In this study, lipo-chitooligosaccharide (NodBj-V (C 18:1, MeFuc); LCO) 10-7M, extracted from Bradyrhizobium japonicum, was sprayed on the leaves of soybean cv. OAC Bayfield soybean and Evans x L66-2470 (carrying the rj1 mutation, and unable to nodulate). Leaf SA level and activities of the PR proteins chitinase, beta-1,3-glucanase and guaiacol peroxidase (GPOX) were quantified. Phenylalanine ammonia-lyase 1 (PAL1) and isoflavone synthase 2 (IFS2) relative gene expression levels in the sprayed leaves were quantified using quantitative real-time PCR. Messenger RNA abundance was quantified using microarrays. The treatment caused a transient increase in local salicylate levels 24 h after exposure, and a systemic increase in GPOX activity 48 h after exposure, in both soybean types. Of the selected 38 genes affected by the LCO treatment, 25 were stress-related. There were no significant differences in (A) chitinase and beta-1,3-glucanase activity, or (B) in PAL1 and IFS2 gene expression.

Soybean (Glycine max L. Merr.) and Bradyrhizobium japonicum can form a nitrogen fixing symbiosis. This symbiosis is important for most sustainable agriculture systems. This thesis examines two ways to enhance nodulation and nitrogen fixation by this symbiosis: coinoculation of plant growth promoting bacteria (PGPB) with B. japonicum, and addition of RNA to a bradyrhizobial culture medium. The optimal coinoculation dose of Serratia proteamaculans 1--102 and S. liquefaciens 2--68 was determined as 108 cells per plant under both optimal and suboptimal root zone temperatures (RZTs). Nodulation dynamics studies indicated that coinoculation of these two PGPB caused earlier nodule initiation and a higher nodulation rate, contributing to the higher nodule number and nodule weight. The coinoculation also increased nitrogen fixation efficiency under both optimal and suboptimal RZTs. A novel inducible activator only produced by the bacteria after addition of flavonoids to the culture system was prepared and evaluated in greenhouse and field experiments. Fourteen non-bradyrhizobial endophytic bacteria (NEB) were isolated from the surface sterilized root nodules, and three of these, designated NEB4, NEW and NEB17, showed soybean plant growth promotion under both greenhouse (with controlled RZTs) and field conditions. Alone, they were neither nodule inducers nor nitrogen fixers. Biolog tests and partial 16S rRNA gene sequence analyses placed the three strains in genus Bacillus: NEB4 and NEB5 are B. subtilis and NEB17 B. thuringiensis. Bradyrhizobium species grow slowly, making the culture process long and the cost of inoculant production higher. Addition of commercial yeast RNA to the bacterial culture medium accelerated the bacterial growth rate, shortened the culture time and increased the lipo-chitooligosaccharide (LCO) yield in flask cultures. Inoculation experiments in the greenhouse also showed that bradyrhizobial inoculant produced in the presence of RNA had better symb

Plants possess highly sensitive perception systems by which they recognize signal compounds originating from microbes. These molecular cues play an important role in both symbiotic and pathogenic relationships. Establishment of the soybean (Glycine max)-Bradyrhizobium symbiosis is orchestrated by specific signal molecules exchanged between appropriate plant and microbe partners: flavonoids as plant-to-bacteria signals, and Nod factor as bacteria-to-plant signals. How this signaling process interacts with stress conditions (abiotic and biotic) is the subject of this thesis. The abiotic stresses were suboptimal growth temperature, low pH, and salinity. Suboptimal growth temperatures affected the ability of the microsymbiont, Bradyrhizobim japonicum, to perceive nod gene inducers (genistein) and produce Nod factor. Nod Bj-V (C18:1, MeFuc) production by B. japonicum strains 523C and USDA110 was strongly affected by suboptimal growth temperature. Nod factor production declined with temperature, from 28 to 15°C. Strain USDA110 was more affected by decreased temperature than strain 532C. Decreased Nod factor production at low temperature was due to both decreased bacterial growth and lower production efficiency (Nod factor per cell). When a 1:1 mixture of Nod factor Nod Bj-V (C18:1, MeFuc) and Nod Bj-V (Ac, C16:0, MeFuc) was applied to soybean roots, root hair deformation increased as Nod factor concentration increased under stressfully low temperature and low pH conditions. High salinity stress strongly reduced the root hair deformation caused by Nod factor, and increasing the concentrations of added Nod factor did not over come this. Exogenous application of Nod Bj-V (C18:1, MeFuc), from strain 532C, to soybean root systems under two root zone temperatures (RZTs---17 and 25°C) reduced the progression of disease (powdery mildew---Microsphaera difussa) development on soybean leaves; this effect increased with Nod factor concentration and was gr

Adult schistosomes depend for growth and development on hormonal signals from the mammalian host, which may include the insulin signalling pathway. In this project, I firstly used microarray analysis to demonstrate that human insulin can be utilised by adult S. japonicum in culture, resulting in the modulation of distinct metabolic effects as reflected in transcriptional levels of parasite genes. The addition of insulin resulted in the differential expression of 1,101 genes with many related to functions corresponding to the biological and metabolic effects of insulin reported for mammalian cells. Those identified genes in male or female S. japonicum worms that were up or down regulated after exposure to insulin were predominantly involved in growth and development, with significant sex-specific responses evident. Insulin appeared to play a similar role in male parasites as those seen in classical mammalian systems including an increase in protein synthesis though gene transcription and the stimulation of mRNA translation and control protein degradation via the ubiquitin proteasome pathway. Microarray analysis indicated that insulin not only leads to increased gene expression of the PI3-K pathway, which enhances parasite growth, but may also play a role in the sexual differentiation and fecundity of female worms by activating the MAPK pathway. As the insulin target proteins, two types of insulin receptors from Schistosoma japonicum were isolated, S. japonicum insulin receptors 1 (SjIR-1) and 2 (SjIR-2), with features similar to insulin receptors from other taxa. The sequences share 70% and 74% identity to S. mansoni insulin receptor 1 and 2 (SmIR-1 and SmIR-2), respectively. SjIR-1 and SjIR-2 are highly conserved in their tyrosine kinase domain to other IRs from Homo, Mus musculus and Drosophila melanogaster. SjIR-2 is located in the parenchyma in males and in the vitelline glands of female worms, which occupy most of male or female tissue and play an important role in growth or fecundity. In contrast, SjIR-1 was located in the tegument and intestinal epithelium of adult worms, representing much smaller cellular regions compared with the voluminous vitelline tissue or parenchyma. This observation was further confirmed by real time PCR showing that SjIR-2 was more abundantly expressed in S. japonicum adult worm than SjIR-1. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are closer to EmIR than to SjIR-1 and SmIR-1, indicating that SjIR-1 and SmIR-1 might perform specific functions in schistosomes, while SjIR-2, SmIR-2 and EmIR might share similar roles in parasite growth and development in the three parasitic flatworms. Structure modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells. Adult worms of S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. In vaccination/challenge trials, there was no significant reduction in adult worm burdens with either of the SjLD vaccines. However, there were significant reductions in mean lengths of adult worms ranging from 22-25% in the SjLD1 vaccinated group to 37-42% in the SjLD2 vaccinated groups, significant reductions in faecal eggs in both the SjLD1 (66%) and SjLD2 (68%) vaccinated groups, and a reduction in liver egg numbers in the SjLD1(33%) vaccinated group. These results show that although the SjLDs vaccines were unable to reduce adult worm numbers by clearing them from the vaccinated mice, nevertheless, they significantly depressed the growth of male and female adult worms and affected female egg production. The protective efficacy obtained in terms of the substantial decrease in faecal eggs exceeded that of many of the recently available schistosome antigens and prototype vaccine formulations, which, at best, elicit 40–50% protection in animals using the standard readouts of reduced worm burden or egg production and viability. Overall, disruption of this insulin pathway leading to parasite starvation through the prevention of glucose uptake thereby affecting parasite growth, development and female fecundity, provides a new intervention target and transmission blocking approach to combat schistosomiasis and may be applicable for the control of other debilitating parasitic infections as well.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 24, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Gary Stacey. Vita. Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on May 27, 2009) Includes bibliographical references.

Verificou-se a influência da temperatura e da agitação ótima para o desenvolvimento da estirpe SEMIA 587 de Bradyrhizobium japonicum em fermentador de bancada. A variação da temperatura, nos estudos cinéticos, influenciou de forma mais marcante o desenvolvimento microbiano do que a variação da agitação. Os melhores resultados a nível de rendimento foram obtidos utilizando-se uma temperatura de 28ºC e uma agitação de 230 rpm. Fez-se também um estudo da competição entre as estirpes SEMIA 587 e SEMIA 5019 em fermentador, inoculante turfoso e por sítio de nodulação. A estirpe SEMIA 587, quando desenvolvida conjuntamente com a SEMIA 5019, atingiu aproximadamente 70% da população total após 120 horas de processo. Esta mistura mais as elaboradas com 30, 50 e 70% da estirpe SEMIA 587, foram utilizadas como tratamentos para a inoculação em soja e também para a impregnação em turfa esterilizada. Nestes experimentos observou-se uma predominância da estirpe SEMIA 587, em relação à SEMIA 5019, em fixar nitrogênio e competir por sítios de nodulação e também no inoculante. Os resultados confirmam a possibilidade de utilização dos cultivos mistos, desde que sejam encontradas condições operacionais para o desenvolvimento ótimo das duas estirpes.

Rizóbios são bactérias aeróbias, Gram-negativas, que fixam nitrogênio atmosférico quando associadas a leguminosas. O gênero Bradyrhizobium é de grande importância na agricultura, pois essas bactérias fixam nitrogênio em simbiose com soja [Glycine max (L.) Merrill]. A caracterização dos rizóbios é fundamental para estudos relacionados à diversidade e à distribuição ecológica desses microrganismos. Três estudos foram conduzidos nesse trabalho: i) uma estratégia baseada em amplificação por PCR para diferenciar Bradyrhizobium japonicum de B. elkanii, utilizando-se seqüências 16S rDNA; ii) a caracterização da variabilidade genética de uma população de bradirrizóbios isolada de um campo experimental, trinta anos após a inoculação de estirpes padrão; iii) a avaliação da variabilidade genética de bradirrizóbios isolados de cinco regiões produtoras de soja no Estado do Rio Grande do Sul (RS) e dos possíveis fatores ambientais que poderiam ser os responsáveis por tal diversidade. Técnicas de biologia molecular foram utilizadas na identificação, ocorrência, distribuição e estudo populacional dos rizóbios, especialmente a amplificação do gene 16S rRNA por PCR, rep-PCR e AFLP.Essas duas últimas técnicas quando combinadas permitiram uma análise mais precisa da variabilidade genética das populações estudadas. O índice de diversidade de Shannon foi utilizado para comparar o grau de diversidade observado nas diferentes populações. Também foi observada uma correlação direta entre o grau de diversidade e o pH do solo. Os resultados obtidos permitiram concluir que as populações de bradirrizóbios que nodulam as lavouras de soja do RS são altamente variáveis, podem persistir nos solos, mesmo na ausência da planta hospedeira, e sofrem influência de fatores bióticos e abióticos. / Rhizobia are Gram-negative aerobic bacteria that fix atmospheric nitrogen in symbiosis with leguminous plants. Bacteria belonging to the Bradyrhizobium genus are very important because they are able to nodulate and fix nitrogen in symbiosis with soybean [Glycine max (L.) Merrill]. Characterization of rhizobia is fundamental in studies concerning the diversity and ecological distribution of these microorganisms. Three studies have been conducted in this work: i) a strategy based on amplification by PCR to differentiate Bradyrhizobium japonicum and B. elkanii using 16S rDNA sequences; ii) the genetic variability assessment of a bradyrhizobial population isolated from an experimental field thirty years after the inoculation with reference strains; iii) the genetic variability characterization of bradyrhizobia isolated from five soybean fields in different regions in Rio Grande do Sul (RS) State and the environmental factors that can influence such diversity. Molecular biology techniques were used for the identification, occurrence, distribution, and studies of rhizobia population, specially the 16S rRNA gene amplification by PCR, rep-PCR and AFLP. When combined, these last two techniques provided an accurate analysis of the genetic diversity of the analyzed populations. Shannon diversity index was used to compare the diversity among different populations.A direct correlation was observed between diversity degree and soil pH. The results obtained have shown that bradyrhizobia nodulating soybean in RS are highly variable and were able to persist in soil even when the host legume was lacking. Besides they have shown to be influenced by abiotic and biotic parameters.

Embora o ferro seja um dos elementos mais abundantes na crosta terrestre, somente uma pequena fração está disponível para ser utilizada pelos organismos vivos. A solubilidade do ferro em solos com pH neutro é muito baixa, aproximadamente 10-18 M. Em condições de baixa disponibilidade de ferro, diversos microrganismos podem produzir e excretar sideróforos, quelantes orgânicos de baixo peso molecular envolvidos na solubilização e seqüestro de Fe3+. A captação de ferro é fator limitante para a fixação biológica do nitrogênio, uma vez que esse elemento está diretamente envolvido em moléculas como a nitrogenase, leg-hemoglobina, ferredoxina e citocromos. Enquanto microrganismos de vida-livre, os rizóbios devem ser capazes de solubilizar ferro e competir por ele no solo. As bactérias pertencentes ao gênero Bradyrhizobium são de grande relevância agronômica devido à capacidade de fixar nitrogênio em simbiose com diversas leguminosas, especialmente a soja [Glycine max (L.) Merrill]. B. japonicum and B. elkanii são duas espécies capazes de nodular soja e quatro estirpes são comumente utilizadas como inoculantes no Brasil: B. elkanii SEMIA 587 e SEMIA 5019 e B. japonicum SEMIA 5079 e SEMIA 5080. Os genes fegA e fhuA, relacionados à síntese de proteínas de membrana transportadoras do complexo Fe3+ - sideróforo já foram identificados em B. japonicum 61A152 e Rhizobium leguminosarum, respectivamente. Ao contrário de B. japonicum, nada se conhece sobre esses genes na espécie B. elkanii. No presente trabalho foram analisadas a habilidade em produzir sideróforos e a presença de genes relacionados à síntese de proteínas receptoras do complexo Fe3+ - sideróforo entre diferentes estirpes de Bradyrhizobium. Uma porção do gene fhuA de B. elkanii foi isolada e apresentou um elevado grau de conservação com outros genes relacionados à captação de sideróforos em diferentes bactérias. Experimentos de Southern-blot demonstraram que existe apenas uma cópia do gene fhuA, e nenhuma do gene fegA, no genoma da estirpe SEMIA 587 de B. elkanii. Através do tradicional método CAS, a capacidade de produzir e captar sideróforos in vitro foi confirmada entre as estirpes de B. elkanii utilizadas como inoculantes comerciais no Brasil. As linhagens de B. japonicum, entretanto, mesmo possuindo receptores de membrana específicos para sideróforos, não são capazes de se multiplicar em meio de cultura deficiente em ferro. Tais resultados indicam a possibilidade de diferenças significativas quanto ao sistema de captação de ferro entre tais estirpes. Estudos que confirmem a função do gene fhuA estão sendo conduzidos para verificação da viabilidade fenotípica de B. elkanii mutante para esse gene. / Although iron is one of the most abundant elements in Earth, only one small fraction is available to be used by living organisms. The solubility of iron in soils with neutral pH is very low, approximately 10-18 M. During iron deficiency, many microorganisms can produce and excrete low molecular weight organic chelators termed siderophores involved in the solubilization and sequestration of Fe3+. The iron uptake is a limiting factor for the biological nitrogen fixation as iron is directly involved in many molecules that are essential to this process, like nitrogenase, leg-hemoglobin, ferredoxin and cytochrome. While free-living microorganism, rhizobia should be able to solubilize iron and compete for it in soil. Bacteria belonging to the genus Bradyrhizobium are of enormous agricultural value since they are able to fix atmospheric nitrogen in symbiosis with several leguminous plants, especially soybean [Glycine max (L.) Merrill]. B. japonicum and B. elkanii strains are two species capable of nodulate soybean, in which four strains are commonly used as inoculant in Brazil: B. elkanii SEMIA 587 and SEMIA 5019 and B. japonicum SEMIA 5079 and SEMIA 5080. In B. japonicum 61A152 and Rhizobium leguminosarum fegA and fhuA genes, which are involved in the synthesis of membrane Fe3+-siderophore uptake proteins were already identified, respectively. In the opposite, almost nothing is known about these genes in B. elkanii species. In this work the siderophore production ability and the presence of genes related to the membrane Fe3+- siderophore uptake were analyzed in several Bradyrhizobium strains. A B. elkanii fhuA DNA region was isolated and it presented a high level of homology with others siderophore uptake related genes from different bacterial species. Southern blot experiments shown that there is a single fhuA gene copy in the B. elkanii SEMIA 587 genome, and no fegA gene was identified in this genome. Through the traditional CAS methodology, the siderophore production and uptake in vitro activities were demonstrated in the B. elkanii strains used as inoculant in Brazil. B. japonicum strains, however, although having siderophores specific membrane receptors, were not able to growth in a medium lacking iron. Such results indicated that significant differences concerning iron uptake system might exist between these two bacterial species. Studies to confirm fhuA gene function are under way aiming to verify the phenotypic viability of B. elkanii fhuA mutants.

Rizóbios são bactérias aeróbias, Gram-negativas, que fixam nitrogênio atmosférico quando associadas a leguminosas. O gênero Bradyrhizobium é de grande importância na agricultura, pois essas bactérias fixam nitrogênio em simbiose com soja [Glycine max (L.) Merrill]. A caracterização dos rizóbios é fundamental para estudos relacionados à diversidade e à distribuição ecológica desses microrganismos. Três estudos foram conduzidos nesse trabalho: i) uma estratégia baseada em amplificação por PCR para diferenciar Bradyrhizobium japonicum de B. elkanii, utilizando-se seqüências 16S rDNA; ii) a caracterização da variabilidade genética de uma população de bradirrizóbios isolada de um campo experimental, trinta anos após a inoculação de estirpes padrão; iii) a avaliação da variabilidade genética de bradirrizóbios isolados de cinco regiões produtoras de soja no Estado do Rio Grande do Sul (RS) e dos possíveis fatores ambientais que poderiam ser os responsáveis por tal diversidade. Técnicas de biologia molecular foram utilizadas na identificação, ocorrência, distribuição e estudo populacional dos rizóbios, especialmente a amplificação do gene 16S rRNA por PCR, rep-PCR e AFLP.Essas duas últimas técnicas quando combinadas permitiram uma análise mais precisa da variabilidade genética das populações estudadas. O índice de diversidade de Shannon foi utilizado para comparar o grau de diversidade observado nas diferentes populações. Também foi observada uma correlação direta entre o grau de diversidade e o pH do solo. Os resultados obtidos permitiram concluir que as populações de bradirrizóbios que nodulam as lavouras de soja do RS são altamente variáveis, podem persistir nos solos, mesmo na ausência da planta hospedeira, e sofrem influência de fatores bióticos e abióticos. / Rhizobia are Gram-negative aerobic bacteria that fix atmospheric nitrogen in symbiosis with leguminous plants. Bacteria belonging to the Bradyrhizobium genus are very important because they are able to nodulate and fix nitrogen in symbiosis with soybean [Glycine max (L.) Merrill]. Characterization of rhizobia is fundamental in studies concerning the diversity and ecological distribution of these microorganisms. Three studies have been conducted in this work: i) a strategy based on amplification by PCR to differentiate Bradyrhizobium japonicum and B. elkanii using 16S rDNA sequences; ii) the genetic variability assessment of a bradyrhizobial population isolated from an experimental field thirty years after the inoculation with reference strains; iii) the genetic variability characterization of bradyrhizobia isolated from five soybean fields in different regions in Rio Grande do Sul (RS) State and the environmental factors that can influence such diversity. Molecular biology techniques were used for the identification, occurrence, distribution, and studies of rhizobia population, specially the 16S rRNA gene amplification by PCR, rep-PCR and AFLP. When combined, these last two techniques provided an accurate analysis of the genetic diversity of the analyzed populations. Shannon diversity index was used to compare the diversity among different populations.A direct correlation was observed between diversity degree and soil pH. The results obtained have shown that bradyrhizobia nodulating soybean in RS are highly variable and were able to persist in soil even when the host legume was lacking. Besides they have shown to be influenced by abiotic and biotic parameters.