En el Valle Inferior del río Negro aproximadamente el 20% de la superficie total bajo riego se destina al cultivo de alfalfa (Medicago sativa L.). Esto contribuye a mantener la oferta anual de forraje con heno de calidad, principal fuente de alimentación de los sistemas de invernada. El elevado potencial productivo de este cultivo en la región ocasiona una alta extracción de nutrientes, particularmente fósforo (P) y azufre (S), los que podrían reponerse mediante una adecuada fertilización con el objeto de maximizar la producción y la calidad del forraje. El trabajo de tesis se llevó a cabo durante 2010-2014 en la Estación Experimental Valle Inferior del INTA en Viedma, provincia de Río Negro, sobre un ensayo productivo de alfalfa bajo riego. Los objetivos fueron: 1. Evaluar la producción de materia seca del cultivo de alfalfa en respuesta a la fertilización fosforada y azufrada en suelos de textura fina bajo condiciones de riego, durante cuatro ciclos de producción. 2. Determinar el contenido de proteína bruta del forraje en cada estación de crecimiento (primavera-verano-otoño). 3. Determinar la concentración y el contenido de nutrientes de alfalfa en el estado fenológico de 10% de floración, en todos los cortes durante los dos primeros ciclos del cultivo. 4. Cuantificar la evolución del carbono orgánico total (COT), el carbono orgánico particulado (COP) del suelo y la concentración de P y S totales en la fracción de la materia orgánica (MO) particulada en los dos primeros ciclos de producción. Se utilizó un diseño en bloques completos aleatorizados con parcelas divididas, donde se distribuyeron los 10 tratamientos con P y S. Se aplicaron cinco dosis de P (0, 20, 40, 80 y 160 kg ha-1) combinados con dos dosis de S (0 y 24 kg ha-¹). En el cultivo se evaluó la producción de forraje por corte, y la acumulada en cada ciclo (Mg MS ha¯¹); la concentración de proteína bruta (%PB) y el contenido de nutrientes en el forraje (kg MS ha¯¹). En el suelo se determinó: COT, COP y contenido de P y S totales en la fracción particulada. En el período correspondiente a los cinco cortes de primavera y verano se produjo el 89% del forraje total del ciclo. Sólo los cortes 1, 4 y 6 mostraron diferencias respecto al tratamiento sin fertilizante, con altas dosis de P, con respuestas entre 33 y 40%. La fertilización combinada con P+S incrementó la producción anual acumulada de forraje a partir del 2º ciclo de estudio; en el 3º y 4º ciclo únicamente las dosis más altas de P (con y sin S) produjeron los mayores aumentos de forraje. El porcentaje de PB fue de 18% en los cortes de primavera y verano y del 23% en otoño. Los tratamientos con P y S produjeron mayor % PB en forraje respecto al testigo en los cortes de primavera y otoño. En la época estival cuando se presentaron los menores valores de PB, la fertilización no generó cambios en los porcentajes. Los mayores valores proteicos se observaron en otoño. El forraje producido luego de la fertilización fósforo azufrada, mostró niveles suficientes de N, Ca y B y ligeras deficiencias de P, K y S. Cuando los resultados se expresaron en kg ha-1, no se detectaron diferencias significativas de los contenidos de N, S y B entre tratamientos. Sólo en el primer ciclo los valores de P, K y Ca fueron más altos con dosis iguales o mayores a 20 kg P ha-1 (con y sin S). En los dos ciclos estudiados los niveles de COT fueron semejantes en todos los tratamientos fertilizados. La fertilización no modificó el contenido de COP, Pt y St de la fracción gruesa en ambos ciclos con diferencias entre ciclos. La disminución de la MOP en 19% durante el segundo ciclo productivo explicaría el menor contenido de COP, Pt y St de la fracción gruesa en ese período. El contenido de St de la fracción gruesa presentó diferencias entre los dos ciclos mientras que el Pt se mantuvo. La aplicación de 24 kg S ha-1 disminuyó el contenido de Pt de dicha fracción como consecuencia de una mayor extracción de P por parte del cultivo. La relación S:P mostró un enriquecimiento de S. La relación de estratificación indica una mayor acumulación de COP en la profundidad de suelo 0-10 cm respecto a 0-5 cm, debido a la mayor acumulación de raíces en este espesor. En general, en todos los tratamientos, esta relación fue mayor que 2, lo que sugeriría un efecto beneficioso de la pastura sobre la calidad del suelo. / In the Lower Valley of Río Negro (VIRN) approximately 20% of the total area under irrigation is used for alfalfa (Medicago sativa L.) cultivation. This contributes to maintaining the annual supply of forage with better quality, which is the main source of feed for wintering systems. The high productive potential of this crop in the region causes a high extraction of nutrients, particularly phosphorus (P) and sulfur (S), which could be recovered by means of an adequate fertilization in order to maximize forage production and quality. This thesis was carried out during 2010-2014 period at the Lower Valley Experiment Station of INTA in Viedma, Río Negro province, on a productive trial of alfalfa under irrigation. The objectives were: 1. To evaluate the dry matter production of alfalfa in response to phosphorus and sulfur fertilization in fine textured soils under irrigation conditions during four production cycles. 2. Determine the crude protein content (%PB) of the forage at each growing season (spring-summer-autumn). 3. Determine the concentration and nutrient content of alfalfa at 10% flowering phenological state, in all forage cuts during the first two crop cycles. 4. To quantify the total organic carbon (TOC) evolution, particulate organic carbon (POC) and total P and S concentration in the coarse fraction of organic matter (OM) in the first two production cycles. A randomized complete block design with split plots was used to distribute ten P and S treatments. Five doses of P (0, 20, 40, 80 and 160 kg ha-1) were applied in combination with two doses of S (0 and 24 kg ha-1). In the crop, the forage production per cut, the accumulated in each cycle (Mg MS ha-1), the % PB and the nutrient content (kg DM ha-1) were evaluated. In the soil, TOC, POC and total P and S content in the particulate fraction were also determined. The 89% of total forage was produced in the period corresponding to five cuts of spring and summer of the alfalfa cycle. Only 1, 4 and 6 cuts showed differences regarding treatment without fertilizer, when high doses of P were used, which responses were from 33 to 40%. Combined P + S fertilization increased the annual accumulated forage production from the 2nd cycle of study; in the 3rd and 4th cycle only the highest doses of P (with and without S) produced the highest forage increases. The PB content was 18% in the spring and summer cuts and 23% in the corresponding autumn. The P and S treatments produced higher %PB than the control in the spring and autumn cuts. In the summer when the lowest %PB values were obtained, the fertilization did not produce changes in these values. The highest %PB were observed in autumn. The forage produced after sulfur and phosphorus application, showed sufficient levels of N, Ca and B and small P, K and S deficiencies. When the results were expressed in kg ha-1, no significant differences of N, S and B contents between treatments were observed. Only in the first cycle the P, K and Ca values were higher with doses equal or greater than 20 kg P ha-1 (with and without S). In the two cycles evaluated the TOC levels were similar in all fertilized treatments. The fertilization did not modify the POC, Pt and St contents in the coarse soil fraction in both cycles with differences between them. The 19% decrease of the MOP during the second productive cycle would explain the lower content of POC, Pt and St of the coarse fraction in that period. The St content of the coarse fraction showed differences between the two cycles while the Pt remained constant. The application of 24 kg S ha-1 decreased the Pt content of this fraction as consequence of higher extraction of phosphorus by the crop. The S: P ratio showed an S enrichment. The stratification ratio indicated a higher accumulation of POC in the 0-10 cm compared to 0-5 cm soil depth, due to the greater roots accumulation in this thickness. In all treatments this ratio was greater than 2, which would suggest a beneficial effect of pasture on soil quality.

Both alfalfa weevil, <i>Hypera postica</i> (Gyllenhal), (Coleoptera: Curculionidae), and potato leafhopper, <i>Empoasca fabae</i> (Harris), (Homoptera: Cicadellidae), remain key pests of alfalfa in Virginia. Commercial varieties of potato leafhopper-resistant (or glandular-haired) alfalfa were released in the mid-1990s, but the impact of alfalfa weevil on these varieties has not been well documented. In 1999, two large-scale field experiments were initiated to compare the performance of a glandular-haired alfalfa variety against a standard, non-glandular-haired variety under both alfalfa weevil and potato leafhopper pest pressures in the southwestern and Piedmont regions of Virginia over a 3 year period. Results indicated that alfalfa weevil must be managed in potato leafhopper-resistant alfalfa to limit crop loss. Surprisingly, similar densities of potato leafhoppers were found in both the glandular-haired and standard varieties. Both varieties frequently had similar yields and forage quality. In general, the glandular-haired variety did not outperform the standard variety. Results also indicated that insecticide application did not always provide the expected benefits of higher yields and forage quality, despite reducing pest densities for 2-3 weeks after application. These data suggest that the economic thresholds for one or both of these pests in Virginia may require adjustment. The potential impact of glandular-haired alfalfa on the natural enemies of alfalfa weevil was examined as well. <i>Bathyplectes anurus</i> (Thompson) (Hymenoptera: Ichneumonidae) was the dominant parasitoid attacking weevil larvae at both locations. Parasitization of weevil larvae by <i>Bathyplectes</i> spp. did not appear to be adversely affected by the presence of glandular trichomes on the potato leafhopper-resistant variety. Glandular trichomes had little impact on the infection of weevil larvae by the fungus <i>Zoophthora phytonomi</i> as well, but this was not unexpected. The genetic variation of <i>B. anurus</i> was surveyed at both study sites using RAPD-PCR to establish or eliminate the possibility that differences in parasitization levels between the Piedmont and southwestern regions could be attributed to the presence of different parasitoid strains. Most of the detected phenotypic variation was attributed to within population variation, with very little variation occurring between the two populations. However, the between population variation was statistically significant in 2000, but not in 2001. / Ph. D.

To make use of the information on infestation estimates of forage insect pests obtained by the conventional methods of sampling, it is important to estimate the actual losses taking place in association with definite numbers of the insect. Six month old red clover, alfalfa, and birdsfoot trefoil plants were exposed for a month to spittlebug infestations of 0, 50, 100, and 150 adults. Although the spittlebug mortality was lowest when reared on alfalfa and highest on birdsfoot trefoil, yellowing appeared on alfalfa and birdsfoot trefoil. On red clover there was a consistent increase in protein and nitrogen free extract and a reduction in crude fiber and ash content indicating slight reduction in growth. None of these differences were significant. On alfalfa the loss of xylem sap due to feeding caused a consistent general reduction of proteins, fat and ash content and an increase in dry matter and nitrogen free extract. Of these constituents only the increase in dry matter was significant with an infestation of 150 spittlebugs per cage. No differences in dry matter, proteins and fat were significant. Another test on 4 month old alfalfa with infestation levels of 0, 100, 200 and 300 showed similar consistent patterns but the difference in dry matter was significant only when the infestation was 200 spittlebugs or more. On birdsfoot trefoil there was a consistent increase of dry matter, proteins, and ash and reduction of fat and crude fiber. Only the difference of dry matter and fats was consistent showing that an infestation of 100 or more spittlebugs per sq ft will affect the general growth of the plants. The survival of 1st instar nymphs of the pea aphid (a phloem feeder) was very poor on birdsfoot trefoil. There was a fair amount of survival on red clover and those on alfalfa survived fully. There was no visible symptoms on birdsfoot trefoil and red clover but the alfalfa showed retarded growth. On red clover there was a consistent increase of dry matter, fat, crude fiber and nitrogen free extract but a reduction of proteins. The difference in crude fiber was significant when the infestation was 1000 pea aphid nymphs or more. A consistent reduction of proteins and crude fiber was also recorded on alfalfa but these differences were not significant. On birdsfoot trefoil, there was a decrease in dry matter and an increase in fat and ash content. The difference in ash content was significant when the infestation of pea aphid nymphs was 1000 or more. Potato leafhopper (phloem feeder) injury was very characteristic on birdsfoot trefoil. The terminal 1 to 2 inches of the sterns became wilted and bent down. On alfalfa there was gradual yellowing and the retarded growth was evident. The red clover did not show any characteristic symptoms. Infestations of 20 or more caused a consistent increase in protein, fat, crude fiber and a reduction of nitrogen free extract. On alfalfa there was consistent decrease in dry matter. On birdsfoot trefoil, there was consistent increase in dry matter, fat, and nitrogen free extract but a reduction of crude fiber showing that feeding on birdsfoot trefoil probably retards growth and condensation of carbohydrates to form crude fibers. The increase in fat was significant when the infestation was 40 potato leafhoppers or more. Alfalfa weevil adult feeding caused characteristic stripping of leaves, girdling of stem and drying of the entire plant. Feeding of 10 or more weevils for 18 days on a two month growth of alfalfa after the 1st cutting, cause a consistent reduction of proteins, fat and crude fiber and an increase of dry matter, ash, and nitrogen free extract. Increase of percent dry matter was significant when the infestation of alfalfa weevil adults was 20 or more. In another test, the dry matter, fat and ash gave similar consistent results, when 40, 60, and 80 adults were fed on 4 month old alfalfa for 18 days. Reduction of percent fat was significant when the infestation was 40 or more alfalfa weevil adults. Alfalfa weevil larval feeding showed stripping of the leaves and buds. Infestation of 30 or less, 2nd to 4th instar larvae for 12 days caused a consistent reduction of fat and ash and an increase of dry matter, proteins and crude fiber. The infestation was not high enough to show a significant change in plant constituents. / Ph. D.

Six combinations of grazing, tillage, and herbicides were studied during 2 yr in a randomized block design to determine alfalfa (Medicago sativa) survival, corn (Zea mays) plant populations and corn growth in an alfalfa-corn rotation. Alfalfa was overseeded with rye (Secale cereale) in autumn prior to planting corn in spring of each year. Treatments were 1) non-grazed control with standard chemical treatments for no-till establishment of corn, 2) grazing alfalfa, then grazing rye until corn planting followed by dicamba application, 3) alfalfa grazing plus glyphosate prior to corn planting, 4) treatment 3 plus grazing prior to rye jointing, and 5) alfalfa grazing plus autumn application of glyphosate, 6) alfalfa grazing plus autumn disking (no herbicides). Grazing until corn planting decreased (P<0.05) alfalfa stands and rye biomass, but increased (P<0.05) corn plant populations and soil bulk density compared to shorter grazing periods. Grazing prior to rye jointing increased (P <0.05) soil bulk density and corn plant populations, and decreased (P<0.05) rye biomass compared to no spring grazing. Autumn disking decreased (P<0.05) bulk density, but generally provided less (P<0.05) control of alfalfa compared to autumn application of glyphosate. Autumn disking and glyphosate application (Trts. 5 and 6) increased (P<0.05) rye biomass compared to no autumn treatment (Trts. 3 and 4). Applying glyphosate prior to corn planting (Trts. 3 and 4) improved (P<0.05) corn populations and growth compared to autumn glyphosate or disking (Trts. 5 and 6) and was similar to conventional no-till establishment (Trt. 1). Herbicides were necessary to completely kill alfalfa, but grazing reduced TNC in alfalfa roots and alfalfa plant persistence. / Master of Science

’Kentucky 31’ endophyte-free tall fescue fertilized with 160 kg N ha⁻¹yr⁻¹ was compared to similar fescue grown with ’Cimarron’ alfalfa in a randomized block pasture experiment with four replications. Quality and yield of stockpiled forages and performance and serum minerals of grazing steers were investigated during 1991-92 and 1992-93. Effects of grazing and timing of initiation of stockpiling forages were investigated during autumn of 1992. Rate and extent of release of Ca, Mg, P, S, Cu, and Fe from stockpiled forages were determined in a dacron bag study in 1992-93. In 1991-92, stockpiled N-fertilized fescue improved steer performance over stockpiled fescue-alfalfa (P<0.05); in 1992-93, this result was reversed (P<0.05) due to forage availability. Blood urea nitrogen was higher (P<0.07) and serum Ca and S were higher (P<0.05) in steers which grazed stockpiled fescue-alfalfa. September stockpiling of fescue-alfalfa improved botanical composition, yield, and forage quality, compared to August stockpiling. September stockpiling of N-fertilized fescue improved quality but lowered yield, compared to August stockpiling. Fescue-alfalfa had higher (P<0.05) yield and improved botanical composition when grazing occurred. Nitrogen- fertilized fescue had higher (P<0.05) yield when mechanically harvested. Alfalfa released P, Ca, Mg, S, and Fe to a greater extent and rate than either type of fescue at 24 h and P, Mg, S, and Fe at 72 h (P<0.05). Fescue grown with alfalfa released S to a greater extent and rate than N-fertilized fescue, Fescue-alfalfa produces animal performance as good or better than N-fertilized tall fescue, while eliminating need for N-fertilization. / Master of Science

Sinorhizobium meliloti is a nitrogen-fixing endosymbiont of the legume Medicago sativa commonly known as alfalfa. It uses flagellar rotation and chemotaxis to seek roots of host plants to inhabit. This symbiosis serves as a great model system for studying biological nitrogen fixation and plant-microbe interactions. Since alfalfa brings enormous economic value to the USA, investments into the knowledge of the chemotaxis process that initiates symbiosis have the ability to mitigate deterioration of the environment and significantly increase food supply. The chemotaxis system in the enteric bacteria Escherichia coli is well studied and has been a great resource to understanding the process in other bacterial systems including our model organism S. meliloti. This dissertation compares and contrasts the chemotaxis features in E. coli and S. meliloti and investigates their molecular functions. Based on the understanding gained so far, we attempt to offer plausible explanations for the underlying mechanisms of the S. meliloti chemotaxis pathway. Chapter 1 describes why biological nitrogen fixation is important for agriculture and the health of our environment. This chapter also sheds light on the symbiotic relationship between alfalfa and S. meliloti, which culminates in the formation of nitrogen fixing nodules. We expound on the chemotaxis systems in E. coli and other bacteria including S. meliloti and Bacillus subtilis. In chapter 2, we compare the distribution of C-terminal pentapeptide-bearing receptors and the adaptation proteins that they tether in E. coli and S. meliloti. The stoichiometry data show that the ratio of pentapeptide-bearing chemoreceptors to chemotaxis protein (Che)R and CheB molecules are approximately 500- and 160-fold higher in S. meliloti than in E. coli, respectively. Since not all chemoreceptors in chemotactic bacteria have and utilize the pentapeptide moiety, we investigated the S. meliloti system and observed a strong interaction between CheR, activated CheB and the isolated pentapeptides via in-vitro binding studies. On the contrary, unmodified CheB showed weak binding to the pentapeptide. Through in-vivo studies, we highlighted the physiological necessity of the pentapeptide for chemotaxis. S. meliloti strains with substitutions of the conserved tryptophan residue to alanine in one or all four pentapeptide-bearing Methyl-accepting Chemotaxis Proteins (MCPs) resulted in diminished or loss of chemotaxis to glycine betaine, lysine, and acetate, ligands sensed by pentapeptide-bearing McpX and pentapeptide-lacking McpU and McpV, respectively. The flexible linker connecting the pentapeptide to the MCPs together with the pentapeptide itself were shown to be functional on pentapeptide-lacking chemoreceptors and provided adaptational assistance to other chemoreceptors that lacked a functional pentapeptide. Based on these results, we concluded that S. meliloti employs a pentapeptide-dependent adaptation system with MCPs possessing a consensus pentapeptide motif (N/D)WE(E/N)F). Finally, we postulated that the higher abundance of CheR and CheB in S. meliloti compared to E. coli compensates for the lower number of pentapeptide-bearing chemoreceptors in the chemosensory array. In chapter 3, we explored the putative phosphatase function of a novel protein, CheT, on phosphorylated S. meliloti response regulators. The kinase CheA phosphorylates both the sink response regulator, CheY1, and the flagellar motor interacting response regulator, CheY2. CheY1 competes with CheY2 for these phosphate groups, but we have discovered another layer of complexity to the story. Sequence comparison of S. meliloti CheT and the E. coli phosphatase CheZ shows little sequence homology. However, both proteins share a DXXXQ phosphatase motif. Phosphorylation assays performed using radiolabeled [γ-32P]-ATP revealed that CheT acts as a phosphatase of CheY1~P and accelerates dephosphorylation of CheY1~P by at least two-fold. Interestingly, we also discovered that CheT interacts with CheR, but this interaction did not affect the enzymatic activity of either protein under the examined conditions. Unexpectedly, a cheT deletion strain and strains carrying mutations in the phosphatase motif exhibit an increased swimming speed, a phenotype that does not conform with the model that the absence of CheT or its activity results in increased CheY2~P levels and reduced swimming speed. We concluded that a revised S. meliloti signal termination pathway should include CheT enhancing dephosphorylation of CheY1~P and sensory adaptation involving the yet unknown function of CheT on CheR. While the adaptation system in S. meliloti is unexplored, this work provides first insights into fascinating deviations and similarities to the known paradigm. We have also delivered evidence that the S. meliloti signal termination system requires a dedicated phosphatase. The knowledge gained here takes us a step closer to enhance the S. meliloti chemotaxis pathway towards improved symbiosis with alfalfa and to reduce our dependence on environmentally deleterious synthetic fertilizers. / Doctor of Philosophy / Like all living things, bacteria inhabit a constantly changing environment, hence the need to take up and process this information. Bacterial cells have evolved sophisticated biological tools to tackle this challenge of detecting, responding and adapting to environmental signals like nutrients, toxins, temperature changes, light, metabolites, etc. Motile bacteria such as Escherichia coli, a gut resident microbe, and Sinorhizobium meliloti, a soil dwelling bacterium, direct their swimming behavior in response to chemical gradients within the milieu through a process termed chemotaxis. Generally, this vital process enables a bacterium to escape harmful chemicals and gravitate towards beneficial ones. However, S. meliloti specifically employs chemotaxis to locate the roots of its plant host (alfalfa) and to establish a symbiotic relationship through which the bacteria provide essential nitrogen for plant growth in exchange for nourishment. The biological tools employed by S. meliloti for chemotaxis include environmental sensing receptors called Methyl-accepting Chemotaxis Proteins (MCPs) and proteins inside the bacterial cell that transfer information from the sensors to long, helical rotating propeller structures, called flagella. Importantly, the efficiency of this process hinges on a timely termination of information flow and the ability to adapt to prevailing stimuli while maintaining sensitivity to increasing concentration gradients. This work investigates the function of the C-terminal five amino acid motif of MCPs known to be critical for adaptation in E. coli and the phosphatase activity of a novel protein, CheT, in signal termination of S. meliloti chemotaxis system.

A pesar de los beneficios a corto plazo que se obtienen al utilizar insecticidas sintéticos convencionales para el control de plagas agrícolas, su uso continuo puede generar varios problemas a largo plazo (contaminación ambiental, eliminación de entomofauna benéfica, selección de individuos resistentes y daños a la salud humana). Esta situación lleva a una búsqueda permanente de métodos alternativos de control. En la presente Tesis se analizó la resistencia de cultivares de Medicago sativa L., la actividad insecticida, el efecto sobre la reproducción y la actividad repelente de los aceites esenciales de Eucalyptus globulus Labill. y de Mentha x piperita L. en adultos de Acyrthosiphon pisum H., Aphis craccivora K. y Therioaphis trifolii M. (Hemiptera: Aphididae). En 23 cultivares de M. sativa se evaluaron la resistencia por antixenosis y por antibiosis y la tolerancia. Los cultivares ACA 605, Brava y CW 194 resultaron antixénoticos. Los cultivares CW 194, CW 1010, EBC 90, Pampa Flor, Sirosal y Venus, poseen resistencia por antibiosis. Finalmente, Carmina, CW 194, CW 830 y SPS 6550 fueron altamente tolerantes al ataque de estos áfidos. La actividad insecticida de los aceites esenciales de E. globulus y de M. x piperita se evaluó a través de ensayos de inmersión propuestos por la FAO y por exposición a hojas tratadas. Los aceites esenciales de E. globulus y de M. x piperita producen toxicidad por inmersión y por contacto. El efecto subletal de los aceites esenciales fue evaluado utilizando el método de inmersión de hoja. Ambos aceites disminuyeron la progenie en las tres especies de áfidos evaluadas. Por otra parte, el efecto repelente se analizó mediante una prueba de elección foliar. Ambos aceites produjeron un efecto repelente en adultos de A. pisum, A. craccivora y T. trifolii. En consecuencia, los cultivares resistentes y los aceites esenciales de E. globulus y de M. x piperita tienen una potencial aplicación como herramientas dentro del manejo integrado de plagas. / Although the use of synthetic insecticides is really advantageous for the control of agricultural pests at short periods, their continuous use can generate several longterm problems (environmental contamination, elimination of beneficial entomofauna, generation of resistance and damage to human health). This situation leads to a permanent search for alternative methods of control. In this thesis were assessed the resistance of cultivars of Medicago sativa L., the insecticidal activity, the effect on the reproduction and the repellent activity of essential oils of Eucalyptus globulus Labill. and Mentha x piperita L. on adults of Acyrthosiphon pisum H., Aphis craccivora K. and Therioaphis trifolii M. (Hemiptera: Aphididae). Twenty-three cultivars of M. sativa were evaluated for their antixenosis and antibiosis resistance and tolerance. Cultivars ACA 605, Brava and CW 194 were antixenotic. Cultivars CW 194, CW 1010, EBC 90, Pampa Flor, Sirosal and Venus, had resistance by antibiosis. Finally, Carmina, CW 194, CW 830 and SPS 6550 were highly tolerant to the attack of these aphids. The insecticidal activity of both essential oils was evaluated through two methods: toxicity by inmersion using the FAO dip test protocol and exposition to a pretreated leaf. Essential oils of E. globulus and M. x piperita produced toxicity by inmersion and by contact. The sublethal effect of essential oils on reproduction was evaluated using the leaf-dipping method. Both essential oils decreased progeny production of all the three aphid species evaluated. On the other hand, the repellent effect was analyzed by foliar choice test. Both oils produced a repellent effect on adults of A. pisum, A. craccivora and T. trifolii. Therefore, M. sativa resistance cultivars and essential oils of E. globulus and M. x piperita have potential applications for integrated management of this pest.

Selected insecticides were evaluated to determine effects on the potato leafhopper and its predators. Three types of tests were conducted. First, the common predators of the potato leafhopper in alfalfa were examined to determine their response to the stages of the leafhopper. Second, under field conditions, insecticides were tested to detect effects on the populations of the insects. Finally, LC₅₀ toxicities of selected insecticides were investigated under laboratory conditions. In the predation tests, two studies were conducted, namely, predation on egg stage and predation on nymphal and adult stages of the potato leafhopper. An indirect method of determining egg predation was employed because potato leafhopper eggs are not visible, being concealed in the xylem and phloem tissues. Using two sets of treatments (with and without predators), the potato leafhopper females were allowed to oviposit in predation cages and the suspected predators introduced thereafter. Hatching nymphs were counted and used as indications of the number of eggs laid. Nabis americoferus and Orius insidiosis were found to be predaceous on eggs of the potato leafhopper. In the predation tests, using nymphal and adult stages of the potato leafhopper, the following adult and larval stages of insects were confirmed as predators: Hippodamia convergens, Coccinella novemnotata, and Chrysopa carnea. Two field tests, one in summer of 1977 and one in summer of 1978, were conducted to determine the influence of insecticides on populations of potato leafhoppers and its predators in alfalfa. Samples were taken using a 38 cm sweep net employing a 180° motion at 2 days, 1 week, and 2 weeks after treatment. In both tests, significant control was provided by the chemicals against potato leafhoppers 48 hours following application. A gradual population recovery occurred one week and two weeks after treatment. Most of the entomophagous insects sampled were adversely affected by insecticides. No statistical differences in yield and percent dry matter were obtained with various insecticidal treatments. The acute toxicities of selected insecticides on potato leafhopper, tarnished plant bug, and damsel bug were investigated. The bioassay method used was the immersion technique and toxicity was expressed in LC₅₀. The relative toxicities of Nabis americoferus to 50° Empoasca fabae and Lygus lineolaris were obtained by deriving the selectivity ratios. Selectivity ratios are calculated by dividing the LC₅₀ of the non-target organism divided by the LC₅₀ of the pest. The ratios indicated that most of the insecticides tested were highly selective. Among the organophosphates, azinphosmethyl was the most selective, while among the carbamates, carbaryl was the most selective. / Master of Science

Experiments were conducted to investigate the fermentation characteristics and nutritional value of mixtures containing different proportions of alfalfa (Medicago sativa L.) and wheat (Triticum aestivum L.) straw ensiled with and without molasses, microbial inoculant and molasses + microbial inoculant. In experiment 1, chopped alfalfa harvested at 1/10 bloom and chopped wheat straw were ensiled in the following proportions (DM basis): 0:100, 25:75, 50:50, 75:25, 100:0, 0:100 (urea-treated), with O and 5% dry molasses, and 0 and .1% microbial inoculant (Lactobaci]lus plantarum and Streptococcus faecium) in a 6 x 2 x 2 completely randomized design with a factorial arrangement. Each mixture was ensiled in 3.8-liter cardboard containers double lined with polyethylene. The pH values for the ensiled mixtures of 0:100, 25:75, 50:50, 75:25 and 100:0 alfalfa:straw without additives were 4.70, 4.76, 4.65, 4.76 and 4.82, respectively. Urea treatment of the 0:100 mixture (wheat straw) increased (P < .01) pH to 8.84. Overall lower pH (P < .01) occurred when molasses was added, compared to untreated and inoculated silages. Lactic acid concentration decreased (P < .01) linearly with decreased level of alfalfa, over all additives. Molasses increased (P < .01) lactic acid concentration. Overall, water soluble carbohydrates (WSC) in pre-ensiled mixtures increased (P < .01) linearly with increases in alfalfa. The WSC decreased (P < .01) after ensiling. Microbial inoculant did not affect pH or lactic acid content in the silages. In vitro dry matter digestibility (IVDMD) increased (P < .01) linearly with level of alfalfa, and molasses increased (P < .01) IVDMD. In experiment 2, chopped alfalfa (1/10 bloom, third cutting) and wheat straw were ensiled in the following respective proportions (DM basis): 0:100, 25:75, 50:50, 75:25, 100:0, 100:0 + 5% dry molasses, and 0:100 (urea-treated) in 210-liter metal drums double lined with .08 mm polyethylene bags. Increased proportions of wheat straw to alfalfa increased (P < .01) DM content and lactic acid concentration, and decreased pH and total VFA content. The CP, Ca, P, Mg and K contents decreased (P < .01) with increases in wheat straw. Addition of molasses to alfalfa increased (P < .01) WSC and lactic acid, and decreased (P < .01) pH and total VFA. Urea treatment of wheat straw increased (P < .01) pH and acetic acid, and decreased (P < .01) lactic acid, compared to untreated wheat straw. The NDF (P < .05) and hemicellulose (P < .01) were decreased with urea treatment. Two metabolism trials were conducted, each with 21 crossbred wethers. The diets were alfalfa and wheat straw silages made in 210-liter metal drums described above. Wheat straw ensiled alone was supplemented with soybean meal because of insufficient consumption when the straw was fed alone. Linear increases (P < .01) in DM, OM, and CP digestibilities were observed with increased level of alfalfa. Adding molasses increased (P < .05) DM digestibility. A linear increase (P < .01) in N retention was observed with increasing alfalfa level. Ruminal NH₃-N and blood urea-N increased (P < .01) with increased proportion of alfalfa. Feeding urea-treated straw increased (P < .01) ruminal NH₃-N and blood urea-N, compared to untreated wheat straw. A palatability trial was conducted with 42 sheep fed the same diets as in the metabolism trials. Daily DM intake increased linearly (P < .01) with increased proportion of alfalfa. It is concluded that ensiling cereal crop residues with leguminous forage has potential in improving fermentation characteristics and nutritional value of alfalfa silage and improving the nutritional value and utilization of cereal crop residues. / Ph. D.

La verticilliose de la luzerne cultivée (Medicago sativa L.) est une maladie de flétrissement vasculaire causée par le champignon du sol Verticillium alfalfae. C’est une des maladies les plus dévastatrices et les plus difficiles à contrôler. Les symptômes sont un jaunissement des feuilles suivi de flétrissement et défoliation. Les structures de dormance produites en fin de cycle de maladie constituent une source de contamination pour plusieurs années. Aucun traitement fongicide n’est efficace, la seule méthode de contrôle reste la production de variétés résistantes. En raison de sa nature tétraploïde et de son allogamie, il est difficile de réaliser des études génétiques sur M. sativa. Un pathosystème entre la légumineuse modèle Medicago truncatula et V. alfalfae a été mis au point pour étudier les mécanismes mis en place au cours de l’interaction entre V. alfalfae et son hôte. Les lignées A17 et F83005.5 ont été identifiées comme étant respectivement résistante et sensible à la souche V31-2 de V. alfalfae. La première partie de ce travail de thèse est une étude comparative du processus d’infection de V. alfalfae V31-2 au cours d’une interaction compatible et incompatible. Nous avons étudié la cinétique de colonisation des racines d’A17 et F83005.5 avec la souche V31-2 exprimant le gène marqueur GFP ce qui confère une fluorescence verte au champignon. Les observations en microscopie confocale ont montré que le champignon se développait dans les racines des deux lignées contrastées de façon similaire pendant les premières étapes d’infection. Quelques jours plus tard, il n’était plus détectable dans la lignée résistante, tandis qu’il colonisait les vaisseaux du xylème dans la lignée sensible et avançait vers les parties aériennes. La lignée résistante A17 était donc capable d’inhiber totalement le développement du pathogène dans la partie racinaire. Ce résultat a été confirmé par la quantification de l’ADN du pathogène dans la racine et dans les parties aériennes. Nous avons conclu que la lignée A17 exprime une résistance totale à V. alfalfae. Dans la deuxième partie de cette thèse, nous avons cherché à identifier le rôle des hormones dans les mécanismes de défense de M. truncatula en réalisant des traitements exogènes avec l’acide salicylique (SA), le méthyl jasmonate (MeJA), l’éthylène (ET), l’auxine et l’acide abscissique (ABA). Ces traitements n’avaient aucun effet sur la résistance d’A17, mais toutes les hormones, à l’exception du MeJA, protégeaient la lignée sensible contre les symptômes de la maladie. La quantification de l’ADN du champignon in planta a montré que seule l’ABA inhibait significativement le développement du pathogène. Dans la troisième partie, nous avons cherché à identifier des acteurs moléculaires impliqués dans la résistance et la sensibilité en comparant le transcriptome de la lignée F83005.5 et A17 dans la phase précoce de l’infection. L’analyse des gènes différentiellement exprimés en réponse à l’inoculation montre que les deux lignées induisent des gènes impliqués dans la production de métabolites secondaires, et des gènes des voies de signalisation hormonale. Mais seule la lignée résistante montre une induction de l’expression de gènes de résistance et de gènes impliqués dans les voies de signalisation tels que des gènes de la synthèse de l’ABA et des facteurs de transcription. Ces résultats renforcent l’hypothèse que l’ABA serait un facteur important dans la résistance à V. alfalfae chez M. truncatula. L’analyse des réseaux de gènes coexprimés a montré une désorganisation de la réponse de la lignée F83005.5. En revanche, dans la lignée A17, on observe une réponse organisée et orientée vers la défense. Ce travail décrit pour la première fois les mécanismes de défense de M. truncatula contre V. alfalfae. L’ensemble des résultats montre que la résistance exprimée chez la lignée A17 est différente des mécanismes de résistance contre la verticilliose décrits chez la tomate et le coton. / Verticllium wilt of alfalfa (Medicago sativa L.) is a vascular disease caused by the soil fungus Verticillium alfalfae. It is one of the most devastating diseases and most difficult to control. Symptoms are leaf yellowing followed by wilting and defoliation. Survival structures which are produced at the end of the disease cycle are a source of inoculum for many years. Fungicide treatment is not efficient, and the only way to control this disease is to breed resistant cultivars. Genetic studies are difficult in M. sativa because it is tetraploid and outcrossing. A pathosystem has been set up in our laboratory in order to study the mechanisms involved in the interaction between V. alfalfae and its host. It involves the model legume plant M. truncatula and strain V31-2 of V. alfalfae. The lines A17 and F83005.5 were identified as respectively resistant and susceptible to V31-2. The first part of this thesis is a comparative study of the infection process of V. alfalfae V31-2 in a compatible and incompatible interaction. The time course of root colonization in lines A17 and F83005.5 was studied with a GFP-expressing strain which confers green fluorescence to the fungus. Observations by confocal microscopy showed that the fungus developed in a similar way in roots of both lines during the first stage of the interaction. Some days later the fungus was not detectable anymore in roots of the resistant line, but has colonized the xylem vessels and grew towards the aerial part of the plant in the susceptible line. Quantification of fungal DNA in roots and aerial parts confirmed these results. This showed that the resistant line A17 was able to suppress the pathogen’s development in the root. It can be concluded that line A17 presents total resistance towards V. alfalfae. The second part of the thesis concerns the role of phytohormones for defence mechanisms against V. alfalfae in M. truncatula. Susceptible and resistant plants were treated with salicylic acid (SA), methyl jasmonate (MeJA), ethylene (ET), auxine and abscissic acid (ABA). Resistance of line A17 was not affected by these treatments, but all hormones except MeJA protected the susceptible line against disease symptoms. However, when fungal DNA was quantified in planta in these assays, only ABA inhibited the pathogen’s development significantly. The third part of this thesis aims at identifying molecular factors involved in resistance and susceptibility. To address this topic, the transcriptome of lines A17 and F83005.5 was compared during the early stages of infection, in inoculated or mock-inoculated plants. A bioinformatics analysis of differentially expressed genes showed that both lines responded to inoculation by inducing genes involved in secondary metabolism and hormone signaling pathways. However, only resistant line A17 showed induction of the expression of putative resistance and signaling genes, genes involved in ABA synthesis and transcription factors. This result confirms our hypothesis that ABA might be an important factor in M. truncatula resistance against V. alfalfae. Gene network analysis of co-expressed genes showed a disorganised response in the susceptible line, whereas in the resistant line the response was highly organised and turned to defence. Taken together, this work describes for the first time defence mechanisms against V. alfalfae in M. truncatula. The results show that resistance of line A17 is different from resistance mechanisms Verticillium resistance described in tomato and cotton. Several approaches for future research are presented in order to test our hypotheses concerning genes and molecules putatively involved in this interaction. With regard to applied research, defence and signaling genes identified in this work may be useful for the improvement of alfalfa, after functional validation.