The histone 2A (H2A) variant Z (H2A.Z) regulates gene expression, replacing H2A predominately at +1 nucleosomes. Whereas, H2A.Z can act as either a positive or negative transcriptional regulator, this research focuses on its transcriptional activation role. This thesis focuses on examining the Glycine max HTA9 gene family (H2A.Z9). The Arabidopsis thaliana HTA9 protein sequence was used to query the G. max proteome resulting in identification of five H2A.Z9 (H2A.Z9-1-5) paralogs. H2A.Z9-1, H2A.Z9-2, H2A.Z9-4, and H2A.Z9-5 are expressed within Heterodera glycines-parasitized root cells undergoing defense. All 5 paralogs, including H2A.Z9-3, were studied in transgenic-functional analyses. Data demonstrates that H2A.Z9 overexpression leads to a 60-70% reduction in H. glycines- parasitism with no effect on root growth. In contrast, H2A.Z9 knockdown by RNAi results in a 3.5-5.0-fold increase in H. glycines-parasitism with no effect on root growth. These results demonstrate that H2A.Z9 is important to G. max defense toward H. glycines-parasitism and indicate possible redundant or specific roles for each paralog.

Fusarium virguliforme, the causal agent of sudden death syndrome (SDS) in North America, and Heterodera glycines, soybean cyst nematode (SCN), are significant threats to the production of soybean. In 2014, two field trials were established to evaluate seed treatments and their efficacy in managing SDS and SCN. The locations were selected because each has a history of SDS and SCN. Plots were 3.04 meters wide by 6.1 meters in length with row spacing of 0.76 meters. Each plot received 2.45 grams of infested sorghum per 30.5 centimeters of row. At each location, two soybean varieties and ten different seed treatments were tested in 2014. In 2015, twelve seed treatments were tested on the same two varieties at the same two locations. Seed treatments were evaluated for phytotoxicity, vigor, stand count, SDS foliar ratings and soybean yield. Soil samples were collected at planting and at harvest to determine SCN reproduction. Root samples were collected from each plot to quantify the amount of F. virguliforme DNA in the soybean roots using a real-time quantitative polymerase chain reaction (qPCR) protocol. ANOVA and Fisher’s LSD Test were used to separate treatment means. Analysis of the qPCR was done by comparing the quantification cycle (Cq) values and separating means using ANOVA. There were significant differences between varieties for soybean yield and disease index. Treatments containing fluopyram had more phytotoxicity than treatments lacking fluopyram at both locations. Several treatments allowed for higher SCN reproduction than treatments with Clariva. At Ina, a fluopyram treatment and one of the Clariva treatments had higher soybean yield than the non-treated control. At Shawneetown, one fluopyram treatment had higher soybean yield than all other treatments. In the qPCR analysis, seed treatments with fluopyram had a lower amount of F. virguliforme DNA in the roots than the non-treated control.

The secretory pathway of G. max performs a prominent role in its defense to H. glycines parasitism, a finding reinforced by the identification of the SNARE-associated gene ALPHA-SNAP-5 being rhg1. Other proteins performing important roles in secretion are Sec61-ALPHA, Sec61-BETA and Sec61-GAMMA which bind to form a trimeric complex that imports proteins into the ER for their eventual secretion. Comparative analyses to the Saccharomyces cerevisiae Sec61ALPHA, Sec61BETA and Sec61GAMMA proteins, respectively, shows G. max has 4 Sec61-ALPHA, 9 Sec61-BETA and4 Sec61-GAMMA proteins. At least one paralog from each gene family is expressed in H. glycines-parasitized root cells in G. max, but while undergoing a defense process. The overexpression of one selected Sec61-ALPHA, Sec61-BETA and Sec61-GAMMA in the H. glycines-susceptible genotype G. max[Williams 82/PI 518671] leads to an engineered defense response. In contrast, RNAi of the same selected Sec61-ALPHA, Sec61-BETA and Sec61-GAMMA genes in the H. glycines-resistant genotype G. max[Peking/PI 548402] leads to an engineered susceptible response. The combined opposite outcomes of the Sec61-ALPHA, Sec61-BETA and Sec61-GAMMA gene overexpression and RNAi provides evidence that they function in the defense process, consistent with the hypothesis that the G. max secretion system plays a role in its defense to H. glycines parasitism.

Made available in DSpace on 2017-07-10T17:37:32Z (GMT). No. of bitstreams: 1 Anna Paula Comerlato.pdf: 310606 bytes, checksum: 263346497f6f6a5c056dbda7677bb798 (MD5) Previous issue date: 2009-05-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to evaluate the effect of liquid waste, produced by the cassava industry in Western Paraná, on the soybean cyst nematode Heterodera glycines Ichinohe through in vitro and in vivo essays. In vitro and in vivo essays were performed in randomized experimental designs with 5 replications for each treatment as follows: water (positive control), nematicide (negative control) and cassava waste at 5%, 10%, 15%, 20%, 25%, 50%, 75% and 100% for the in vitro test and cassava waste at 10%, 25%, 50%, 75% and 100% for the in vivo test. The in vitro tests were performed with eggs, J2 and cysts of H. glycines, which were immersed into each solution for 24 hours and then inoculated into pots containing soybean plants with two mature leaves. The evaluation occurred 30 days after inoculation (dai). The in vivo essays were carried out with one application and two applications of cassava waste. The in vivo essays started with the inoculation of 5,000 eggs and/or J2 of H. glycines on soybean plants. Forty five dai, liquid waste was applied in the pots and soybean plants had their shoot cut. Twenty four hours after the first application, new soybean plants were transplanted to the pots. To the essay with two applications of liquid waste, the second one was run 75 dai but keeping the same plants after application. The evaluation for the in vivo essays occurred 60 days after the transplanting. The variables assessed were plant height, shoot length, root length, number of cysts, number of females and eggs per root system, fresh weight and dry weight of plants and the reproduction factor of H. glycines on soybean plants. The cyanide content for the cassava waste was determined as 40 mg.L-1 in manipueira 100%, using the kit Microquant (Merck). The in vitro essays showed that the number of J2 decreased significantly and progressively (R2=0,82) from cassava waste 15% to 100%. For eggs and cysts, the cassava waste treatments from 10% to 100% were more efficient than the control water. Moreover, cassava waste at 25% concentration showed the lowest values for number of eggs and cysts for the in vitro essays. For the in vivo essays, two applications of cassava residue allowed a better development of soybean plants, allied to a decrease on the number of females and cysts of H. glycines, when compared to one application alone. The treatments cassava waste at 50%, 75% and 100% were superior to the others but cassava waste at 50% showed the lowest values for the number of females and cysts / Objetivou-se com o presente estudo avaliar o efeito de manipueira industrial, coletada em fecularia do oeste do Paraná, sobre o nematóide de cisto da soja Heterodera glycines Ichinohe, mediante ensaios in vitro e in vivo. Os ensaios in vitro e in vivo foram realizados no delineamento experimental inteiramente casualisado com os tratamentos água (controle positivo), nematicida (controle negativo) e manipueira a 5%, 10%, 15%, 20%, 25%, 50%, 75% e 100% para o ensaio in vitro e manipueira a 10%, 25%, 50%, 75% e 100% para o ensaio in vivo em 5 repetições cada. Os ensaios in vitro foram realizados com ovos, J2 e cistos de H. glycines, os quais permaneceram imersos nas diferentes soluções por 24 horas, sendo posteriormente inoculados em vasos contendo plantas de soja com duas folhas definitivas. A avaliação ocorreu aos 30 dias após a inoculação (dai). No tratamento in vivo, foram realizados ensaios com uma aplicação e duas aplicações de manipueira. Para o ensaio com uma aplicação, plantas de soja foram inoculadas com 5.000 ovos e/ou J2 de H. glycines com aplicação de manipueira aos 45 dai. Após 24 horas da aplicação, a parte aérea das plantas foi cortada, tendo sido plantada nova muda de soja. Para o ensaio com duas aplicações, manipueira foi aplicada aos 45 e aos 75 daí, mantendo-se as plantas após a segunda aplicação. A avaliação dos ensaios in vivo ocorreu 60 dias após a última aplicação de manipueira. As variáveis avaliadas para os ensaios realizados foram altura de plantas, comprimento da parte aérea, comprimento do sistema radicular, número de cistos, fêmeas e ovos por sistema radicular, massa seca e massa fresca de plantas e fator de reprodução (FR). O teor de cianeto em manipueira 100% foi de 40 ppm, o qual foi determinado antes de cada ensaio. Para J2 tratados in vitro, houve redução significativa e progressiva (R2=0,82) a partir de manipueira a 15% de concentração. Para ovos e cistos tratados in vitro, manipueira a partir de 10% de concentração foi superior à testemunha somente água, sendo que manipueira 25% apresentou os valores mais baixos para número de ovos e cistos. Para os ensaios in vivo, manipueira em duas aplicações permitiu um melhor desenvolvimento das plantas de soja com maior redução de fêmeas e cistos por planta, quando comparado ao ensaio com apenas uma aplicação. Os tratamentos manipueira 50%, 75% e 100% foram significativamente superiores aos demais quando aplicados via solo em tubetes, sendo que manipueira 50% apresentou os valores mais baixos para número de fêmeas e cistos

Master of Science / Department of Agronomy / William T. Schapaugh Jr / The soybean cyst nematode (Heterodera glycines) (SCN) is an important pathogen of soybean in the United States. Annual yield losses from SCN are estimated to be over $2 billion worldwide. However, SCN virulence or the ability of a nematode to grow on resistant soybean genotypes varies widely among SCN populations. Fortunately there are several genetic sources of resistance to decrease the virulence of the pathogen on soybean. The objectives of this research were to: 1) characterize the genetic diversity of soybean cyst nematode populations in Kansas, 2) determine the frequency of Kansas SCN populations virulent on PI88788, 3) determine which plant introductions used in the HG Type Test provide the best level resistance, and 4) compare the performance of commercial soybean cultivars to the plant introduction from which their SCN resistance was derived. Soil samples were collected from SCN-infested fields across the state. Each soil sample was taken to the greenhouse and planted to a susceptible soybean cultivar to increase SCN population. Following an SCN population increase, a HG Type Test was planted. H. glycines field populations were highly variable, not only in population densities, but also in their abilities to develop on soybean genotypes. Collected from a diverse range of environments, ten HG types were identified. About 50% of the H. glycines populations were virulent on PI 88788, and most of the populations were virulent on commercial SCN resistant lines which derived their resistance from PI 88788. The commercial lines tended to be more susceptible to SCN than the lines from which they derived their resistance, but few HG populations were virulent on PI 437654 or the commercial line that derived its resistance from PI 437654. These results suggest that sources other than PI 88788 should be used in the development of H. glycines resistant cultivars for Kansas. One possible source of resistance is PI 437654. Information about SCN diversity in Kansas will improve decisions regarding cultivar development and selection for SCN management.

Submitted by Carlos Cesar De Oliveira Guarnieri (carloscesarguarnieri@gmail.com) on 2018-08-02T18:26:04Z No. of bitstreams: 1 EFICÁCIA DE TIODICARBE, CADUSAFÓS E CONDICIONADOR DE SOLO VIA TRATAMENTO DE SEMENTES E OU SULCO DE PLANTIO NO CONTROLE DE NEMATOIDES NA CULTURA DE SOJA.pdf: 756636 bytes, checksum: 89d88ef49a1b212168332c38507a27e0 (MD5) / Approved for entry into archive by Neli Silvia Pereira null (nelisps@fcav.unesp.br) on 2018-08-03T17:20:53Z (GMT) No. of bitstreams: 1 guarnieri_cco_me_jabo.pdf: 756636 bytes, checksum: 89d88ef49a1b212168332c38507a27e0 (MD5) / Made available in DSpace on 2018-08-03T17:20:53Z (GMT). No. of bitstreams: 1 guarnieri_cco_me_jabo.pdf: 756636 bytes, checksum: 89d88ef49a1b212168332c38507a27e0 (MD5) Previous issue date: 2018-06-14 / Os principais métodos de controle de nematoides na cultura de soja atualmente utilizados são a resistência ou tolerância genética da planta, a rotação/sucessão de culturas resistentes/não hospedeiras e poucos são os estudos com o uso de produtos químicos/biológico e condicionadores de solo. Um dos métodos adotados para a aplicação de nematicida químico nas lavouras de soja é a aplicação via tratamento de sementes, porém os estudos de controle com este método, na grande maioria dos casos, apontam período de proteção de até 30 dias após a emergência das plantas, e na maioria das vezes tem trazido resultados insatisfatórios. A aplicação de nematicida químico, no sulco de plantio, ou associação do tratamento de sementes com a aplicação no sulco de plantio, pode melhorar os resultados de eficácia e eficiência, todavia esta técnica é pouco estudada no Brasil para a cultura de soja, consequentemente são poucos trabalhos que comprovem essas características nos produtos disponíveis atualmente. Também, o uso de um condicionador de solo poderá contribuir com um melhor desenvolvimento radicular, da planta e na eficácia e eficiência do produto nematicida químico. O objetivo deste estudo foi avaliar a eficácia e eficiência do nematicidas químicos tiodicarbe, cadusafós e do condicionador de solo Maskio, usados em tratamento de sementes e/ou aplicados no de sulco de plantio, no controle dos nematoides de galha (Meloidogyne incognita), de cisto da soja (Heterodera glycines) e das lesões radiculares (Pratylenchus brachyurus) na cultura de soja. Foram instalados ensaios a campo em áreas naturalmente infestadas pelos nematoides, e em vasos onde foram avaliados o desenvolvimento e produtividade da cultura e a redução na população de nematoides. Nenhum dos tratamentos testados causou sintomas visuais de intoxicação na cultura. Isoladamente, o tratamento de sementes com tiodicarbe (600 mL 100 kg sementes-1) é tão eficaz na redução da população total de P. brachyurus nas raízes da soja, quanto o tratamento de sulco com cadusafós (4,0 L ha-1) ou cadusafós + Maskio (4,0 L ha-1 + 2,0 L ha-1). Porém nenhum tratamento aumentou significativamente a produtividade da cultura. Para que haja incremento significativo em produtividade foi necessário associar a aplicação de tiodicarbe na semente e no sulco de plantio para áreas infestadas por P. brachyurus. Para áreas com M. incognita os tratamentos, de semente ou de sulco, podem ser eficazes no controle e incrementar em produtividade a cultura da soja se utilizados em área com alta infestação de nematoides de galha. A aplicação de tiodicarbe nas sementes foi tão eficaz na redução de cistos de Heterodera glycines quanto a aplicação no sulco de plantio de tiodicarbe, cadusafós, Maskio e suas associações. A associação de tiodicarbe nas sementes e uma aplicação no sulco melhorou a eficácia de controle de cistos de H. glycines, saindo de 27% de eficácia para tratamento de sementes isolado, para 75% de eficácia para o tratamento com tiodicarbe nas sementes associado ao uso da mesma molécula no sulco de plantio. / The main methods of nematode control in soybean crop currently used are resistance or genetic tolerance, rotation / succession of resistant / non - host crops, and few studies with the use of chemical / biological and soil conditioners. One of the methods adopted for the application of chemical nematicide in soybean crops is the application through seed treatment, but the studies in the control of this method, in the great majority of cases, indicate protection period up to 30 days after the emergence of the plants, in the most of the time it has brought unsatisfactory results. The application of chemical nematicide, in the planting groove, or association of the treatment of seeds with the application in the planting groove, can improve the results of efficacy and efficiency, however this technique is little studied in Brazil for the soybean crop, consequently are few works that prove the effectiveness and efficiency of the currently available products. Also, the use of a soil conditioner may contribute to a better root development of the plant and to the effectiveness and efficiency of the chemical nematicide product. The objective of this study was to evaluate the efficacy and efficiency of the chemical nematicides thiodicarb, cadusafos and the Maskio soil conditioner, used in seed treatment and / or applied to the planting groove, in the control of root-knot nematode (Meloidogyne incognita), soybean cist nematode (Heterodera glycines) and root-lesion nematode (Pratylenchus brachyurus) on soybean crop. Field trials were carried out in areas naturally infested by nematodes, and in pots where the development and productivity of the crop and the reduction in the nematode population were evaluated. None of the treatments tested causes visual symptoms of intoxication in the culture. Seed treatment with thiodicarb (600 mL 100 kg seed-1) is as effective in reducing the total population of P. brachyurus in soybean roots as in grouper treatment with cadusafos (4.0 L ha-1) or cadusafos + Maskio (4.0 L ha-1 + 2.0 L ha-1). However, no treatment significantly increases the productivity of the crop. In order to have a significant increase in productivity it is necessary to associate the application of thiodicarb in the seed and in the planting groove to areas infested by P. brachyurus. For areas with M. incognita, seed or planting groove treatments may be effective in controlling and increasing soybean productivity if used in an area with high root-knot nematode infestation. The application of thiodicarb in the seeds is as effective in the reduction of Heterodera glycines cysts as the application in the planting groove of thiodicarb, cadusafos, Maskio and their associations. The association of thiodicarb in seeds and a planting groove treatment improves the control efficacy of H. glycines. Leaving 27% efficacy for isolated seed treatment, to 75% efficacy for treatment with thiodicarb in seeds associated with the use of the same molecule in the planting groove.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-09-21T11:29:40Z No. of bitstreams: 1 texto completo.pdf: 414321 bytes, checksum: 1217b68cf39d80cb3ce2b85b1eae2eea (MD5) / Made available in DSpace on 2016-09-21T11:29:40Z (GMT). No. of bitstreams: 1 texto completo.pdf: 414321 bytes, checksum: 1217b68cf39d80cb3ce2b85b1eae2eea (MD5) Previous issue date: 1999-07-14 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Neste trabalho foram sintetizados a timoquinona [15] e os éteres derivados do timol, 2-benziloxi-1-isopropil-4-metilbenzeno [8; 35,8%], 2-etoxi- 1-isopropil-4-metilbenzeno [9; 18,8%], 1-isopropil-4-metil-2-octiloxibenzeno [10; 23,6%], 1-isopropil-4-metil-2-octadeciloxibenzeno [11; 33,0%], 2- (2’,3’,4’,5’,6’-pentafluorobenziloxi)-1-isopropil-4-metilbenzeno [12; 32,0%] e 1- isopropil-4-metil-2-metoxibenzeno [13; 75,8%]. Foram também sintetizados os sais derivados de piperazina: cloreto de piperazina [17], citrato de piperazina [18], fosfato de piperazina [19], oxalato de piperazina [20] e sulfato de piperazina [21]. Os éteres derivados do timol foram preparados através da síntese de Williamson, entre o timol e os compostos halogenados aromáticos e alifáticos. A timoquinona [15] foi obtida como produto da nitração do timol, utilizando ácido sulfúrico/nitrato de sódio. Os sais derivados da piperazina foram sintetizados mediante reação entre a piperazina e um ácido, apresentando rendimento quantitativo. Os ensaios biológicos dos produtos sintetizados e do timol foram realizados in vitro, com os nematóides Meloidogyne incognita raça 3 e Heterodera glycines, utilizando-se doses de 5 e 50 μg mL-1 dos compostos sintetizados. Nestes bioensaios, os índices de mortalidade para o Meloidogyne incognita variaram de 1,8 a 81,8% na concentração de 5 μg mL-1 e de 5,3 a 86,7% na concentração de 50 μg mL-1. Para Heterodera glycines estes índices variaram de 0 a 83% na concentração de 5 μg mL-1 e de 0 a 88% na concentração de 50 μg mL-1. Estes resultados permitiram observar a influência dos diferentes grupos substituintes, dos ânions e da concentração dos compostos sintetizados sobre sua atividade nematicida. Observou-se também diferença entre os mecanismos de ação destes compostos nas espécies Meloidogyne incognita e Heterodera glycines, sugerindo que uma abordagem diferente deve ser feita para a síntese de nematicidas que visam ao controle de Heterodera glycines. / In this work were synthesized the thymoquinone [15] and the derivatives ethers of the thymol, 2-benzyloxy-1-isopropyl-4-methylbenzene [8; 35.8%], 2- ethoxy-1-isopropyl-4-methylbenzene [9; 18.8%], 1-isopropyl-4-methyl-2- octyloxybenzene [10; 23.6%], 1-isopropyl-4-methyl-2-octadecyloxybenzene [11; 33.0%], 2-(2',3',4',5',6'-pentafluorobenzyloxy)-1-isopropyl-4-methylbenzene [12; 32.0%] and 1-isopropyl-4-methyl-2-methoxybenzene [13; 75.8%]. they were also synthesized the derived salts of piperazine: piperazine chloride [17], piperazine citrate [18], piperazine phosphate [19], piperazine oxalate [20] and piperazine sulphate [21]. The derived ethers of the thymol were prepared through the Williamson synthesis, between the thymol and the halogenates compounds aromatics and aliphatics. The thymoquinone [15] was obtained as product of the nitration of the thymol, using sulfuric acid/sodium nitrate. The derived salts of the piperazine were synthesized by means of reaction between the piperazine and an acid, presenting quantitative yeld. The biological assays of the synthesized products and of the thimol were carried in vitro, with the nematodes Meloidogyne incognita strain 3 and Heterodera glycines, using doses of 5 and 50 mg mL-1 of the synthesized compounds. In these bioassays, the mortality rate for Meloidogyne incognita varied from 1.8 to 81.8% at a 5 mg mL-1 concentration and of 5.3 to 86.7% at a 50 mg mL-1 concentration. For Heterodera glycines these rates varied from 0 to 83% at a 5 mg mL-1 concentration and from 0 to 88% at a 50 mg mL-1 concentration. These results allowed to observe the influence of the different substituents groups, of the anions and of the concentration of the synthesized compounds, on its nematicidal activity. It was also observed difference among the action mechanism of these compounds in Meloidogyne incognita and Heterodera glycines, suggesting that a different approach should be made for the synthesis of nematicides that seek the control of Heterodera glycines.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-10-05T18:17:05Z No. of bitstreams: 1 texto completo.pdf: 1361747 bytes, checksum: 02f07a1a422620aeb4025cd1437df02b (MD5) / Made available in DSpace on 2016-10-05T18:17:05Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1361747 bytes, checksum: 02f07a1a422620aeb4025cd1437df02b (MD5) Previous issue date: 2005-01-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os extratos brutos de nim foram preparados a partir de sementes extraídas seqüencialmente com hexano, metanol e água. O extrato metanólico foi particionado com acetato de etila e água seguido por tratamento com carvão ativo, repetindo-se 3 vezes e obtendo-se o extrato concentrado enriquecido com 60% de azadiractina A (Aza- A 60). Primeiramente, isolou-se azadiractina A através da injeção de 700 mg de Aza-A 60 no cromatógrafo em contracorrente (CCC), com coluna de politetrafluoretileno e 116mL de capacidade, utilizando-se o sistema bifásico hexano:butanol:metanol:água (1:0.9:1:0.9, v/v) eluído a 2mL/min, na direção cabeça-cauda. Coletaram-se 30 frações recuperando-se 95% do material injetado. Após monitoramento por cromatografia em camada delgada (CCD) usando-se tolueno:acetato de etila:metanol (10:1:2, v/v/v) como sistema eluente, as frações (9-13) foram purificadas por cromatografia líquida de alta eficiência de fase reversa (CLAE-FR) preparativa, utilizando-se uma coluna LC-8 (250 x 21 mm di, 5 μm) e metanol:água (50:50 v/v) a 6mL/min, sendo os efluentes monitorados num comprimento de onda de 217 nm. Assim, obteve-se Aza-A com 96% de pureza, segundo CLAE-FR analítica e sua estrutura foi confirmada por RMN 13 C. Num segundo estudo foram isolados azadiractina B (Aza-B), azadiractina H (Aza-H), desacetilnimbim (Dnim), desacetilsalanim (Dsal), nimbim (Nim) e salanim (Sal) injetando-se 4.2 g de Aza-A 60 (700 mg/corrida) no CCC seguindo as mesmas condições relatadas no isolamento de Aza-A. Após as 6 injeções, obtiveram-se duas frações principais que foram reunidas após monitoramento por (CCD), havendo recuperação de 97% do material injetado. A primeira delas, correspondendo à soma das frações (9-13) de cada injeção, foi purificada por CLAE-FR preparativa nas mesmas condições do isolamento de Aza-A, obtendo-se Aza-B e Aza-H, ambas com 97% de pureza, segundo análises por CLAE. A segunda fração, referente à soma das frações (17-30) de cada injeção, foi purificada utilizando-se metanol:água (60:40 v/v) a 8 mL/min, obtendo-se Dnim, Dsal, Nim e Sal, todos 97% puros após análises por CLAE analítica, sendo todas estruturas confirmadas por RMN 13 C. Todos os compostos purificados bem como extratos brutos de sementes de nim foram submetidos a ensaios de atividade nematicida sobre Heterodera glycines. Avaliando-se a mobilidade de juvenis do segundo estádio (J2), seguindo a metodologia do tubo invertido, verificou-se que ocorreu menor recuperação (P 0,05) dos J2 tratados com os extratos brutos aquoso (1/2 e 1/10 v/v) e metanólico (1000 ppm), sendo que o extrato hexânico não apresentou efeito nematicida. A atividade dos extratos brutos aquoso e metanólico foi atribuída, respectivamente, à presença de saponinas e triterpenóides (limonóides). Nos ensaios em casa de vegetação, plantas de soja inoculadas com juvenis tratados com os extratos aquoso (1/2 e 1/10 v/v) ou metanólico (1000 ppm) produziram menos fêmeas e ovos (P 0,05), se comparada à testemunha. O extrato de Aza-A 60 (70 ppm) causou maior imobilidade (P 0,05) dos J2 (92%), sendo esta proporcional às concentrações estudadas. A mortalidade dos J2 foi avaliada para todos os triterpenóides isolados nas concentrações de 0,8, 8 e 80 ppm, havendo redução do número de J2 recuperados com aumento das dosagens aplicadas. Nim e Sal a 80 ppm foram os mais efetivos causando 99% de mortalidade. Plantas de soja inoculadas com J2 tratados com os triterpenóides a 80 ppm apresentaram um efeito nematicida médio de 95% na redução do número de fêmeas e ovos (P 0,05), se comparados à testemunha. Com relação à eclosão dos ovos, os triterpenóides testados a 100 ppm apresentaram uma atividade ovicida média (P 0,05) de 50 %, em relação à testemunha, e não se observou ação nematostática até 96 horas após o tratamento. / Crude extracts were prepared from Neem seeds by sequential extraction with n- hexane, methanol and water. The methanolic extract was partitioned three times between ethyl acetate and water and treated with activated charcoal to obtain a concentrate (Aza-A 60) containing 60% azadirachtin A (aza-A). In the first study, aza- A (the major triterpenoid) was isolated by countercurrent chromatography (CCC) fitted with polytetrafluorethylene coil (116 mL volume) using the biphasic solvent system n- hexane:butanol:methanol:water (l:0.9:l:0.9,v/v) by injecting 700 mg of Aza-A 60 and eluting with 2mL/min, in the head to tail direction. Thirty fractions (3 mL each) were collected with 95% of the material being recovered. After monitoring by thin-layer chromatography (TLC) using toluene:ethyl acetate:methanol (10:1:2, v/v) as the developing solvent, the fractions (9-13) were purified by preparative reverse phase high performance liquid chromatography (RP-HPLC) using the LC-8 stainless-steel column (5 μm, 250 x 21 mm id, Supelco) and methanol:water (50:50 v/v) as eluent at a flow rate of 6mL/min. The effluents were monitored at 217 nm. Aza-A thus obtained was about 96% pure according to HPLC analysis. The structure of aza-A was confirmed by NMR (13C). In the second study, six minor triterpenoids, i.e., azadirachtin B (aza-B), azadirachtin H (aza-H), desacetylnimbin (Dnim), desacetylsalannin (Dsal), nimbin (Nim) and salannin (Sal) were isolated by injecting 4.2 g of Aza-A 60 (700 mg/run) in CCC, using the same conditions as described for the isolation of aza-A. After the six injections, two main fractions were obtained and monitored by TLC with 97% of the injected material being recovered. The first fractions (9-13) were purified by RP-HPLC as described above to obtain aza-B and aza-H, both 97% pure as attested by analytical HPLC. The second fraction (17-30) was purified using methanol:water (60:40 v/v) at a flow rate of 8 mL/min, with dnim, dsal, nim and sal being obtained (each 97% pure) according to HPLC analysis and their structures being confirmed by NMR (13C). The nematicidal activity against Heterodera glycines of all the above-purified compounds and crude extracts was evaluated. The mobility of the second stage juveniles (J2) was evaluated by the inverted tube methodology. A smaller recovery (P 0.05) of J2 treated with the aqueous crude (l/2 and 1/10 v/v) and methanolic (1000 ppm) extract was obtained while the hexane extract did not present activity. The activity of the aqueous crude and methanolic extracts was likely due to the presence of saponins and triterpenoids (limonoids), respectively. In the greenhouse assay, soy plants inoculated with juveniles treated with the aqueous (l/2 and 1/10 v/v) or methanolic (1000 ppm) extract produced less females and eggs (P 0,05) as compared to the control. The extract Aza-A 60 (70 ppm) caused larger immobility (P 0,05) of J2 (92%), proportional to the studied concentrations. The mortality of J2 for all the isolated triterpenoids at concentrations of 0.8, 8 and 80 ppm was evaluated with a reduction in the number of the J2 recovered, observed with increase of the applied dosages. Nim and Sal at 80 ppm were the most effective causing 99% mortality. Soy plants inoculated with J2 treated with the triterpenoids at 80 ppm presented medium nematicidal effect of 95%, reducing the number of females and eggs (P 0,05) as compared to the control. In regard to egg hatching, the triterpenoids tested at 100 ppm showed medium ovicide activity (P 0,05) of 50% as compared to the control with no nematostatic activity being observed 96 h after treatment.

The understanding of plant defense response in plant-pathogen interaction provides useful information to combat the disease. Soybean cyst nematode (SCN), Heterodera glycines, and charcoal rot causing fungus, Macrophomina phaseolina, are major pathogens of soybean, Glycine max, and infestation with SCN has found to aggravate the fungal disease severity. The gene expression analysis in resistant and susceptible interactions between H. glycines and G. max has identified some candidate resistance genes and signaling pathways but they are yet to be fully characterized. This dissertation aims to characterize one such gene, Conserved Oligomeric Golgi (COG) complex. The COG complex is important in structural and functional integrity of Golgi complex among eukaryotes, but very little is understood about its role in plants. This study demonstrated a defensive role of the COG complex in G. max against H. glycines. The transgenic plants for overexpression of COG genes, which were originally susceptible to the pathogen, showed reduced parasitism. In contrast, the RNA interference (RNAi) in originally resistant soybean lines showed a marked increase in parasitism. Further, these COG genes were found to be inducible by harpin elicitor molecules. In another study, the already proven resistance genes (NDR1, NPR1, EDS1 and TGA2) against H. glycines were investigated against the fungal pathogen. The transgenic plants for overexpression of these genes showed reduced disease severity, while the RNAi resulted increased severity compared to control lines. In addition, the H. glycines parasitism study and the candidate gene expression analysis in M. Phaseolina susceptible and moderately resistant G. max indicate that there are some cross communications between the defense processes of G. max to H. glycines and M. phaseolina.

Plant-parasitic nematodes represent a substantial constraint on global food security by reducing the yield potential of all major crops, including soybean (Glycine max L.). The soybean cyst nematode (SCN) (Heterodera glycines I.) is widely distributed across all soybean production areas of the US, and is the major yield-limiting factor, especially in the Midwestern US. Double cropping (DC) is defined as producing more than one crop on the same parcel of land in a single growing season. Compared to conventional single annual crops, DC provides many advantages, including improving soil health, enhanced nutrient provisioning to plants, improvement of soil physical properties, control of erosion, decrease in tillage requirements, and enhanced profitability. In some double-cropping systems, soybean is planted following winter wheat (Triticum aestivum L.), and several reports suggest the potential of wheat to suppress SCN populations. Field trials were conducted from 2017 to 2018 to investigate the effect of wheat on SCN populations in double-cropping soybean. Nine fields with three levels of initial SCN populations (low, moderate, and high) were selected in Illinois. Wheat was planted in strips alternating with strips-maintained weed-free and under fallow over winter and early spring. Soybean was planted in all strips after the wheat harvest. Soybean cyst nematode egg densities were acquired at four time points: wheat establishment, post-wheat/pre-soybean, mid-soybean (R1 growth stage or beginning of flowering), and post-soybean harvest. Wheat strips reduced SCN egg densities compared with fallow strips at the R1 stage (−31.8%) and after soybean harvest (−32.7%). Field locations with noted SCN suppression were selected for a metagenomics study. The structure of fungal communities differed significantly between DC and fallow plots at soybean planting and after harvest (P<0.001). Fungal populations were affected by location at all sampling times and by treatments before planting and after soybean harvest. Several enriched fungal and bacterial taxa in wheat plots, including Mortierella, Exophiala, Conocybe, Rhizobacter spp., and others, were previously reported to parasitize SCN and other plant-parasitic nematodes. Knowing that phytocompounds with potential nematicidal activity are released via the root system of plants, we implemented a gas chromatography–mass spectrometry (GC-MS) pipeline to investigate the profile of phytochemicals in soil samples collected from these fields and reviewed the potential nematicidal activity of compounds with higher concentration in double cropping fields. A total of 51 compounds were detected with the GSMS analysis, 8 with unknown identification. Several compounds, including multiple fatty acids, had greater relative peak areas in double-cropping samples compared to fallow. This multi-approach study provides a better understanding of the mechanisms governing wheat's effect on SCN populations. Rather than a single mechanism, the suppression of SCN in soybean fields double-cropped with winter wheat is potentially linked to enriched microbial communities, increased populations of beneficial organisms, and higher concentrations of phytochemicals with nematicidal activity. This is the first study implementing metagenomics and GCMS to characterize soil microbial and chemical profiles in soybean fields double-cropped with winter wheat. Finally, a set of studies were conducted to evaluate the effect of two seed-applied succinate dehydrogenase inhibitors (SDHI) compounds, fluopyram and pydiflumetofen, on SCN population densities, plant injury, and plant growth. Next-generation sequencing was later employed to identify transcriptomic shifts in gene expression profiles of soybean seedlings treated with fluopyram and pydiflumetofen. Fluopyram reduced both SCN egg and cyst counts in comparison to pydiflumetofen and the untreated control. RNA expression patterns of seed treatments clustered by sampling time (5 DAP vs 10 DAP). Multiple DEGs identified in soybean seedlings treated with fluopyram are reported to be linked to systemic resistance, suggesting a role of systemic resistance on the suppression of SCN by fluopyram. The non-target inhibition of soybean succinate dehydrogenase genes by fluopyram may be the origin of the phytotoxicity symptoms observed and potentially the cause of the systemic resistance activation reported in the current study. To our knowledge, this is the first report of systemic resistance being activated by fluopyram in addition to the suppression of soybean succinate dehydrogenase (SDH) and ubiquinone oxidoreductase genes. This work helps to elucidate the mechanisms of suppression of SCN by fluopyram