. Powdery mildew control by quinoxyfen was good, similar to, or only slightly less, than that provided by myclobutanil and boscalid in all three years. The frequency of QLR in vines not treated with quinoxyfen declined only slowly over the three years, from 65% to 46%. Information about the mode of action of quinoxyfen is limited; previous research suggests that quinoxyfen interferes with the signal transduction process. We profiled the transcriptomes of QLR and sensitive isolates in response to quinoxyfen treatment, providing support for this hypothesis. Additional transcriptional targets of quinoxyfen were revealed to be involved in the positive regulation of the MAPK signaling cascade, pathogenesis, and sporulation activity. Grape downy mildew (Plasmopara viticola), another important grape pathogen, is commonly controlled by phosphite fungicides. A field trial and laboratory bioassays were conducted to determine whether P. viticola isolates from vineyards with suspected control failures showed reduced sensitivity against phosphite fungicides. Prophyt applied at 14-day intervals under high disease pressure provided poor downy mildew control in the field. Next-generation sequencing technologies were utilized to identify 391,930 single nucleotide polymorphisms (SNPs) and generated a draft P. viticola genome assembly at ~130 megabase (Mb). Finally, field isolates of P. viticola collected from a Virginia vineyard with suspected mandipropamid control failure were bioassayed. The EC50 values of the isolates were >240 μg.ml-1 for mandipropamid, well above the field rate. The PvCesA3 gene of two resistant isolates was sequenced revealing that these isolates had a GGC-to-AGC substitution at codon 1105, the same mutation that has been found associated with CAA resistance elsewhere.
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    Characterization of Fungicide Resistance in Venturia inaequalis Populations in Virginia
    Marine, Sasha Cahn (Virginia Tech, 2012-03-28)
    Apple scab (causal organism: Venturia inaequalis) is an economically devastating disease of apples that is predominantly controlled with fungicides. Of the chemical classes currently available, the sterol-inhibiting (SI) and strobilurin (QoI) fungicides are the most commonly used. Recent observations indicate that V. inaequalis populations in Virginia have developed resistance to myclobutanil and other SIs. However, little is known about the frequency and distribution of SI and QoI resistance in Virginia's scab populations. The first objective of this research was to evaluate V. inaequalis populations in Virginia for SI and QoI resistance. Fungal isolates were collected from experimental orchards at the Alson H. Smith Jr., Agricultural Research and Extension Center (AHS AREC) and from commercial orchards in Virginia and Maryland. Sensitivities were determined by assessing colony growth at 19°C on potato dextrose agar (PDA) amended with 0 or 1.0 µg ml-1 of myclobutanil (SI) (N=87) or trifloxystrobin (QoI) (N=25) at 28 days. A range of fungicide sensitivity was observed for both chemical classes. The second objective of this research was to monitor the temporal dynamics of SI resistance over five sequential field seasons. To monitor shoot growth, neon rubber bands were placed over actively growing shoot tips following myclobutanil application or sample collection. Fungal isolates were collected from the same trees from 2007 through 2010 (N=176) and compared with isolates collected from wild apple seedlings (N=3). A continuum of SI resistance was observed for each year, and the V. inaequalis population exhibited a baseline shifted toward reduced sensitivity. The third objective of this research was to examine the spatial distribution of SI fungicide resistance within the tree canopy in a lower-density orchard (less than 150 trees A-1). Leaves collected from larger trees (>8m) in a lower-density orchard at the AHS AREC were analyzed for manganese deposition, pre- and post-mancozeb application. Fungal isolates (N=105) were collected from several locations within the canopy in replicated trees in the same orchard. Weather sensors also monitored the microclimates within those tree canopies. Spray deposition, microclimate and SI resistance were influenced by canopy location. The fourth objective of this research was to investigate potential SI resistance mechanisms. Previously classified isolates were screened for point mutations within the CYP51A1 gene (Appendix C), differences in polymorphic bands (alleles) (Appendix D), and differences in metabolism of myclobutanil (Appendix E). The consensus sequences for the CYP51A1 gene were identical for all isolates tested (N=9), and results from amplified fragment length polymorphism experiment (N=82) were inconclusive. There were, however, significant differences among incubation time and myclobutanil concentration in the bioassay (N=11). Our results indicate that myclobutanil is still an effective compound for control of apple scab in many areas of Virginia.
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    Characterization of Hulled and Hulless Winter Barley, Hordeum vulgare L., Through Traditional Breeding and Molecular Techniques
    Berger, Gregory Lawrence (Virginia Tech, 2012-11-28)
    Phenotypic and genotypic characterization of hulled and hulless winter barley (Hordeum vulgare L.) is necessary for improvement using traditional and molecular breeding techniques.  Identification of genomic regions conferring resistance to Fusarium head blight (caused by Fusarium graminearum), leaf rust (caused by Puccinia hordei G. Otth), powdery mildew [caused by Blumeria graminis (DC.) E.O. Speer f. sp. hordei Em. Marchal], net blotch (caused by Pyrenophora teres) and spot blotch [caused by Cochliobolus sativus (Ito & Kuribayashi) Drechsler ex Dastur] will greatly aid in breeding for improved resistance.  Determining factors that contribute to yield differences between hulled and hulless genotypes, and identification of markers associated with yield and yield related traits will greatly aid in improvement of hulled and hulless genotypes.  The hulled cultivar Nomini, hulless cultivar Eve, and hulless line VA06H-48 were consistently resistant to Fusarium head blight (FHB) and had low deoxynivalenol (DON) accumulation.  Screening with molecular markers on chromosomes 2H and 6H for FHB and DON identified quantitative trait loci (QTL) which may confer resistance in Virginia Tech germplasm.  Evaluation of hulled and hulless full-sibs from four populations indicated  that  grain volume weight and protein concentration were significantly (P d 0.05) higher for hulless genotypes, while seedling emergence and grain ash concentration were significantly (P d 0.05) higher for hulled genotypes.  In linear regression analysis, none of the assessed traits explained yield variation in all populations and environments.  Identification of hulless genotypes having yield potentials similar to those of their hulled sibs should be possible after adjusting for hull weight.  A genome wide association study was used to identify chromosome regions governing traits of importance in six-rowed winter barley germplasm and to identify single nucleotide polymorphisms (SNPs) markers for use in a marker-assisted breeding program. Significant SNPs associated with previously described QTL or genes were identified for heading date, test weight, yield, grain protein, polyphenol oxidase activity, and resistance to leaf rust, powdery mildew, net blotch, and spot blotch.  Novel QTL also were identified for agronomic, quality, and disease resistance traits.  These SNP-trait associations provide the opportunity to directly select for QTL contributing to multiple traits in breeding programs.
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    Characterizing resistance of Erysiphe necator to fungicides belonging to the quinone outside inhibitors and demethylation inhibitors
    Rallos, Lynn Esther E. (Virginia Tech, 2013-01-21)
    Practical resistance of Erysiphe necator to quinone outside inhibitors (QoIs) is now widespread, and resistance to demethylation inhibitors (DMIs) has also developed.  The goal of this research was to characterize fungicide resistance by elucidating resistance mechanisms and determining its stability.  QoI resistance persisted for several years in a field population after QoI application ended.  Resistant isolates were highly competitive in mixed populations in competition assays under laboratory conditions, indicating a lack of fitness cost.  In one competition trial under field conditions, resistance frequency declined, possibly due to spore migration and influx of background inoculum, but in a second trial, it did not decline.  Double resistance to QoI and DMI was detected and DMI application may have been partially responsible for maintaining QoI resistance in the field.  One isolate with QoI resistance but an undetectable level of the major QoI mutation was shown to be heteroplasmic -- resistant strains could be selected from this isolate. DMI resistance mechanisms in E. necator included the Y136F mutation in CYP51 and cyp51 over-expression.  The first mechanism was present in almost all isolates with substantial levels of resistance, and cyp51 expression level was correlated with resistance level.  Three cyp51 genotypes were detected.  Wildtype isolates with the TAT genotype were sensitive to DMIs, while isolates with increased resistance had either a TTT or TWT genotype; TWT indicated the presence of both wildtype and mutant alleles.  Cyp51 was expressed 1.4 to 19 times more in mutants than in wildtype.  It is not known whether the significant differences in cyp51 expression level among isolates and among genotype groups are due to gene copy number variation.  DMI resistance was found to decline after years of subculturing, and the decline appeared to occur after a few culture transfers of field samples on fungicide-free host leaves.  The observed decline, together with the finding that isolates could be "trained" to increase resistance, and may be slightly induced in cyp51 expression when successively challenged to grow in increasing fungicide concentration, indicate instability of DMI resistance.
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    A Comprehensive Analysis of Rust Disease Resistance in the Bioenergy Plant Switchgrass (Panicum virgatum L.)
    Frazier, Taylor Price (Virginia Tech, 2016-01-14)
    Switchgrass is a C4 perennial grass that is currently being developed for use as a second generation lignocellulosic biofuel crop. For switchgrass to be fully utilized as a bioenergy crop, large-scale plantings of elite switchgrass germplasm, possibly in monoculture, are likely to occur. This practice may increase the selection pressure on plant pathogens, such as switchgrass rust, which could result in devastating disease epidemics. The identification and deployment of quantitative trait loci (QTLs) and major plant disease resistance genes (R) in switchgrass breeding programs could offer broad spectrum and durable disease resistance in commercial switchgrass cultivars. 'Alamo', a lowland cultivar, is generally resistant to switchgrass rust whereas 'Dacotah', an upland cultivar, is highly susceptible. I hypothesized that major R genes and/or QTLs were contributing to the differences in disease phenotypes of these two cultivars. In this dissertation, bioinformatics and molecular biology approaches were employed to dissect the genetic mechanisms underlying switchgrass rust disease resistance. Novel pseudo-F2 mapping populations were created from a cross derived from 'Alamo' and 'Dacotah'. RNA-sequencing of the pseudo-F2 progenies of 'Alamo' x 'Dacotah' was used to construct a genetic linkage map and to identify potential QTLs correlating with disease resistance. In addition, a homology-based computational method was used to identify 1,011 potential NB-LRR R genes in the switchgrass genome (v 1.1). These potential R genes were characterized for polymorphism and expression differences between 'Alamo' and 'Dacotah'. Moreover, I found that some NB-LRR genes are developmentally regulated in switchgrass. One of the major objectives of switchgrass breeding programs is to develop cultivars with improved feedstock quality; however, changes in the components of the plant cell wall may affect disease resistance. I hypothesized that genetically modified switchgrass plants with altered cell wall components will respond differently than the wild-type to switchgrass rust. Transgenic switchgrass plants overexpressing AtSHN3, a transcription factor with known functions in epicuticular wax accumulation and cell wall deposition, were created. I found that AtSHN3-overexpressing transgenic switchgrass lines were more susceptible than wild-type plants in their response to switchgrass rust. Overall, the results of this dissertation provide a platform for elucidating the molecular mechanisms underlying resistance of switchgrass to switchgrass rust. These findings will help breeders create switchgrass cultivars with improved disease resistance, and will ultimately allow switchgrass to be used for sustainable biomass production.
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    Comprehensive characterization of an aspen (Populus tremuloides) leaf litter sample that maintained ice nucleation activity for 48 years
    Vasebi, Yalda; Mechan Llontop, Marco Enrique; Hanlon, Regina; Schmale, David G. III; Schnell, Russell; Vinatzer, Boris A. (European Geosciences Union, 2019-04-24)
    Decaying vegetation was determined to be a potentially important source of atmospheric ice nucleation particles (INPs) in the early 1970s. The bacterium Pseudomonas syringae was the first microorganism with ice nucleation activity (INA) isolated from decaying leaf litter in 1974. However, the ice nucleation characteristics of P. syringae are not compatible with the characteristics of leaf litter-derived INPs since the latter were found to be sub-micron in size, while INA of P. syringae depends on much larger intact bacterial cells. Here we determined the cumulative ice nucleation spectrum and microbial community composition of the historic leaf litter sample 70-S-14 collected in 1970 that conserved INA for 48 years. The majority of the leaf litter-derived INPs were confirmed to be sub-micron in size and to be sensitive to boiling. Culture-independent microbial community analysis only identified Pseudomonas as potential INA. Culture-dependent analysis identified one P. syringae isolate, two isolates of the bacterial species Pantoea ananatis, and one fungal isolate of Mortierella alpina as having INA among 1170 bacterial colonies and 277 fungal isolates, respectively. Both Pa. ananatis and M. alpina are organisms that produce heat-sensitive sub-micron INPs. They are thus both likely sources of the INPs present in sample 70-S-14 and may represent important terrestrial sources of atmospheric INPs, a conclusion that is in line with other recent results obtained in regard to INPs from soil, precipitation, and the atmosphere.
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    The Contribution of Within-Field Inoculum Sources of Gibberella zeae to Fusarium Head Blight in Winter Wheat and Barley
    Keller, Melissa Dawn (Virginia Tech, 2011-04-26)
    Fusarium head blight (FHB) is one of the most economically important diseases of small grains and continues to impact crops when environmental conditions are favorable to Gibberella zeae (Fusarium graminearum), the causal agent of the disease. Corn residues are considered to be primary sources of inoculum for epidemics of FHB. Therefore, knowledge of the movement of Gibberella zeae from a local source of infested corn residue is critical to the management of FHB in wheat and barley. Previous research made significant progress in defining the spatial dissemination of inoculum sources of G. zeae within agricultural fields, but was unable to clearly distinguish between within-field and background sources. Using amplified fragment length polymorphism, released clones of G. zeae were tracked within wheat and barley fields. This strategy allowed the distinction between the contributions of released clones to FHB, compared to that of background inocula. Corn residue infested with clones of G. zeae was placed into small replicated plots in winter wheat fields in New York and Virginia in 2007 and 2008 and wheat spikes were collected at 0, 3, 6, and ≥24 m from the inoculum sources. Recovery of released clones decreased an average of 90% between 3 and 6 m from inoculum sources. Various amounts of corn residue infested with a single clone of G. zeae were placed into small replicated plots in winter wheat and barley fields in Virginia from 2008 to 2010. The use of minimal or conventional tillage and a moderately resistant cultivar of wheat or barley may reduce the contribution of within-field inocula to FHB; however, environmental conditions play an important role in the effectiveness of these management strategies. With the increase of corn production due to incentives for ethanol-based fuel, overwintering sites for G. zeae on corn residue are likely to increase. Our work contributes to an increased understanding of the influence of overwintered corn residue to FHB which will also direct future research on how to reduce the inoculum potential from within-field sources.
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    Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases
    Khatibi, Piyum A.; Montanti, Justin; Nghiem, Nhuan P.; Hicks, Kevin B.; Berger, Gregory L.; Brooks, Wynse S.; Griffey, Carl A.; Schmale, David G. III (2011-09-02)
    Background The trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation) when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON), and may reduce its toxicity in plants and animals. Results Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (Saccharomyces cerevisiae) during a series of small-scale ethanol fermentations using barley (Hordeum vulgare). DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash. Conclusions This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.
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    Coordinated Sampling of Microorganisms Over Freshwater and Saltwater Environments Using an Unmanned Surface Vehicle (USV) and a Small Unmanned Aircraft System (sUAS)
    Powers, Craig W.; Hanlon, Regina; Grothe, Hinrich; Prussin, Aaron J. II; Marr, Linsey C.; Schmale, David G. III (Frontiers, 2018-08-15)
    Biological aerosols (bioaerosols) are ubiquitous in terrestrial and aquatic environments and may influence cloud formation and precipitation processes. Little is known about the aerosolization and transport of bioaerosols from aquatic environments. We designed and deployed a bioaerosol-sampling system onboard an unmanned surface vehicle (USV; a remotely operated boat) to collect microbes and monitor particle sizes in the atmosphere above a salt pond in Falmouth, MA, United States and a freshwater lake in Dublin, VA, United States. The bioaerosol-sampling system included a series of 3D-printed impingers, two different optical particle counters, and a weather station. A small unmanned aircraft system (sUAS; a remotely operated airplane) was used in a coordinated effort with the USV to collect microorganisms on agar media 50 m above the surface of the water. Samples from the USV and sUAS were cultured on selective media to estimate concentrations of culturable microorganisms (bacteria and fungi). Concentrations of microbes from the sUAS ranged from 6 to 9 CFU/m3 over saltwater, and 12 to 16 CFU/m3 over freshwater (over 10-min sampling intervals) at 50 m above ground level (AGL). Concentrations from the USV ranged from 0 (LOD) to 42,411 CFU/m3 over saltwater, and 0 (LOD) to 56,809 CFU/m3 over freshwater (over 30-min sampling intervals) in air near the water surface. Particle concentrations recorded onboard the USV ranged from 0 (LOD) to 288 μg/m3 for PM1, 1 to 290 μg/m3 for PM2.5, and 1 to 290 μg/m3 for PM10. A general trend of increasing concentration with an increase in particle size was recorded by each sensor. Through laboratory testing, the collection efficiency of the 3D-printed impingers was determined to be 75% for 1 μm beads and 99% for 3 μm beads. Additional laboratory tests were conducted to determine the accuracy of the miniaturized optical particle counters used onboard the USV. Future work aims to understand the distribution of bioaerosols above aquatic environments and their potential association with cloud formation and precipitation processes.
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    Coordinated Unmanned Aircraft System (UAS) and Ground-Based Weather Measurements to Predict Lagrangian Coherent Structures (LCSs)
    Nolan, Peter J.; Pinto, James; González-Rocha, Javier; Jensen, Anders; Vezzi, Christina N.; Bailey, Sean C. C.; de Boer, Gijs; Diehl, Constantin; Laurence, Roger; Powers, Craig W.; Foroutan, Hosein; Ross, Shane D.; Schmale, David G. III (MDPI, 2018-12-15)
    Concentrations of airborne chemical and biological agents from a hazardous release are not spread uniformly. Instead, there are regions of higher concentration, in part due to local atmospheric flow conditions which can attract agents. We equipped a ground station and two rotary-wing unmanned aircraft systems (UASs) with ultrasonic anemometers. Flights reported here were conducted 10 to 15 m above ground level (AGL) at the Leach Airfield in the San Luis Valley, Colorado as part of the Lower Atmospheric Process Studies at Elevation—a Remotely-Piloted Aircraft Team Experiment (LAPSE-RATE) campaign in 2018. The ultrasonic anemometers were used to collect simultaneous measurements of wind speed, wind direction, and temperature in a fixed triangle pattern; each sensor was located at one apex of a triangle with ∼100 to 200 m on each side, depending on the experiment. A WRF-LES model was used to determine the wind field across the sampling domain. Data from the ground-based sensors and the two UASs were used to detect attracting regions (also known as Lagrangian Coherent Structures, or LCSs), which have the potential to transport high concentrations of agents. This unique framework for detection of high concentration regions is based on estimates of the horizontal wind gradient tensor. To our knowledge, our work represents the first direct measurement of an LCS indicator in the atmosphere using a team of sensors. Our ultimate goal is to use environmental data from swarms of sensors to drive transport models of hazardous agents that can lead to real-time proper decisions regarding rapid emergency responses. The integration of real-time data from unmanned assets, advanced mathematical techniques for transport analysis, and predictive models can help assist in emergency response decisions in the future.
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    Detection of a Surrogate Biological Threat Agent (Bacillus globigii) with a Portable Surface Plasmon Resonance Biosensor
    Adducci, Benjamin Augustus (Virginia Tech, 2015-06-08)
    New methods and technology are needed to detect biological agents that threaten the health of humans and domestic animals. The bacterium Bacillus anthracis, causal agent of anthrax, has been used as a biological warfare agent. Here, we extend the work of Chinowksy et al. (2007) to the detection of a surrogate of B. anthracis, B. globigii (also known as B. atrophaeus, B. subtilis var. niger, B. subtilis var. subtilis) in a mixed sample containing two different species of Bacillus using a portable surface plasmon resonance (SPR) biosensor (SPIRIT 4.0, Seattle Sensor Systems). Two methods (direct capture and antibody injection) were used to determine the limit of detection for spores of B. globigii and to detect spores of B. globigii in a mixed sample containing at least one other Bacillus spp. Spores of B. globigii were detected on freshly coated sensors (not previously exposed to spores) with direct capture at a minimum concentration of 10^7 spores/mL, and with antibody injection at a concentration of 10^5 spores/mL. Spores of B. globigii were also detected when mixed with B. pumilus spores in the same sample at equal concentrations (107 spores/mL) using antibody injection. An SPR method using synthetic miRNA was adapted to the portable SPR unit (SPIRIT), and preliminary experiments suggested that the target sequence could be detected. SPR methods using nucleic acids have an exciting future in the detection of biological agents, such as B. anthracis. With the availability of portable instrumentation to accurately detect biological warfare agents such as B. anthracis, emergency responders can implement emergency protocols in a timely fashion, limiting the amount of people and domestic animals exposed.
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    Development of an Autonomous Unmanned Aerial Vehicle for Aerobiological Sampling
    Dingus, Benjamin Ross (Virginia Tech, 2007-05-08)
    The ability to detect, monitor, and forecast the movement of airborne plant pathogens in agricultural ecosystems is essential for developing rational approaches to managing these habitats. We developed an autonomous (self-controlling) unmanned aerial vehicle (UAV) platform for aerobiological sampling tens to hundreds of meters above agricultural fields. Autonomous UAVs have the potential to extend the range of aerobiological sampling, improve positional accuracy of sampling paths, and enable coordinated flight with multiple aircraft at different altitudes. We equipped a Senior Telemaster model airplane with two spore-sampling devices and a MicroPilot autonomous system, and we conducted over 60 autonomous microbe-sampling flights at Virginia Tech's Kentland Farm. To determine the most appropriate sampling path for aerobiological sampling, we explored a variety of different sampling patterns for our autonomous UAVs including multiple GPS waypoints plotted over a variety of spatial scales. We conducted a total of 25 autonomous aerobiological sampling flights for five different aerobiological sampling patterns. The pattern of a single waypoint exhibited the best flight characteristics with good positional accuracy and standard deviations in altitude from 1.6 to 2.8 meters. The four point pattern configured as a rectangle also demonstrated good flight characteristics and altitude standard deviations from 1.6 to 4.7 meters.