The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields. Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor. Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates. Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline. In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar. These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields. / Ph. D.

The purpose of this investigation was to test three insecticides on all the life stages of the alfalfa weevil. The objectives were to obtain information on the toxicity of certain insecticides on the alfalfa weevil in the laboratory, to establish standard susceptibility curves of the life stages of the alfalfa weevil to the insecticides tested, to establish a basis for insect resistance, to compare the speed of action of certain classes of insecticides and to observe the effects of certain insecticides on molting, feeding and other processes that may be affected. Dibrom, heptachlor epoxide, and Indian were tested against various life stages of the alfalfa weevil. These three insecticides were chosen because of the differences in their chemistry and activity. Dibrom, a volatile organophosphate, could be expected to act fast but have relatively limited residual life.Heptachlor epoxide as a cyclodiene compound should have exhibited the characteristic latent period of from two to six hours before symptoms of its activity appeared. The residual activity should be great. Indian, a more stable organophosphate than Dibrom, would be expected to be somewhat intermediate between the other two in speed of action and residual activity. Each insecticide was impregnated in filter paper at the dosage of 100 micrograms per square centimeter of filter paper surface. The insects were introduced on to the filter paper for continuous exposure to the insecticide residue. Mortality and certain other effects were recorded according to time of exposure. In general, responses of the alfalfa weevil to the three insecticides were comparable. One insecticide may have been quicker acting than the other two against a particular life stage, but the differences in the effects of the insecticides were not great. In every case, the three insecticides inhibited feeding, molting, or maturation to another life stage. / Master of Science

Bathyplectes anurus (Thomson) and B. curculionis (Thomson) (Hymenoptera: Ichneumonidae) are specialist parasitoids introduced to the United States for classical biological control of alfalfa weevil, Hypera postica (Coleoptera: Curculionidae). Adults of both species are morphologically similar, generally described as wasps ≈3-4 mm long with black, robust bodies. The Bathyplectes spp. occupy sympatric ranges throughout much of the United States and often coexist within alfalfa fields. In Virginia, B. anurus is the primary biological control agent, accounting for more than 90% of alfalfa weevil parasitization. A previous study, however, found that the levels of control by B. anurus varied among the three alfalfa-growing regions of Virginia, and noted that little is known about the mechanisms or factors responsible for the differential levels of parasitization. Of the factors that may affect host parasitization, three were addressed in this study: population density and spatial distribution of host and parasitoid, the effectiveness of parasitoids in response to host density as measured by the population functional response, and genetic variation among and between populations of parasitoids. GPS-referenced quadrat sampling was conducted in alfalfa fields in the Piedmont and southwestern region to assess alfalfa weevil population levels and spatial distribution, and the level and distribution of parasitization by B. anurus and B. curculionis. Results showed that there was no significant difference in alfalfa weevil densities among alfalfa fields in the Piedmont and southwestern region, and also no significant difference between regions in the level of parasitization. Mean alfalfa weevil density per 0.2-m2 quadrat per sampling date was 11.0 in the Piedmont and 8.0 in the southwestern region; mean percent parasitization per 0.2-m2 quadrat per sampling date was 9.1% in the Piedmont and 9.9% in the southwestern region. The spatial distributions of alfalfa weevil larvae and parasitization were found to be aggregated in all alfalfa fields sampled. The population functional response of B. anurus attack on alfalfa weevil was determined qualitatively to be Type II in all alfalfa fields sampled, except the field located at Blacksburg in the southwestern region. The Type II functional response suggests that, in general, the parasitoid has an inverse density dependent attack response to the density of alfalfa weevil larvae in the two regions of Virginia. Molecular analyses were conducted to 1) derive genetic sequences for B. anurus and B. curculionis, which could be used to identify and differentiate the two species, and 2) test for genetic variation within and between regional populations of Bathyplectes. Sequences for each species were obtained for the D2–D3 region of the 28S ribosomal DNA (28S rDNA) gene; sequence identity between B. anurus and B. curculionis was 96.6%. The 28S rDNA sequences were also obtained for another Bathyplectes species, possibly B. stenostigma, and for four taxa of Hymenoptera that hyperparasitize B. anurus and B. curculionis cocoons. Sequences were obtained for B. anurus and B. curculionis for a portion of the mitochondrial gene cytochrome oxidase subunit 1 (COI); sequence identity between B. anurus and B. curculionis was 89.7%. Genetic variation in the COI gene was observed for both species. One variation of the COI sequence expressed by 25% of B. anurus specimens (Haplotype 2) might indicate regional genetic variation correlated to temperature, in particular the average number of days per year that a region experiences temperatures above 32°C. Two major COI sequence haplotypes found in B. curculionis specimens might be indicative of incipient speciation. / Ph. D.

Soil acidity is a major cause of low yields of alfalfa (Medicago sativa L.) in the southeastern United States. Two field experiments were conducted on an Ernest silt loam soil (fine—loamy, mixed, mesic Aquic Fragiudult) to determine what conditions are necessary for optimal alfalfa growth in an acid soil environment. In the first experiment alfalfa was no—till planted to evaluate the influence of surface applied dolomitic limestone at 0 and 6.7 Mg/ha either 8 or 20 months before planting and at planting on alfalfa performance under acidic conditions. Yield increases, greater than two fold, resulted from surface application of limestone regardless of time of application. In the second field experiment treatments included surface and incorporated dolomitic limestone at either 6.5 or 13.0 Mg/ha, gypsum at 13.0 Mg/ha, foliar Mo at 560 g/ha and N as a split. Yields increased in response to surface and incorporated lime, Ca, Mo and N application, possibly as a result of Al activities in soil solution being as low as 0 and 0.05 uM in the surface and 1 m depth samples, respectively, for the surface limed soils. Low activity of Al in soil solution may explain why subsurface acidity was not toxic to alfalfa grown on these soils. This study showed that surface limestone at half the recommended rate is adequate for the growth of alfalfa under acidic conditions. The implanted soil mass technique was used to evaluate the influence of subsurface amendments (Ca, N, Ca and N, KOH and Ca(OH)2) on alfalfa root growth and N fixation on soils that received either 0 or 6.7 Mg/ha surface applied limestone. Of the various subsurface amendments the Ca(OH)2 treatment produced the most root growth and N fixation. This study showed that both the reduction in acidity and the availability of Ca are necessary for optimal N fixation and root growth. In vitro studies conducted in growth pouches showed that pH 4.5 nutrient solution was not ·detrimental to alfalfa growth unless A1 was present. Addition of 3.0 mM Ca to a nutrient solution containing 0.08 mM Al alleviated toxic effects of Al on alfalfa growth. No-till alfalfa can thus be grown on acid soils with surface lime by providing Ca and reducing acidity. / Ph. D. / incomplete_metadata

Corn (Zea mays) and alfalfa (Medicago sativa) silages are used extensively in Virginia. A survey was conducted to determine chemical composition of these two forage silages grown in five geographical regions of Virginia; Eastern Virginia (EV), Northern Piedmont (NP), Southern Piedmont (SP), Shenandoah Valley (SV), and South-Western Virginia (SWV). A total of 889 samples of corn silage, 106 of ammoniated corn silage and 247 of alfalfa silage collected during 1988 and 1989 from 76 counties, were analyzed for fiber, N, and macro- and micro-nutrients. Chemical composition of the silages was correlated with S applied in fertilizer or manure. Data were compared with critical levels of mineral requirements of various classes of livestock. Alfalfa silage was higher (P < 0.05) in crude protein (CP), P, K, Ca, Mg, S, Mn, and Fe than com silage. Ammoniated corn silage was higher (P < 0.01) in CP and NS ratio, and lower in P, S (P < 0.01) and K (P < 0.05) concentrations than non-ammoniated corn silage. For lactating dairy cows, 86 and 95% of corn silage and ammoniated corn silage, respectively, grown throughout the State were deficient in P. Information supplied by farmers suggested that manure application increased P concentration of these forages. Over 90% of all corn silage would not have met the Ca requirements of dairy cows, however, 97% of the alfalfa silage was excessive in Ca concentration for dairy cows and could have served as a Ca supplement to the diet. Nitrogen:S ratio indicated S deficiency (N:S ratio > 12) in 34, 89 and 41% of samples of corn silage, ammoniated corn silage and alfalfa silage for dairy cattle and in 85, 96 and 91% of the respective silages for sheep (N:S ratio > 10). Based on S concentrations, 96% of corn silage and ammoniated corn silage grown throughout Virginia were S deficient for dairy cows while 72% of corn silage and 86% of ammoniated corn silage were deficient in S for sheep. Sulphur concentrations in silages did not indicate S deficiencies for plant growth. Over 60% of corn and alfalfa silages would not have met nutritional requirements for Zn and Cu in lactating dairy cows but requirements for Mg and Mn would have been supplied by more than half of the silages produced in Virginia. Regional/ geographical variations in almost all the nutrients were observed for both forages. Generally, corn silage grown in EV was lower in CP, TDN, Mg, and Mn and was higher in ADF compared to silage grown in the rest of the State. Lower CP, Ca, and S were observed in alfalfa silage grown in EV compared to the mean of other regions. Generally, higher N:S ratio in corn and alfalfa silages and lower P were found in alfalfa silage grown in Western Highlands compared to Piedmont region. Also CP and Ca were lower in corn silage grown in SWV compared to SV while Mg was lower in either silage grown in SV compared to SWV region. In general, concentrations of P, Ca, S, Zn, and Cu in corn silage and ammoniated corn silage were widely deficient ( > 70% samples deficient) for dairy cattle, and deficient in S for sheep. Magnesium deficiencies were less frequent. In alfalfa silage concentrations of Zn, and Cu were low for dairy cows. Nitrogen:S ratios indicated S deficiency for livestock, particularly in sheep and lactating dairy cows. / Master of Science

No-till alfalfa (Medicago sativa L.) establishment is the only environmentally sound procedure for alfalfa production on erodable land. Surface limestone application is the only reasonable method of placement in no-til1 systems with pH below 6.5. The purpose of this research was to evaluate surface limestone application vs. incorporation throughout the plow layer for alfalfa production in acidic soils. In this study limestone placement included: 1. incorporation during tillage operations, 2. surface application after tillage and preparation of seedbed, and 3. surface application without tillage. Limestone was applied at 0, 1.25, 2.50, and 5.00 tons per acre with each placement method. Limestone treatments were imposed on 30 Sept. 1986 in Montgomery County, VA (37° 1lâ N, 80° 25â W and 1950 ft. elevation) on a Groseclose silt loam (clayey, mixed, mesic Typic Hapludult) having a pH of 5.6 in the top 9 inches. Limestone treatments were imposed at a second site on 25 Nov. 1986 in Orange County, VA (38° l3â N, 70° 7â W and 515 ft. elevation) on a Davidson clay loam (clayey, oxidic, thermic Rhodic Paleudult) having a pH of 5.7 in the top 9 inches. 'Cimmeronâ alfalfa was planted using no-till procedures on 23 March 1987 in Montgomery County and on 26 Aug. 1987 in Orange County. Soil samples were taken about 2 years after limestone application at depths of 0 to 1, 1 to 2, 2 to 3, and 3 to 6 inches for surface applied treatments and 0 to 3, 3 to 6, and 6 to 9 inches for incorporated treatments. Soil samples w0ere also taken at a depth of 18 to 24 inches where limestone was incorporated during tillage at 5 tons per acre. Four harvests were made in 1988. At both locations, limestone application increased yields by 114 to 300% as compared with the check where no limestone was applied. Yields from plots receiving surface limestone application were equal to plots with incorporation at both locations. Tillage did not increase yields as compared with no tillage except in Orange County on plots where no limestone was applied. Soil pH increased from 5.6 to 6.8 at the 1- to 2-inch depths and from 5.6 to 6.3 at the 2- to 3-inch depths where 5 tons per acre of limestone were surface applied in Montgomery County. In Orange County, soil pH increased from 5.5 to 6.5 at the 1- to 2-inch depths and from 5.6 to 6.1 at the 2- to 3-inch depths where 5 tons per acre limestone were surface applied. Above pH 5.5, Al saturation was below 2.7% in Montgomery County and 0.7% in Orange County. Aluminum saturation averaged 9.0% and 4.6% where pH was between 5.0 and 5.5 in Montgomery and Orange Counties, respectively. At both locations, in the 18- to 24-inch depths a pH of 4.8 was not influenced by limestone incorporated at 5 tons per acre. Aluminum saturation at 18- to 24-inch depths was 51% and 62% in Montgomery and Orange Counties, respectively. These data indicate that surface applied limestone can produce first year alfalfa yields similar to yields obtained with incorporated limestone. / Master of Science

Sclerotinia crown and stem rot (SCSR) incited by Sclerotinia trifoliorum Eriks. causes severe losses in some fall-seeded, no-tillage plantings of alfalfa (Medicago sativa IL.) in Virginia. A mycelial plug inoculation technique was used to detect differences between cultivar (cv) responses of two alfalfa cvs, Arc and Vertus, under greenhouse conditions. A six dia plug from the margin of a 5-day-old culture of S. trifoliorum was placed near the crown area of a plant and incubated for a pre-determined period in a dew chamber at 18 C and 100% RH. Differences in isolate virulence were detected; cv Vertus was less susceptible than Arc to the less virulent isolates while the more virulent isolate (TAL 4) was equally severe on both. An incubation period of 96 hr produced significantly higher disease severity than 72, 48 or 24 hr, however, cv differentiation was best after 72 hr. Eight-, and nine-week-old plants were found to be most suitable for cv evaluation tests since younger seedlings were severely damaged and more mature plants did not develop sufficient symptom expression. Evaluation of twelve cvs with the virulent isolate (TAL 4) and the less virulent isolate (LAL 3) after 96 hr incubation produced significant differences between the mean disease severity ratings (MDSRs). Disease severity increased up to 20 days and then stabilized. Cultivar Anstar followed by WL 320, Vertus and Saranac AR were less susceptible in a majority of the tests; Endure and Euver performed well in some tests while Pioneer Brand 526 and Raidor performed poorly in all tests. This inoculation technique may act as the primary step in the selection of disease resistant germplasm for propagation, re-evaluation, and mass selection before field testing. / Master of Science

Master of Science

The enhancing effect of K on alfalfa (Medicago sativa L.) yield has frequently been reported, but the nature of this influence has not been clearly shown. It was assumed that K contributed either to increased leaf expansion, thereby resulting in a larger photosynthetic surface, or to higher CO₂ assimilation rates per unit leaf area. Sand culture technique for growing plants was used in two greenhouse experiments, while field plants were grown in plots with soil differing in available K. Yield increases were consistently obtained with high K. Added K increased plant height and leaves per plant and per plot, The rate of leaf accumulation was higher as K increased. Leaf size and weight per unit area also increased, as did stomatal number and aperture. Larger epidermal cells and greater numbers per leaf were observed with high K nutrition. Percent K in plants was associated with rate of K application. Net photosynthesis rates of excised leaves increased with potassium application, but all K levels had similar light compensation points. Leaves from plants with added K had lower CO₂ compensation points, indicating higher efficiency of CO₂ assimilation. Based on these data, K appears to function both to increase the effective photosynthetic surface through greater leaf initiation and development and to increase the rate of CO₂ utilization per unit leaf area. The latter increase probably results from greater CO₂ diffusion into substomatal cavities. / Ph. D.

The arid conditions found in the west allow for the production of high quality hay for export. However, hay production in this region is highly dependent upon irrigation. There is significant interest in developing a hay export market in the eastern U.S. Therefore, a greater understanding of the challenges and opportunities with containerizing hay in high humidity environments is needed. The objective of this study was to evaluate the impact of hay preservatives on the stability of containerized hay. Treatments included 1) propionic acid at baling, 2) propiopnic acid at baling + surface applied propionic acid at container loading, 3) propionic acid at baling + ammonization of the container after loading, and 4) no preservatives. Propionic acid was applied at a rate of 2.5 kg Mg-1 of hay at baling to all treatments except the control. Hay was then stored for 5 weeks before compressing to a density of 320.0 kg m-3. Immediately before containerizing hay, treatment 2 received a surface application of propionic acid at a rate of 3.4 g bale-1. After loading hay into containers, treatment 3 was ammoniated at a rate of 1.5 kg NH3 Mg-1 DM. Temperature and relative humidity in the containers were monitored for the 45-day storage period. Hay was sampled at compression and immediately after opening the containers. There were no treatment effects on nutritive value parameters after containerization (P > 0.05). Neither propionic acid at harvest, nor treatments at containerization had an effect on mold development in this study (P > 0.05). / Master of Science / Hay exports from the western U.S. have more than doubled since 2000. The arid conditions found in the west allow for the production of high quality hay for export. However, hay production in this region is highly dependent upon irrigation. There is significant interest in developing a hay export market in the humid east. The objective of this study was to evaluate the impact of two hay preservatives on the stability of containerized hay. Treatments included 1) propionic acid at baling, 2) propionic acid at baling + surface applied propionic acid at container loading, 3) propionic acid at baling + ammoniaization of the container after loading, and 4) no preservatives. Propionic acid was applied at a rate of 2.5 kg Mg⁻¹ of hay at baling to Treatments 1, 2, and 3. Hay was then stored for 5 weeks before double compressing. After compression, bales were placed into containers with treatment 2 receiving a surface application of propionic acid at a rate of 3.4 g bale⁻¹ . Treatment 3 was ammoniated in the container after loading at a rate of 1.5 kg NH₃ Mg⁻¹ DM. Temperature and relative humidity in the containers were monitored for the 45 day storage period. Hay was sampled prior to compression, at compression, and immediately after opening the containers. Samples were analyzed for neutral detergent fiber, acid detergent fiber, and crude protein. Subsamples were collected and sent for mold analysis. Results suggest that no treatments are needed in order to export quality alfalfa hay from the Mid-Atlantic.