A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues(R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA was used for relative quantification of viral protein in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity.

No description available.

Diagnostics is important in the development and implementation of pest management strategies. The virus diagnostic capabilities of several plant pathology collaborators within the Integrated Pest Management Collaborative Research Support Program (IPM CRSP) host countries were evaluated with the aid of a survey. Very few plant disease diagnostic clinics had funds to cover daily operations despite over half of the responding clinics receiving an operational budget. Academically and government affiliated clinics within the developing host countries had little access to molecular tools and equipment, relying mostly on biological and serological methods. Clinics affiliated with private companies and within the USA relied more upon molecular assays. Ten CMV isolates identified by tissue blot immunoassay (TBIA) were collected from a garden at the Historic Smithfield Plantation on the Virginia Tech campus, and from Painter, Virginia on the Eastern Shore. Three CMV isolates from Smithfield were biologically compared to six early CMV isolates stored since the 1970s, while all isolates were compared serologically and molecularly. Sequences obtained after reverse transcription-polymerase chain reaction (RT-PCR) assigned the CMV isolates into subgroups, with eleven to subgroup 1A and three to subgroup 2. The subgroup assignments were confirmed by TBIA using CMV subgroup-specific monoclonal antibodies (Agdia Inc). At Smithfield Plantation, another virus, Turnip mosaic virus (TuMV) was identified from Dame's Rocket (Hesperis matronalis L.). This is the first report of TuMV in Virginia.  In TBIA virus-infected plant samples are blotted onto nitrocellulose membranes, dried, and processed. Membranes can be stored for long periods of time and transported safely across borders without risk of introducing viruses into new environments, but virus remains immunologically active for several months. Methods were developed with CMV and three potyviruses, using the same membranes, for detecting viral RNA by RT-PCR and direct sequencing of PCR products.. Amplification by RT-PCR  was possible after membrane storage for up to 15 months. The membranes also performed well with samples sent from IPM CRSP host countries and within the USA. This method should improve molecular diagnostic capabilities in developing countries, as samples can be blotted to membranes and sent to a centralized molecular laboratory for analysis. / Ph. D.

The presence of turnip crinkle virus (TCV) in Arabidopsis thaliana plants has previously been shown to suppress the ability of these plants to produce a hypersensitive response (HR) upon inoculation with pathogens that would normally elicit this defense response. The ecotype Colombia-0 was examined using wildtype TCV and non-pathogenic strains of Pseudomonas syringae pv. glycinea Race 4 containing virulence genes avrRpt2, avrRpm1 and avrRps4. Transgenic lines of A. thaliana that express the TCV proteins p8, p9 or CP were also examined in an attempt to determine if these proteins play a role in suppression of the HR. Crosses of these transgenic lines were made in order to determine if binary combinations of these proteins were sufficient for HR suppression. In addition, assays were completed to determine if the inhibition of the HR correlated with suppression of resistance to the virulent Pseudomonas syringae pv. maculicola ES4236 avrRpt2 growth in the plant. Finally, PR-1 protein expression was inspected by visual and quantitative GUS reporter gene assays to determine if TCV also played a role in inhibition of the plants ability to develop systemic acquired resistance (SAR).

Replication of genetic material is the most important and central process during the viral life cycle. Most RNA viruses assign one or more proteins translated from their own genome for replicating genomic RNAs. Understanding the various biochemical activities of these replication proteins is the aim of this dissertation research. The replicase proteins of Turnip crinkle virus (TCV) and Tomato bushy stunt virus (TBSV) were selected for this study. Both viruses have small, messenger-sense, single-stranded RNA genomes. Replicase proteins p28/p88 of TCV and p33/p92 of TBSV- were expressed and purified from E. coli as N-terminal fusions to maltose binding protein. In vitro assays revealed that the recombinant p88 has RNA-dependent RNA polymerase (RdRp) and RNAbinding activities. Deletion of the N-terminal p28 domain in p88 resulted in a highly active RdRp, while further deletions at both N- and C-terminal ends abolished RdRp activity. Comparison of p88, the N-terminal p28-deletion mutant of p88 and a TCV RdRp preparation obtained from infected plants revealed remarkable similarities in RNA template recognition and plus and minus strands synthesis. Contrary to recombinant TCV RdRp activities under similar experimental conditions. p33 preferentially binds to singlestranded (ss) RNA with positive cooperativity in vitro. The RNA binding activity was mapped to arginine/proline-rich motif (RPR-motif) at the C-terminus of p33 and the corresponding sequence in p92. The non-overlapping C-terminal domain of p92 also contained additional RNA-binding regions that flank the conserved RdRp motifs on both sides. Cooperative RNA binding by p33 suggested inter-molecular interactions between p33 monomers. Indeed the yeast two-hybrid and surface plasmon resonance assays revealed interactions between p33 and p33 and also between p33 and p92. The sequence involved in the protein-protein interactions was mapped to the C-terminal region in p33, proximal to RPR-motif. Within this region, mutations introduced at two short stretches of amino acid residues were found to affect p33:p33 and p33:p92 interactions in vivo and also decreased the replication of a TBSV-defective interfering RNA in yeast, a model system, supporting the significance of these protein interactions in tombusvirus replication.

The relative amount of viral RNA of two comoviruses (RaMV and TuRSV) in different parts of plant during infection was determined. The two viruses were compared according to their ability to spread and multiply in plants.

Purple nutsedge (Cyperus rotundus) is a troublesome weed that outcompetes crops and contributes to poor yields. In the past, agriculturalists controlled purple nutsedge by fumigating soil with methyl bromide but the fumigant has since been classified as a controlled substance under the Montreal Protocol. This study evaluated the effectiveness of several alternative purple nutsedge control techniques and compared them with results obtained from the application of Roundup. Concentration treatment effects for the allelopathic seed powders of watercress and turnip were tested in a field trial while seed powders of yellow mustard and sunflower were tested in a potted trial. The allelopathic amendments significantly delayed weed emergence but several factors interfered with long-term effectiveness. Roundup was determined to be the most effective season-long weed control among the treatments consistently leaving the least amount of surviving weeds and underground organs.

Em sistemas de plantio direto, o material vegetal incorporado libera fósforo solúvel no solo, porém, sua absorção por plantas subseqüentes ainda é pouco conhecida. Parte do fósforo nos tecidos vegetais está na forma de compostos insolúveis, que precisam ser mineralizados para serem absorvidos pelas plantas. Os objetivos deste trabalho foram estabelecer a melhor época de aplicação de 32P para marcação de plantas de adubo verde; determinar as formas de P nas plantas; determinar as principais formas de fósforo no solo após a incorporação de diferentes espécies de adubo verde; estimar a eficiência dos adubos verdes no fornecimento de P para plantas de arroz, e determinar a relação entre as formas de P nas plantas de adubo verde e sua mineralização no solo. Os experimentos foram conduzidos em duas etapas. Na Etapa 1 foram marcadas com 32P plantas de aveia-preta, milheto e nabo forrageiro na semeadura e aos 14, 28 e 42 dias. Estas plantas foram incubadas e utilizadas como adubo verde no cultivo de arroz. As plantas de arroz apresentaram eficiência de utilização de P semelhantes para adubos verdes marcados até 28 dias, podendo ser utilizada uma atividade 75% menor para marcação dessas espécies. Na etapa 2 foram cultivadas plantas de aveia-preta, crotalária e nabo forrageiro marcadas com 32P aos 14 dias. Foi verificado que a maioria do P nas plantas está na forma solúvel, correspondendo em média 67% do 32Pt. Essas plantas foram incubadas por 20, 40 e 60 dias e cultivadas plantas de arroz. A eficiência de utilização do P proveniente de adubo verde foi maior para incubação por 20 e 60 dias, diminuindo consideravelmente para incubação por 40 dias. Foram determinadas as frações de P no solo incubado com adubo verde nos diferentes períodos de incubação, utilizando o método de Hedley. A fração Pi-resina e P-NaHCO3, que correspondem ao P-lábil, apresentaram as maiores atividades específicas, demonstrando que a maior parte do P solúvel do adubo verde são encontrados nessas frações. A fração Po-NaOH apresentou as maiores concentrações de P, enquanto as menores foram encontradas na fração P-HCl / In no-tillage systems, plant materials incorporated into soil release soluble phosphorus, but its absorption by subsequent plants is not completely clarified. A fraction of phosphorus in vegetal tissues is in the form of insoluble species, which require mineralization to be absorbed by plants. The aims of this work were to establish the best period for 32P application for green manure labeling; to determine the forms of P in plants and in soil after incorporation of different green manures; to estimate the efficiency of green manures for supplying phosphorus to rice plants and to determine the relationship between the P species in green manure and their mineralization in soil. The experiments were carried out in two steps. In the first, plants of black oat, millet and turnip were labeled with 32P at sowing and after 14, 28 and 42 days. These plants were incubated and used as green manure for rice plants. The rice plants showed similar utilization efficiencies of P form green manure plants labeled up to 28 days, making possible the use of a 75% lower activity to label these plants. In step two, black oat, sunnyhemp and turnip plants labeled with 32P after 14 days from sowing were grown. It was verified that the more significant fraction of P in plants is in the soluble form, corresponding in average 67% of 32Pt. These plants were incubated for 20, 40 and 60 days before culturing rice plants. The utilization efficiency of P from green manure sources was higher when the material was incubated for 20 and 60 days, being considerably reduced for 40 days incubation. The P-fractions in the soil incubated with green manure in different periods were determined by the Hedley method. The Pi-resin and P-NaHCO3 fractions, which correspond to labile P, showed the highest specific activities, because the most significant part of the soluble P was found in these fractions. The Po-NaOH fraction showed the highest concentrations of P, while the lowest amounts were in the P-HCl fraction

Plant viruses have been instrumental in our understanding of the biophysical properties pertaining to non-enveloped icosahedral virus particles. A substantial amount of research has been performed over five decades on Turnip yellow mosaic virus (TYMV), arguably one of the most extensively studied icosahedral plant viruses and the type-member of the Tymovirus plant virus genus. Even with a substantial body of published scientific literature, little is known about the role of specific coat protein (CP) residues in TYMV assembly, disassembly and disencapsidation. We have shown through our mutagenesis studies that the N-terminal region of the CP that is involved in the formation of an annulus structure and is disordered in A-subunit pentamers is not essential in vivo, but annulus-forming residues are critical in ensuring virion stability and low accessibility after virus is purified (Chapter 2). We have shown that a range of amino acid residue types is tolerated within the CP N-terminus in vivo, although they can greatly affect the stability of virions and empty particles, most notably at low pH (Chapter 3). Unlike full-length CP, N-terminal deletion and substitution mutants fail to reassemble into particles in vitro (Chapter 2, 3) suggesting a critical determinant for the N-terminus in reassembly (discussed Chapter 7). This is the first documented in vitro reassembly reported for a member of the Tymoviridae family and should provide a framework for further studies. We have identified a new way to create empty artificial top component (ATC)-particles through treatment with EDTA (Chapter 6) and we also show that tymoviruses can be engineered with altered pH-dependent enhanced stability (Chapter 4). In collaboration with the Qian Wang laboratory from the University of South Carolina we have shown that an RGD (Arg-Gly-Asp) motif can be genetically engineered within the CP of TYMV, resulting in infectious particles with attractive stem-cell adhesion properties (Chapter 5). With focus on basic viral mechanisms, we have crystallized the TYMV virion and ATC particle at pH 7.7 and collected data to less than 5 Å resolution (Chapter 4, supplementary). These structures represent the first tymovirus-based structures solved above pH 5.5 and will provide insight into the N-terminal conformations within the TYMV particle. Finally, we have characterized an N-terminal CP cleavage seen after ATC formation (Chapter 4) suggesting an additional and yet uncharacterized feature associated with decapsidation. / Graduation date: 2012

Thesis (Ph.D.). / Written for the Dept. of Plant Science. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.