Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-19Bitstream added on 2014-06-13T20:10:49Z : No. of bitstreams: 1 monteiro_nk_me_sjrp.pdf: 687247 bytes, checksum: 5856bbc04fe8d3e52218193110c45156 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Bactérias do gênero Rhizobium são conhecidas por manterem uma relação simbiótica com determinadas plantas leguminosas e atuam como fixadores biológicos de nitrogênio. Elas vêm sendo estudadas em relação à produção de exopolissacarídeos (EPS), o qual acredita-se estar relacionado com o processo de reconhecimento e invasão do microrganismo na planta hospedeira. Estas moléculas apresentam propriedades físico-químicas e reológicas interessantes para a indústria alimentícia, podendo ser aplicadas como estabilizantes, emulsificantes ou gelificantes. Estudos da estrutura química realizados em quatro EPS produzidos por Rhizobuim tropici (em dois meios de cultivos diferentes), Mesorhizobium e Rhizobium sp os caracterizaram como sendo heteropolímeros. Esses polímeros são constituídos principalmente por glucose e galactose, com traços de manose, apresentando ácido urônico, acetila e piruvato com substituintes. Por cromatografia de gel permeação e eletroforese em gel de poliacrilamida apenas os polímeros produzidos por Rhizobium tropici (R2) e Mesorhizobium (R3) comportaram-se como moléculas homogêneas com baixo grau de polidispersividade. Estes EPS foram paracialmente caracterizados utilizando-se técnicas de FT-IR, RMN de 1H e 13C / Bacteria of the Rhizobium genus are known to maintain a symbiotic relationship with certain leguminous plants and they act as biological nitrogen fixation. They have been studied as exopolysaccharides (EPS) producers, which may be related to the recognition process and to the microorganism invasion into the host plant. These molecules exhibit interesting physical-chemical and rheological properties for the food industry since they can be used as stabilizers, emulsifiers or gelling agents. Studies of the chemical structure of four EPS produced by Rhizobium tropici (in two different culture media), Mesorhizobium and Rhizobium sp have characterized them as heteropolymers. The EPS are constituted mainly by glucose and galactose with traces of mannose besides uronic acid, pyruvate and acetyl as substituents. Gel permeation chromatography and polyacrylamide gel electrophoresis showed that the polymers produced by Rhizobium tropici (R2) and Mesorhizobium (R3) behaved as homogeneous molecules with low polydispersity. Thus these two EPS were partially characterized by the techniques of FT-IR, 1H and 13C NMR

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-03-07T12:57:02Z No. of bitstreams: 1 PGCS14MA146.pdf: 911298 bytes, checksum: 15d2a151b2d75b3012172eac62b96c08 (MD5) / Made available in DSpace on 2018-03-07T12:57:02Z (GMT). No. of bitstreams: 1 PGCS14MA146.pdf: 911298 bytes, checksum: 15d2a151b2d75b3012172eac62b96c08 (MD5) Previous issue date: 2014-08-29 / The present study aimed to evaluate the influence and residual effect of using the dregs, the residue of pulp industry and dolomitic limestone on nodulation, nitrogen accumulation and dry matter of shoot for beans. The study was conducted in a greenhouse in CAV / UDESC campus in Lages SC, using alítico Haplumbrept, taken from an area where in 2004 were applied superficially in an experimental design in randomized blocks, four replications of dimensions 8 m x 8 m . Two separate experiments, one experiment appointed without any fertilization and the second experiment, where there was a fertilizer of potassium chloride (KCl) and triple superphosphate (STF) were implanted. The soil was collected from 0-20 cm in treatments with lime at a dose of 10.5 mg ha-1 (a full dose of corrective recommended by SMP), dregs at a dose of 13 mg ha-1 (a full dose corrective recommended by SMP) and the control treatment without application of corrective. In addition to these treatments were tested two levels of inoculation factor, namely rhizobia inoculation and treatment without inoculation. The experiments were conducted in a 3 x 2 factorial (three levels of correction factor and two levels of soil inoculation), with 4 replicates, for a total of 6 treatments and 24 sampling units in a completely randomized design. The plants were harvested 40 days after emergence and variable dry matter of shoot (DMS), total nitrogen accumulated per plant (Total N), number of nodules per plant (NNP) and dry weight of nodules per plant (DWNP). Data were subjected to analysis of variance, and when significant, were compared by Tukey test at 5% probability. The use of rhizobia did not result in accumulation of nitrogen in the shoot. The application of corrective as dregs of soil acidity had a beneficial residual effect on dry weight of shoots of bean / O presente estudo visou avaliar a influência e efeito residual do uso do dregs, resíduo da indústria de celulose e do calcário dolomítico, sobre a nodulação, acúmulo de nitrogênio e massa seca da parte aérea em feijoeiro. O estudo foi conduzido em casa de vegetação no campus CAV/UDESC em Lages SC, utilizando Cambissolo Húmico alítico, retirado de uma área onde em 2004 foram aplicados superficialmente em um delineamento experimental em blocos ao acaso, quatro repetições e parcelas de dimensões 8 m x 8 m. Foram implantados dois experimentos distintos, nomeados experimento 1, sem qualquer tipo de adubação e o experimento 2, onde houve a adubação de cloreto de potássio (KCl) e superfosfato triplo (STF). O solo foi coletado da camada 0-20 cm nos tratamentos que receberam calcário na dose de 10,5 mg ha-1 (uma dose completa de corretivo recomendada pelo índice SMP), dregs na dose de 13 mg ha-1 (uma dose completa de corretivo recomendada pelo índice SMP) e o tratamento testemunha sem aplicação de corretivo. Adicionalmente a estes tratamentos testaram-se dois níveis do fator inoculação, a saber, inoculação com rizóbio e a testemunha sem inoculação. Os experimentos foram instalados em um fatorial 3 x 2 (três níveis do fator correção de solo e dois níveis de inoculação), com 4 repetições, perfazendo um total de 6 tratamentos e 24 unidades amostrais, em delineamento experimental inteiramente casualizado. As plantas foram coletadas 40 dias após a emergência e foram avaliadas as variáveis matéria seca de parte aérea (MSPA), nitrogênio total acumulado por planta (NTotal), número de nódulos por planta (NNP) e massa seca de nódulos por planta (MSNP). Os dados foram submetidos à análise de variância, e quando significativos, foram comparados pelo Teste de Tukey ao nível de 5% de probabilidade. O uso do rizóbio não resultou em acúmulo de nitrogênio na parte aérea. A aplicação do dregs como corretivo da acidez do solo apresentou efeito residual benéfico na massa seca da parte aérea do feijoeiro

A mesofauna edáfica, compreendendo ácaros e colêmbolos, é sensível a alterações no solo, sendo considerada um indicador de qualidade, uma vez que realiza a fragmentação de matéria orgânica, controle populacional de determinados grupos da biota do solo e ingestão de microrganismos. A colonização de plantas por micro-organismos, como a de leguminosas por rizobios, pode levar a mudanças na rizosfera vegetal e influenciar a mesofauna. Este estudo teve por objetivo avaliar o efeito da inoculação de plantas de trevo vermelho com rizóbios sobre os ácaros e colêmbolos edáficos e avaliar se colêmbolos alimentados com rizóbios poderiam inocular plantas hospedeiras. Para isso, plantas de trevo vermelho, inoculadas com as estirpes SEMIA 2081, SEMIA 2082 e SEMIA 222, e plantas controle sem inoculação foram plantadas em tubos-armadilha e inseridas em campo. A seguir, a mesofauna atraída foi extraída e identificada. Outro experimento em laboratório utilizou plantas de trevo vermelho que foram colocadas em contato com colêmbolos da família Hypogastruridae e dos gêneros Proisotoma, Onychiurus e Orthonychiurus, os quais haviam sido alimentados com um substrato impregnado com o rizóbio da estirpe SEMIA 2081 As plantas inoculadas com as três estirpes de rizóbios atrairam ácaros edáficos mas não colêmbolos, quando comparadas com as plantas que não foram inoculadas nem receberam fonte de nitrogênio. Considerando os números totais de ácaros e colêmbolos, somente as plantas inoculadas com a estirpe SEMIA 222 apresentaram atratividade quando comparadas ao controle sem inoculação e sem fonte de nitrogênio. A atratividade de ácaros e colêmbolos observada no tratamento controle com nitrogênio foi similar aos tratamentos inoculados. No experimento de laboratório, os colêmbolos vivos e o macerado de colêmbolos da família Hypogastruridae, e gêneros Proisotoma e Orthonychiurus foram capazes de transferir os rizóbios que receberam pela alimentação às plantas, formando nódulos radiculares. As plantas que receberam o macerado de colêmbolos da família Hypogastruridae apresentaram maior número de nódulos comparadas às plantas que receberam os mesmos colêmbolos vivos. / The soil mesofauna comprising mites and springtails, is sensitive to changes in soil, and it is considered a quality indicator since it performs fragmentation of organic matter, population control of certain soil biota groups and ingestion of microorganisms. The plants colonized by microorganisms, such as legumes by rhizobia can lead to changes in plant rhizosphere and influence mesofauna. This study aimed to evaluate the effect of inoculation of red clover plants with rhizobia on edaphic mites and springtails and assess if springtails fed with rhizobia could inoculate host plants. For this, red clover plants inoculated with strains SEMIA 2081, SEMIA 2082 and SEMIA 222, and control plants without inoculation were planted in trap-tubes and inserted on field. Then, the attracted mesofauna was submitted to extraction and identification. Another laboratory experiment used red clover plants put in contact with springtails. These springtails of Hypogastruridae family and Proisotoma, Onychiurus and Orthonychiurus genera, had been fed with a substrate impregnated with rhizobia strain SEMIA 2081. Plants inoculated with three Rhizobia strains attracted edaphic mites, but not Collembola, when compared to uninoculated plants with no nitrogen source Considering the total numbers of mites and springtails, only plants inoculated with SEMIA 222 strain showed attractiveness when compared to the control without inoculation and no nitrogen source. The attractiveness of mites and springtails observed in the control treatment with nitrogen was similar to the inoculated treatments. In the laboratory experiment, the living springtails and the macerate of springtails of Hypogastruridae family, and Proisotoma and Orthonychiurus genera were able to transfer the rhizobia that they received by feeding to the plants, forming root nodules. The plants that received the macerate of springtails of the Hypogastruridae family had a higher number of nodules compared to the plants that received the same living springtails.

As espécies forrageiras alfafa e cornichão são importantes plantas utilizadas durante o período hibernal nas pastagens do Rio Grande do Sul, reunindo características desejadas, como alto valor nutritivo, elevada produção de forragem, boa aceitabilidade e digestibilidade. No entanto, no Brasil, existe uma carência de trabalhos que visem à seleção e caracterização de estirpes de rizóbio em alfafa e cornichão que sejam mais competitivas e tenham alta capacidade de nodulação e fixação de nitrogênio. Desta forma, o objetivo deste trabalho foi selecionar rizóbios eficientes na fixação biológica de nitrogênio em simbiose a Lotus corniculatus e Medicago sativa. Especificamente, os objetivos são: (a) isolar e caracterizar genética e fenotipicamente rizóbios isolados de alfafa e cornichão; (b) avaliar a produção de ácido indol-acético e sua influência na germinação de sementes de arroz; (c) selecionar rizóbios mais eficientes em alfafa; (d) selecionar a campo rizóbios para cornichão; (e) avaliar o efeito do nitrogênio na simbiose de rizóbio e alfafa. Foram realizados experimentos, em casa de vegetação, em laboratório e a campo, com plantas de L. corniculatus e M. sativa. Os rizóbios isolados de alfafa foram identificados como pertencentes às espécies de Sinorhizobium meliloti e Rhizobium sp. Alguns isolados de alfafa foram capazes de produzir ácido indol-acético e estimular o crescimento de sementes de arroz. Os isolados UFRGS Ms98, Ms63, Ms208 e Ms204 apresentaram índice de eficiência relativa da fixação de nitrogênio superiores aos das estirpes recomendadas para inoculação de M. sativa, indicando um bom potencial destes isolados para estudos a campo. O isolado de rizóbio nativo UFRGS Lc322 e a estirpe U510 foram mais eficientes na fixação biológica de nitrogênio, em condições de campo, do que a estirpe SEMIA 816, recomendada para cornichão no país. O aumento da dose de nitrogênio afetou a interação entre rizóbios e alfafa, no entanto foi possível selecionar o isolado UFRGS Ms208 como mais tolerante ao nitrogênio mineral. / Alfalfa and birdsfoot forages are important plants used during the pastures’ hibernal period of Rio Grande do Sul, gathering desired traits such as high nutritional value, high forage production, good acceptability and digestibility. However, in Brazil, there is a lack of studies aiming the selection and characterization of Rhizobium strains in alfalfa and birdsfoot that are more competitive and have high capacity for nodulation and nitrogen fixation. Thus, the objective of this study was to select Rhizobia efficient in biological nitrogen fixation in symbiosis with Lotus corniculatus and Medicago sativa. Specifically, the objectives were: (a) to isolate and characterize genetically and phenotypically Rhizobia from alfalfa and birdsfoot; (b) to evaluate the production of indole acetic acid and its influence on germination of rice seeds; (c) to select the most efficient Rhizobia in alfalfa; (d) to select Rhizobia for birdsfoot in field; (e) to evaluate the effect of nitrogen in the symbiosis of Rhizobium and alfalfa. Experiments were performed, in a greenhouse, in laboratory and in field conditions, with L. corniculatus and M. sativa. The Rhizobia isolated from alfalfa were identified as belonging to the species of Sinorhizobium meliloti and Rhizobium sp. Some strains of alfalfa were able to produce indole acetic acid and stimulate the growth of rice seeds. The isolates UFRGS Ms98, Ms63, Ms208 and Ms204 showed efficiency index for nitrogen fixation higher than the strains recommended for inoculation of M. sativa, indicating a good potential of these isolates for field studies. The isolated of the native Rhizobium UFRGS Lc322 and the strain U510 were more efficient in nitrogen fixation in field conditions than SEMIA 816 strain, recommended for birdsfoot in the country. The increasing in the nitrogen dose affected the interaction between Rhizobia and alfalfa, but it was possible to select UFRGS Ms208 isolate as the most tolerant to mineral nitrogen.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-29T14:32:08Z No. of bitstreams: 1 Carla Regine Reges Silva Franca.pdf: 474566 bytes, checksum: b95c31472908cbb435a486697979ad3b (MD5) / Made available in DSpace on 2016-06-29T14:32:08Z (GMT). No. of bitstreams: 1 Carla Regine Reges Silva Franca.pdf: 474566 bytes, checksum: b95c31472908cbb435a486697979ad3b (MD5) Previous issue date: 2009-05-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study had for objective to evaluate the growth kinetics and characteristics morphophysiological and symbiotic strains of rhizobia of four, front the conditioning different liquids. The experiment was composed of four strains (two of the strains of Rhizobium tropici (SEMIA 4077-SEMIA 4080) and two of Bradyrhizobium japonicum (SEMIA 5079-SEMIA 5080), respectively for bean (Phaseolus vulgaris) and soybean (Glycine max), three conditioning (NaCl 0,1%, glicerol 20% and carboximetilcelulose 0,2%) more fresh bacteria, that you/they were grown close to each evaluation period. After the growth period in liquid middle YM 0,5 mL of the bacterial broth of each strains was transferred for tubes with capacity for 2 mL, containing 1,5 mL of each conditioning liquid and maintained in room temperature by 180 days. After the accomplishment of the different formulations for the conservation of the strains, it was made the counting of UFC that contained 108 cells of the rhizobia culture approximately using serial dilutions and the method of the drop. During the experiment, readings were accomplished in the time 0, 1, 7, 15, 21 days after the conservation, following for monthly samples 30, 60, 90, 120, 150, 180 days. The efficiency symbiotic was evaluated in the periods of 15, 60 and 120 days after the conservation in bean plants and soy. The results allow to observe that the concentration of viable cells varied among the preservantes. The preservante NaCl 0,1% maintained the concentration of viable cells around 107 and 108 UFC mL-1 for the strains SEMIA 4077, 5080 and 5079 for up to 180 days of conservation and it nodulation to the 15 and 120 days of conservation for the strains SEMIA 4077 and SEMIA 4080, when inoculated in bean seeds. The conditioning Glicerol 20%, didn't present desirable characteristics for a good conservation method with the strains SEMIA 4080, SEMIA 5079 and SEMIA 5080. The carboximetilcelulose was promising for the conservation of the ancestries SEMIA 4077 and SEMIA 5079 in room temperature. Nodulation was influenced by the preservative used. In all of the preservantes there was decrease in the number of nodules during the conservation period. With base in the results the conditioning liquids NaCl 0,1% and Carboximeticelulose 0,2% were the one that got maintain the cells of the strains for a period of short period, besides preserving the characteristics morfofisiológicas of the strains of Rhizobium and Bradyrhizobium. / Este estudo visou avaliar a cinética de crescimento e características morfofisiológicas e simbiótica de quatro estirpes de rizóbio, frente a diferentes condicionadores líquidos. O experimento foi composto de quatro estirpes (sendo duas de Rhizobium tropici (SEMIA 4077 – SEMIA 4080) e duas de Bradyrhizobium japonicum (SEMIA 5079 – SEMIA 5080), recomendadas respectivamente para feijão (Phaseolus vulgaris) e soja (Glycine max), três condicioadores líquidos (NaCl 0,1 % , glicerol 20 % e carboximetilcelulose 0,2 %). Após cultivo em meio líquido YM 0,5 mL do caldo bacteriano de cada estirpe foi transferido para tubos com capacidade para 2 mL, contendo 1,5 mL de cada condicionador líquido e mantidas em temperatura ambiente por 180 dias. Em seguida, fez-se a contagem utilizando diluições seriadas e o método da gota das UFC que foi de aproximadamente 108 células. Durante o experimento, foram realizadas determinações da população bacteriana aos 0, 1, 7, 15, 21 dias após a conservação, seguido por amostras aos 30, 60, 90, 120, 150, 180 dias. A eficiência simbiótica foi avaliada nos períodos de 15, 60 e 120 dias após a conservação em plantas de feijão e soja. Os resultados permitem observar que a concentração de células viáveis variou entre os preservantes. O preservante NaCl 0,1% manteve a concentração de células viáveis entre 107 e 108 UFC mL-1 para as estirpes SEMIA 4077, 5080 e 5079 por até 180 dias de conservação e produziu efetiva nodulação aos 15 e 120 dias de conservação para as estirpes SEMIA 4077 e SEMIA 4080, quando inoculados em sementes de feijão. O preservante Glicerol 20%, não apresentou características desejáveis para um bom método de preservação com as estirpes SEMIA 4080, SEMIA 5079 e SEMIA 5080. O carboximetilcelulose foi promissor para a conservação das estirpes SEMIA 4077 e SEMIA 5079 em temperatura ambiente. A nodulação foi influenciada pelo preservante utilizado. Em todos os preservantes houve diminuição no número de nódulos durante o período de conservação. Com base nos resultados osxii condicionadores líquidos NaCl 0,1 % e Carboximeticelulose 0,2 % foram os que conseguiram manter as células das estirpes por um período de curto prazo, além de preservar as características morfofisiológicas das estirpes de Rhizobium e Bradyrhizobium.

Memoria para optar al Título Profesional de Ingeniero Agrónomo Mención: Fitotecnia

With the high rate of human population growth in Africa, it is imperative that food production be increased through improving yields and bringing more land under cultivation. Nitrogen is a key element required for plant growth and with the low input of fertilizers into African farming-systems, it is necessary to harness biological nitrogen fixation (BNF) to its fullest extent. To maximize nitrogen fixation in crop and pasture legumes under various conditions and cropping systems requires intensified research. In this study the diversity of root nodule bacteria associated with Lotononis species in South Africa was investigated. To our knowledge, no past attempts have been made to investigate the diversity of rootnodule-bacteria associated with the genus Lotonollis. Eighty nodulated plants representing thirty-two Lotononis spp. were collected from all the main geographical and climatological regions in South Africa. Isolates obtained from rootnodules were purified and characterized with sodium dodecyl-sulphate gel polyacrylamide gel electrophoresis (SDS-PAGE), supplemented at the genomic level with 16S rDNA sequence data of selected strains. Initial screening (SDS-PAGE) showed that most isolates obtained from the same plant species, grouped into various clusters within the dendrogram. A few of the isolates from similar host plants seemed to cluster with high similarity. It is thus reasonable to conclude that host specificity in the symbiotic interaction is less applicable for most species of Lotononis. When considering the geographical origins of the isolates, their diverse nature was clearly illustrated. It was showed that isolates from similar geographical regions were evenly distributed throughout the dendrogram. Some of the isolates obtained from arid environments formed closely related electrophoretic groups. It was subsequently shown that root-nodule bacteria associated with Lotononis species are not restricted to a particular rhizobial genus, but that heterogeneity is evident. Some of the isolates were also related to genera outside the Rhizobiaceae, namely Methylobacterium and Burkholderia. / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

The projected exponential growth of the human population necessitates a concomitant increase in food supplies, and by implication an increase in fixed nitrogen for crops and pastures. This can to a large extent be supplied by biological nitrogen fixation (BNF). However, to achieve this goal improved effectivity of the legume-rhizobium symbiosis is required, implicating improvement in the macro- as well as the micro symbiont. Therefore the search for more effective microsymbionts is a sine qua non to provide better matching and tolerance to stress conditions. The aim of this study was to investigate the range of rhizobia associated with the exotic Australian Acacia species (A. meamsii, A. dealbata and A. decurrens) in South Africa and to determine whether these species could be useful to provide rhizobial strains for application in the South African inoculant industry in order to improve local existing biological nitrogen-fixing systems. Although these Acacia species are geographically widespread throughout South Africa, their root nodule bacteria have never been investigated in depth. Their widespread occurrence and presumed promiscuity suggested that they might form nitrogen-fixing symbioses with a wide range of indigenous rhizobial strains with different ecological adaptations. In this study nodulated plants of the three Acacia spp. were collected from diverse geographic areas with diverse climatic conditions and different soil pH's. Isolates were obtained from root nodules, purified and the putative rhizobial isolates characterized with sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), supplemented at the genomic level with 16S rDNA sequence data of selected isolates. The majority of the isolates investigated were members of the genus Bradyrhizobium, whilst some isolates showed close relationships to the genera Agrobacterium, Mesorhizobium, Rhizobium and Sinorhizobium. As a result of their predominant association with the slow-growing strains of the genus Bradyrhizobium, the legume spp. A. meamsii, A. dealbata and A. decurrens as trap plants would not playa significant role as a source of diverse rhizobia for application in the South African inoculant industry. / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Crown gall disease of plants is caused by introduction of foreign DNA into susceptible plant cells by strains of Agrobacterium tumefaciens. The expression of bacterial virulence genes is triggered by chemicals present in plant wound exudates. The exudates contain a number of phenolic compounds which act as chemical signals inducing expression of a number of genes directing the DNA transfer process. These are the virulence or vir genes, and vir::lac reporter gene fusions have been widely used to assay vir gene induction in Agrobacterium tumefaciens strains. Using such strains to monitor vir gene expression, Stachel et al. (1985) isolated from Nicotiana tabacum two active acetophenones: 3,5-dimethoxy-4-hydroxyacetophenone, ("acetosyringone" or AS), and α-hydroxy-3,5-dimethoxy-4-hydroxy-acetophenone, ("hydroxyacetosyringone" or HO-AS). However, in vitro assay results suggested that other more common compounds also exhibited activity (Spencer and Towers, 1988). This analysis of structure-activity relationships of induced vir expression in A. tumefaciens was presented in a previous thesis (Paul Spencer, M.Sc. thesis). The results revealed that a variety of commonly occurring plant phenolic compounds were capable of activating vir genes. In addition to the acetophenones, a variety of benzoic and cinnamic acid derivatives, and even a few chalcones of appropriate ring substitution were active. This thesis reports the isolation and identification of a number of these compounds in plant wound exudates. Some Agrobacterium tumefaciens strains are restricted in host range to certain grapevine cultivars. Subsequent to the development of a convenient and sensitive plate-bioassay method, a strongly active component in grapevine wound exudates was purified. A newly described vir-inducing phenolic compound was isolated from a number of Vitis cultivars using gel filtration, thin layer and high pressure liquid chromatographies. This was identified as syringic acid methyl ester (3,5-dimethoxy-4-hydroxybenzoic acid, methyl ester), using mass spectrometry. However, the presence of this compound in grapevine wound exudates does not provide a simple explanation for host range limitation of grapevine strains since it induces vir gene expression in both limited and wide host range strains of A. tumefaciens. Interestingly, neither AS nor HO-AS were present in grapevine-derived extracts. A convenient polyamide column chromatographic method was subsequently developed to permit rapid purification of plant-derived vir gene inducing mixtures, which were detected using the newly developed plate bioassay. Derivatized polyamide fractions were then analysed by combined gas chromatography-mass spectrometry (GC-MS). GC-MS proved to be an ideal means for the identification of the phenolic components in partially purified extracts. Examination of wound exudates from a range of host and non-host species revealed that the production of the acetophenones is restricted to members of the Solanaceae. Some experiments focussed on the biosynthetic precursors of the acetophenones in Nicotiana species. Wound exudates of the majority of species belonging to other plant families contained benzaldehydes and/or benzoic and cinnamic acid derivatives. The induction of virE gene expression was examined in the related Agrobacterium species, A. rhizogenes. To do this, the virE::lacZ gene fusion plasmid pSM358cd was introduced into A. rhizogenes A4 by triparental mating and the strain "A4/pSM358cd" was used to analyze vir activation. Acetophenones, chalcones, benzaldehydes, and benzoic and cinnamic acid derivatives were found to activate vir genes in A. rhizogenes. / Science, Faculty of / Botany, Department of / Graduate

Transcriptional analysis was used to investigate the molecular mechanisms which effect cellulase gene expression in the gram-positive bacterium Cellulomonas fimi strain ATCC 484 and the gram-negative bacterium Agrobacterium sp. strain ATCC 21400. The cenA, cex and cenB genes of C. fimi encoding the extracellular β-1,4-endoglucanase, EngA (EC 3.2.1.4; Mr 48,700), the extracellular β-1, 4-exoglucanase, Exg (EC 3.2.1.91; Mr 47,300) and the extracellular β-1,4-endoglucanase EngB (EC 3.2.1.4; Mr 110,000) respectively, were characterised. By northern blot analysis, cenA mRNA was detected in C. fimi RNA prepared from glycerol- and carboxymethylcellulose (CMC)-grown cells but not in RNA from glucose-grown cells. The cex mRNA was found only in RNA from CMC-grown cells. The cenB mRNA was found in all three preparations of RNA. Therefore, the expression of these genes is subject to regulation by the carbon source provided to C. fimi. High resolution nuclease SI protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, were used to map the 5' termini of cenA and cex mRNAs. Two cenA mRNA 5' ends, 11 bases apart, mapped 51 and 62 bases upstream of the cenA start codon, suggesting that in vivo, cenA transcription was directed from two promoters in tandem. The cex mRNA 5' end was found to map 28 bases upstream of the cex start codon. Using SI mapping with unlabeled DNA probes and C. fimi RNA which had been isolatedin vivo but which had been 5'-labeled in vitro with vaccinia virus capping enzyme confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. The SI mapping revealed mRNA 3' termini 1,438, 1,449, and 1, 464 bases from the major cenA start site, and one 3' terminus 1,564 bases from the major cex mRNA start site, in good agreement with the northern blot data. High resolution SI studies were also used to show that abundant mRNA 5' ends mapped upstream of the cenB start codon in RNA prepared from CMC-grown cells, while less-abundant species mapped 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results seem to indicate a tandem promoter arrangement with an ATG-proximal promoter directing low-level constitutive cenB transcription and a more distal promoter directing higher levels of cenB transcription as a result of C. fimi growth on cellulosic substrate. Steady- state levels were determined for cenA, cex and cenB mRNAs with RNA prepared from glycerol-, glucose-, and CMC-grown cultures of C. fimi in slot-blot hybridisations with radiolabeled oligodeoxyribonucleotide probes. A cex-linked gene (clg) was identified by sequence inspection and SI mapping. Transcripts of the abg gene encoding the β-glucosidase (Abg, EC 3.2.2.21/ Mr 50,000) of Agrobacterium sp. strain ATCC 21400 were also characterised. Northern blot analysis of Agrobacterium RNA revealed the size of the in vivo abgmRNA was approximately 1,500 bases in length. High resolution SI mapping determined abg mRNA 5' ends 22 bases upstream of the abg ATG codon and 3' ends 71 bases downstream of the abg stop codon. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate