Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 99-110). Also available via the Internet.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 19-30). Also available via the Internet.

The objectives of this study were to determine the effects the temperature sensitivity of alleles of Rsv1 in soybean (Glycine max (L.) Merr.). Soybean cultivars carrying alleles of Rsv were exposed to 1 several heat treatments designed to induce heat shock protein production prior to inoculation with soybean mosaic virus (SMV). The heat treatment methods were similar to those employed in the research with N gene-tobacco mosaic virus studies. The soybean cultivars used were Lee 69, York, Kwanggyo, Ogden and PI96983, carrying the Rsv, Rsv1-y, Rsv1-k, Rsv1-t, and Rsv1 allles of Rsv1, respectively, and were selected to provide a range of reactions to selected SMV pathotype groups. For example Rsv1-y and Rsv1-k give a necrotic response to SMV G4 and SMV G6, respectively, while both are resistant to SMV G1. To determine the durability of resistance under heat shock conditions, the symptoms were observed for changes in the phenotype of the resistance response. Immunological techniques were employed to determine the vascular movement and localization of the viral antigen in the plant. Heat treatments used were found to induce HSP but to have no effect on the resistance phenotype. A detached leaf assay was used to test the same Rsv alleles at constant 1 high temperatures. Primary trifoliolate leaflets were removed and inoculated, then placed into a continuously lighted incubator at 20 °C or 30 °C. Leaf immunoprint assays were used to determine the localization of the viral antigen. The visible symptoms for necrotic lesions and veins were observed for necrotic phenotype-pathotype combinations but mosaic symptoms were not observed on detached leaves, as expected for inoculated leaves. The detached leaf assay confirmed that no change from the expected resistance response of the Rsv alleles occurred at 30 C. A breakdown 1 o of resistance to SMV at high temperature had been reported in soybean by Tu and Buzzell (1987). The resistance gene in which the high temperature breakdown occurred has been determined to be Rsv . Using cultivars and breeding lines carrying Rsv a similar experiment was attempted in growth 3 3 chambers. Preliminary results suggest that Rsv is temperature sensitive. / Master of Science

Two Virginia isolates of soybean mosaic virus (SHV), VA and OCM, were classified as G1 and G3 strains, respectively, according to a soybean differential cultivar system. G1/VA and G3/OCM differed in the symptom severity induced on soybean (Glycine max [L.]Merr.) plants and by the production by G3/OCM of cytoplasmic strands, containing virus particles, within infected leaf cells. Two Illinois isolates, G1/IL and G3/IL, resembled G3/0CM in mild symptoms; induced in soybean plants and by the production of cytoplasmic strands ultrastructurally. G1/VA differed from the 3 mild strains by the severe symptoms induced on plants and by the absence of cytoplasmic strands ultrastructurally. All 4 strains were closely related serologically. The tolerant reaction of soybean to the 3 mild SMV strains was correlated with production of cytoplasmic strands. Soybean plants were most susceptible to G1/VA and peanut stunt virus (PSV) when the primary leaves were inoculated at 50-75% of their full expansion. Plants developed more severe symptoms with either virus within a shorter incubation period than did plants which were inoculated when their primary leaves were less than or greater than 50-75% expansion. The SMV strain, SMV-VA (G1/VA), was used to determine the cell types SMV moves into, using pinwheel inclusions as indicators of cell infection, at an early stage of infection when virus particles were first detectable in inoculated and noninoculated tissues of prilutry leaves. By using serologically specific electron microscopy (SSEM), virus particles were first detected in inoculated tissues at 6 days after inoculation. Pinwheels were detected within these areas in palisade, paraveinal, spongy, vascular parenchyma, phloem and immature xylem cells. The pinwheels were frequently opposed to the plasmalemma and were associated with plasmodesmata and plasmalemmasomes. Endoplasmic reticulum was associated with the base of those pinwheels found in the cytoplasm. The early stage of SMV pinwheel formation appeared to be similar to that reported for other potyviruses. The observation of pinwheels in the paraveineal mesophyll cells suggested the involvement of these cells in virus translocation within the soybean leaf. A sinusoidal pattern of SMV particle concentration within the inoculated and noninoculated areas of the leaf was detected by SSEM. The appearance of necrotic lesions within the inoculated area was correlated with an increase in the number of detectable particles within the area. A similar correlation was made with an increase in particle numbers in the non-inoculated area and the appearance of vein-clearing on the first trifoliolate leaf. It was also noted that particle numbers increased in the midrib and petiole when particle numbers decreased in the inoculated and noninoculated areas. This pattern was hypothesized to be due to either an alteration in the viral replication rate or to translocation relationships within the leaf. / Ph. D.

The responses of soybean (Glycine max (L.) Merr.) to soybean mosaic virus (SMV) include mosaic, necrosis and no symptoms, and vary with virus strain and allele of the gene for resistance (Rsv) carried by the host. Genetic studies have shown that plants giving mosaic are susceptible, and that plants giving either the necrotic or symptomless response are resistant. The objective of this study was to examine the mechanism of resistance by tracking SMV replication and movement in susceptible responses, and its restriction in necrotic and symptomless responses. Two SMV strains were inoculated at a single spot to Rsv-containing genotypes, selected to give each response. The leaf imprint immunoassay was developed and used to track the rate and extent of invasion by SMV from the tip of a primary, unifoliolate leaf to regions within that leaf and to the stem and younger trifoliolate leaves. In susceptible responses, SMV was detected at the site of inoculation in 6-7 days, and throughout the mid-rib and in the first trifoliate leaf in 8-9 days. In necrotic resistance responses, SMV was detected at and around the inoculation site in 8-9 days and in leaf veins, midrib and the first trifoliolate in 15-17 days, but was restricted to necrotic areas. In symptomless resistance responses, no virus was detected at any time. Greater virus replication and movement was found in unifoliolate and trifoliolate leaves of younger (15 days-old) than of older (18-days-old) plants. It was concluded that: (i) necrotic ally-responding cultivars manifest resistance by reduction and delay in replication, and by restriction of virus movement; (ii) virus replication is restricted in resistant cultivars; and (iii) rate and extent virus replication and movement is affected by stage of plant development. / Master of Science

Soybean mosaic virus (SMV) is the major virus disease reported all over the world in soybean crop. This disease causes reduction in the yield and quality of soybean crop. Three independent genes Rsv1, Rsv3, and Rsv4, were found to provide host resistance in soybean. Rsv1 confers resistance to all but most virulant strains of SMV. Rsv1 has been mapped to soybean molecular linkage group (MLG) F by using molecular markers. The purpose of this study is to investigate the location of Rsv3 gene on soybean map using molecular markers. The Rsv3 gene of soybean confers resistance to the most vurulent strains (G5-G7) of SMV. In order to map the gene, an F2 population was constructed from a cross between L29, an Rsv3 isoline of 'Williams', and 'Lee 68', a susceptible cultivar. Rsv3 genotypes of 183 F2 plants were determined by inoculating F2:3 progeny with the G7 strain of SMV. A preliminary survey of two parental lines, near isogenic lines (NILs), and bulk segregants with 136 restriction fragment length polymorphism (RFLP) markers yielded 36 markers showing variation between the two parents. These polymorphic RFLP markers unable to provided any indication of linkage to Rsv3. As an alternative strategy, amplified fragment length polymorphic (AFLP) marker analysis of the two parental lines, NILs and bulk segregants was performed using 64 primer combinations. Initial breakthrough came in the form of AFLP primer combination of Eco+AAC/Mse+CTG exhibited polymorphism between NILs, bulk segregants, and two parental lines. This AFLP marker was isolated and cloned to convert it into a RFLP clone to further investigate the linkage to Rsv3 by F2 segregation analysis. A mapping population constructed by crossing Glycine max x Glycine soja employed in determining the location AFLP-derived RFLP clone on soybean linkage map. This population has densely mapped molecular marker data that enabled determining the location of AFLP-derived RFLP clone ACR1 on soybean molecular linkage group (MLG) B2 between the markers pA516 and pA519. This finding, made it easy to establish the linkage of markers pA519, pA516, and pA593 in L29 x Lee 68 population by F2 segregation analysis. The closest marker linked pA519, was 0.9 cM away from Rsv3. In another study Rsv4 is reported to be mapped to MLG D1b of soybean. Results of this study are useful in marker-based selection (MAS), pyramiding viral resistance genes and in cloning the Rsv3 gene. / Master of Science

The nucleotide sequence of the 3' terminus of soybean mosaic virus (SMV) VA/G1 RNA has been determined by dideoxynucleotide sequencing of oligo(dT) primed cDNA cloned into a pUC19 cloning vector via EcoR1 linkers. One recombinant plasmid, pSMV49, identified by colony and dot-blot hybridization to ¹²⁵I-SMV RNA, contained an insert of 1443 nucleotides and had an open reading frame of 1119 nucleotides terminating 224 nucleotides from the 3' terminal poly(A) tract. The coat protein cistron identified by a glutamine:serine dipeptide cleavage site 792 nucleotides upstream from the termination sequence could potentially code for a 29.8 kDa protein. The amino acid sequence predicted from the nucleotide sequence of this cistron contains regions identical to other potyvirus coat proteins. / Master of Science

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.

Strain-specific resistance conditioned by a single dominant gene in soybean cv. York inoculated with SMV-G1, revealed no symptoms and no detectable viral replication (R). Unlike the hypersensitive response (HR), the R response did not result in localized virus and induction of a series of defense responses, but in inhibition of virus replication. However, this resistance was overcome by the resistance- breaking strain, SMV-G4, which induced lethal necrosis (N). Unlike HR, G4 strain was not restricted but spread ina restricted pattern along the vein, stem and into upper un-inoculated leaves where it induced necrosis. Like HR, PR proteins were found to accumulate in the N response and were named SPR (soybean pathogenesis-related) proteins. On the basis of major similarities in molecular weight characteristics and enzyme-substrate specificities, SPRs are proposed to be classed into four groups: SPR1; 1a, 1b, 1c, 1d; SPR2: 2a, 2b, 2c; SPR3: 3a, 3a’, 3b, 3c, 3d, 3e; SPR4. The functions of SPR1 and SPR4 groups have not yet been determined. The SPR2 group was identified as β-1,3-glucanases (GLN) and classed two subgroups. The SPR3 group was identified as chitinases (CHN) and classed three distinct subgroups. Like HR, the N response induced a series of defense responses with marked temporal increases, in a bimodal pattern, of phenylalanine ammonia-lyase (PAL) and peroxidase (POX) activities, bimodal pattern of PAL mRNA and chalcone synthase (CHS) mRNA induction, a single pattern of chitinase mRNA induction, and glyceollin and lignin accumulation. These marked increases occurred at or just before the time of initial appearance of necrotic lesions, or following necrosis development for localization of virus. Total RNA isolated from soybean inoculated with SMV-G4 were used for synthesis of cDNA by reverse transcription, and amplified using 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR)-specific primer sets by polymerase chain reaction. Amplified cDNAs were cloned into Bluescript vector (pKS⁻), transformed into E. coli, isolated pSOY HMGR and used as a probe for Northern analysis. We demonstrate that expression of HMGR mRNA is correlated with strain-specific resistance (R). SMV coat protein (CP) degraded in vitro by proteolysis during purification. The CP of SMV purified from infected leaves had an major size of 34 kD in SDS-PAGE with minor peptides of 32 and 31 kD. The minor peptides increased during the storage at 4°C or with trypsin treatment, and reacted with antiserum to intact virions. This heterogeneity of protein was not removed by alkaline phosphatase treatment for varying time intervals, and was not related with phosphorylation and dephosphorylation, or with virus maturation. These studies provided additional evidence of N- or C-terminal exposure on the particle’s surface. / Ph. D.

The field of functional genomics aims to understand the complex relationship between genotype and phenotype by integrating genome-wide approaches, such as transcriptomics, proteomics, and metabolomics. Large-scale "-omics" research has been made widely possible by the advent of high-throughput techniques, such as next-generation sequencing and mass-spectrometry. The vast data generated from such studies provide a wealth of information on the biological dynamics underlying phenotypes. Though functional genomics approaches are used extensively in human disease research, their use also spans organisms as miniscule as mycoplasmas to as great as sperm whales. In particular, functional genomics is instrumental in agricultural advancements for the improvement of productivity and sustainability in crop and livestock production. Improvement in soybean production is especially imperative, as soybeans are a primary source of oil and protein for human and livestock consumption, respectively. The research presented here employs functional genomics approaches – transcriptomics and metabolomics – to discern the transcriptional regulation and metabolic events underlying two economically important agronomic traits in soybean: seed phytic acid content and Soybean mosaic virus resistance. At normal levels, seed phytic acid content inhibits mineral absorption in humans and livestock, acting as an antinutrient and contributing to phosphorus pollution; however, the development of low phytic acid soybeans has helped mitigate these issues, as their seeds increase nutrient bioavailability and reduce environmental impact. Despite these desirable qualities, low phytic acid soybeans exhibit poor seed performance, which negatively affects germination rates and yield and has prevented their large-scale commercial production. Thus, part of the focus of this research was investigating the effects of mutations conferring the low phytic acid phenotype on seed germination. Comparative studies between low and normal phytic acid soybean seeds were carried out and revealed distinct differences in metabolite profiles and in the transcriptional regulation of biological pathways that may be vital for successful seed germination. The final part of this research concerns Rsv3-mediated extreme resistance, a unique mode of resistance that is effective against the most virulent strains of Soybean mosaic virus. The molecular mechanisms governing this type of resistance are poorly characterized. Therefore, the research presented here attempts to elucidate the regulatory elements responsible for the induction of the Rsv3-mediated extreme resistance response. Utilizing a comparative transcriptomic time series approach on Soybean mosaic virus-inoculated Rsv3 (resistant) and rsv3 (susceptible) soybean lines, this final study provides gene candidates putatively functioning in the regulation of biological pathways demonstrated to be crucial for Rsv3-mediated resistance. / Doctor of Philosophy / Soybeans are a crop of great economic importance, being a primary source of oil and protein for human and livestock consumption, respectively. Increasing demand for soybean calls for improvement in its production. An emerging field that has had tremendous impact on this endeavor is the field of functional genomics. Functional genomics approaches generate large-scale biological data that can aid in discerning how specific processes are regulated and controlled in an organism. The research presented in this work utilizes functional genomics approaches to elucidate the biological mechanisms underlying two economically important traits in soybean: seed phytic acid content and Soybean mosaic virus resistance. Phytic acid is a compound found in soybean seeds that causes nutrient deficiencies and phosphorus pollution. Soybeans with reduced to phytic acid content have been developed to mitigate these problems; they have poor seed germination and emergence. The studies in this work employ functional genomics approaches to compare unique sets of low and normal phytic acid soybeans to help establish the relationship between seed phytic acid content and seed performance. These studies resulted in new and promising hypotheses for future studies on investigating the low phytic acid trait. The final focus of this work used a functional genomics approach to discern the molecular mechanisms underlying a unique mode of resistance to Soybean mosaic virus. The study identified genes in soybean that are potentially critical to resistance against Soybean mosaic virus.