Dissertations / Theses on the topic 'NifHDK genes'

This project sought to determine the response of Leptospirillum species subject to conditions without soluble nitrogen. The most significant outcome of this work was the identification of the nifHDK genes in L. ferriphilum and observations of its continued ability to survive in environments without soluble nitrogen. The sustained proliferation of all three Leptospirillum species studied, combined with the structural analysis of these genes, provides evidence of functional nitrogen fixation in each of these species.

Acetobacter diazotrophicus, a nitrogen-fixing bacterial endophyte, is believed to be responsible for biological nitrogen fixation (BNF) in sugarcane. However, no monocot has yet been unequivocally shown to receive fixed N through BNF. The main question addressed in this dissertation is whether A. diazotrophicus promotes plant growth, and if so, whether growth promotion is via BNF. Another question is whether the growth benefits can be extended to other grasses. To answer these questions, the nifHDK genes encoding the protein subunits of the nitrogenase enzyme were first isolated and sequenced. Secondly, Nif⁻ mutant strains were constructed by inserting a gene cassette in nifD. The growth of sugarcane plants inoculated with A. diazotrophicus wild type and Nif⁻ mutant strains were compared in growth chamber, greenhouse, and field experiments. A. diazotrophicus was also tagged with marker genes to investigate the colonization process in sugarcane and other grasses. The effect of A. diazotrophicus on the growth of other grasses was also determined. Analysis of the A. diazotrophicus NifHDK sequence revealed features typical of proteobacterial nifHDK genes and gene products. Phylogenetic analysis established the close relationship of A. diazotrophicus with the α-proteobacteria and the β-proteobacterium, Herbaspirillum seropedicae, another sugarcane endophyte. Nif⁻ mutant strains established endophytically in sugarcane plants equally well as wild type strains. ¹⁵N₂ incorporation experiments demonstrated that wild type strains but not the Nif⁻ mutants fixed N inside sugarcane plants with decreased fixation when plants were grown in medium with fixed N. In N-deficient conditions, sugarcane inoculated with wild type strains grew better and had higher total N content than either uninoculated or plants inoculated with Nif⁻ mutants. When N was not limiting, growth enhancement was observed in plants inoculated with either wild type or the Nif⁻ mutants. These results suggest that depending on the nitrogen condition, A. diazotrophicus promotes sugarcane growth via nitrogen fixation and other growth promoting factor. The results also indicated a possible strain-cultivar specificity in growth promotion. A. diazotrophicus colonized other grasses through different entry sites but was limited in the root. Under N-deficient conditions, wild type strain but not the Nif-- mutant promoted rice seedling growth indicating the beneficial effects of A. diazotrophicus to other grasses.

The microbial life of sub-Antarctic islands plays a key role in the islands ecosystem, with microbial activities providing the majority of nutrients available for primary production. Knowledge of microbial diversity is still in its infancy and this is particularly true regarding the diversity of micro-organisms in the Antarctic and sub-Antarctic regions. One particularly important functional group of micro-organisms is the diazotrophs, or nitrogen-fixing bacteria and archaea. This group have not been well studied in the sub-Antarctic region, but play an important role in the nutrient cycling of the island. This thesis explored the diversity of nitrogen-fixing organisms in the soil of different ecological habitats on the sub-Antarctic Marion Island.

Sequence analysis of the entire 3.5 kb HindIII genomic DNA fragment previously isolated from Nostoc commune UTEX 584 (Defrancesco and Potts 1988), determined the exact locations of the nifU, nifH, and nifD genes and identified two potential stem loop structures, a direct repeat, and an ORF that codes for a protein with a predicted amino acid sequence similar to that of myoglobin from Paramecium caudatum. The N. commune UTEX 584 myoglobin-like protein has a predicted length of 118 amino acids and molecular mass of 12,906 Da. A PCR copy of the gene (glbN) was cloned for overexpression of the protein. The recombinant protein was purified and used for spectral analysis and for the production of polyclonal antisera. Treatment of the recombinant protein with dithionite and CO resulted in spectral shifts characteristic of hemoproteins that bind oxygen. While some of the spectral characteristics are unique to the protein, in general the spectra were more like those of globins than cytochromes. Based on these characteristics and the sequence similarity to the P. caudatum mnyoglobin, we proposed the name cyanoglobin, with the gene designation glbN and the protein designation GlbN. Western analysis of GlbN expression was performed on N. commune UTEX 584 and two species of Anabaena (Anabaena sp. PCC 7120 and Anabaena variabilis). In N. commune UTEX 584 a protein with a molecular mass similar to that predicted for GlbN was detected. This protein was produced in increased amounts under the same growth conditions that resulted in increased production of nitrogenase reductase (the nifH gene product). No proteins of similar size to GlbN were detected in Anabaena sp. PCC 7120 or A. variabilis. A possible function of GlbN may be for oxygen storage, transport, or protection of the nitrogenase system. These functions as well as those of the direct repeat and the potential stem loop structures and their relationship to nitrogen fixation or other physiological processes in N. commune UTEX 584 require further analysis.
Ph. D.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A crescente demanda de alimentos causada pelo aumento populacional, aliado a alto custo de fertilizantes industrializados e impacto ambiental causado por eles leva, mundialmente, à utilização em grande escala de inoculantes de bactérias fixadoras de nitrogênio. Os inoculantes utilizados no Brasil (coleção SEMIA- IPAGRO) ainda não estão suficientemente caracterizados geneticamente. O objetivo deste trabalho foi avaliar e confrontar as seqüências parciais dos genes 16S rDNA e nifH de 26 estirpes padrões já classificadas, com 70 estirpes de rizóbios recomendadas e autorizadas para a produção de inoculantes no Brasil. Para esta finalidade, a partir das amostras de DNA extraídos destas bactérias foram realizadas reações de PCR com oligonucleotídeos iniciadores relativos à região codificadora do gene 16S rDNA e do nifH, sendo então seqüenciadas com o objetivo de detectar diferenças nucleotídicas entre as diferentes bactérias estudadas. Para a comparação dos resultados do seqüenciamento com a consulta de similaridade de nucleotídeos no BLASTn, foram geradas árvores filogenéticas através de ferramentas de bioinformática. Foi observado que a classificação taxonômica das estirpes SEMIA recomendadas para inoculação de leguminosas previamente disponível na FEPAGRO, com base em propriedades morfológicas e especificidade hospedeira, não foi confirmada em todas as estirpes pelos sequenciamentos parciais dos genes estudados. Sugerimos revisão da classificação destas estirpes. Concluímos também que a consulta de similaridade ao BLASTn pelo seqüenciamento parcial dos genes 16S rDNA e nifH é, na maioria dos casos, consistente com a classificação proposta pela construção de árvores filogenéticas destas sequências. Estas ferramentas apresentaram-se muito confiáveis para obtenção de classificação em nível de gênero das estirpes estudadas.
The growing demand for food caused by population growth, combined with high cost of fertilizers and industrial environmental impacts caused by them leading, worldwide, the large scale use of inoculants different nitrogen-fixing bacteria. The inoculants used in Brazil (SEMIA-IPAGRO collection) are not yet sufficiently characterized genetically. The purpose of this study was to evaluate and compare the sequences of partial 16S rDNA and nifH of 26 strains already classified, with 70 strains of rhizobia recommended and authorized for the production of inoculants in Brazil. For this purpose, from DNA samples taken from these bacteria were performed with PCR reactions with primers on the coding region of the gene 16S rDNA and nifH, and sequencing with the aim of detecting nucleotide differences between different bacteria studied. To compare the results of the consultation of similarity of sequences of nucleotides in BLASTn, phylogenetic trees were generated through bioinformatics tools. It was observed that the taxonomic classification of strains SEMIA recommended for inoculation of legumes previously available in FEPAGRO, based on morphological properties and host specificity, it wasn’t confirmed in all strains by partial sequencing of the genes studied. We suggest reviewing the classification of these strains. We concluded that the similarity of the consultation BLASTn by partial sequencing of 16S rDNA and nifH is, in most cases, consistent with the classification proposed by the construction of phylogenetic trees of these sequences. These tools were very reliable for obtaining classified in the genus level of strains studied.

Orientadora: Eliana Gertrudes de Macedo Lemos
Banca: Maria José Valarini
Banca: Jaime Maia dos Santos
Resumo: A crescente demanda de alimentos causada pelo aumento populacional, aliado a alto custo de fertilizantes industrializados e impacto ambiental causado por eles leva, mundialmente, à utilização em grande escala de inoculantes de bactérias fixadoras de nitrogênio. Os inoculantes utilizados no Brasil (coleção SEMIA- IPAGRO) ainda não estão suficientemente caracterizados geneticamente. O objetivo deste trabalho foi avaliar e confrontar as seqüências parciais dos genes 16S rDNA e nifH de 26 estirpes padrões já classificadas, com 70 estirpes de rizóbios recomendadas e autorizadas para a produção de inoculantes no Brasil. Para esta finalidade, a partir das amostras de DNA extraídos destas bactérias foram realizadas reações de PCR com oligonucleotídeos iniciadores relativos à região codificadora do gene 16S rDNA e do nifH, sendo então seqüenciadas com o objetivo de detectar diferenças nucleotídicas entre as diferentes bactérias estudadas. Para a comparação dos resultados do seqüenciamento com a consulta de similaridade de nucleotídeos no BLASTn, foram geradas árvores filogenéticas através de ferramentas de bioinformática. Foi observado que a classificação taxonômica das estirpes SEMIA recomendadas para inoculação de leguminosas previamente disponível na FEPAGRO, com base em propriedades morfológicas e especificidade hospedeira, não foi confirmada em todas as estirpes pelos sequenciamentos parciais dos genes estudados. Sugerimos revisão da classificação destas estirpes. Concluímos também que a consulta de similaridade ao BLASTn pelo seqüenciamento parcial dos genes 16S rDNA e nifH é, na maioria dos casos, consistente com a classificação proposta pela construção de árvores filogenéticas destas sequências. Estas ferramentas apresentaram-se muito confiáveis para obtenção de classificação em nível de gênero das estirpes estudadas.
Abstract: The growing demand for food caused by population growth, combined with high cost of fertilizers and industrial environmental impacts caused by them leading, worldwide, the large scale use of inoculants different nitrogen-fixing bacteria. The inoculants used in Brazil (SEMIA-IPAGRO collection) are not yet sufficiently characterized genetically. The purpose of this study was to evaluate and compare the sequences of partial 16S rDNA and nifH of 26 strains already classified, with 70 strains of rhizobia recommended and authorized for the production of inoculants in Brazil. For this purpose, from DNA samples taken from these bacteria were performed with PCR reactions with primers on the coding region of the gene 16S rDNA and nifH, and sequencing with the aim of detecting nucleotide differences between different bacteria studied. To compare the results of the consultation of similarity of sequences of nucleotides in BLASTn, phylogenetic trees were generated through bioinformatics tools. It was observed that the taxonomic classification of strains SEMIA recommended for inoculation of legumes previously available in FEPAGRO, based on morphological properties and host specificity, it wasn't confirmed in all strains by partial sequencing of the genes studied. We suggest reviewing the classification of these strains. We concluded that the similarity of the consultation BLASTn by partial sequencing of 16S rDNA and nifH is, in most cases, consistent with the classification proposed by the construction of phylogenetic trees of these sequences. These tools were very reliable for obtaining classified in the genus level of strains studied.
Mestre

Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Biological Sciences.
"May 2007." Title from PDF title page (viewed on Dec. 4, 2007). Thesis adviser: Mark A. Schneegurt. Includes bibliographic references (leaves 63-72).

Hydrogen, which is a clean energy source, is one of the alternatives for fossil fuels. Biological hydrogen production is one of the hydrogen production methods. Rhodobacter capsulatus is a photosynthetic bacterium that produces hydrogen via photofermentation. R. capsulatus can also synthesize some valuable by-products such as Poly-beta- hydroxy butyric acid (PHB), which is a biodegradable bioplastic. In a two stage biohydrogen production system, which is combination of dark fermentation and photofermentation, dark fermentor effluents are used for photofermentation by R.capsulatus. Dark fermentor effluents usually contain high amount of acetate. High amount of acetate may decrease the efficiency of hydrogen production by causing high amount of PHB production. Therefore, it is significant to determine optimum acetate concentration for photofermentation. In this study, the effects of acetate concentration on hydrogen and PHB production by R.capsulatus were investigated by growing bacteria at various acetate concentrations (10 mM-65 mM). In addition, gene expression analysis was performed to investigate the effects of acetate at transcriptional level. For this purpose, expression levels of the genes that encode nitrogenase which is the enzyme that catalyzes hydrogen production and PHB synthase, which is the key enzyme of the PHB synthesis pathway, are examined. Optimum acetate concentration for photofermentation with high hydrogen yield and low PHB amount was determined to be in the range 25 mM-50 mM. nifD expression was found to be high at optimum acetate concentrations and phaC expression was found to be the highest at 65 mM.

Chlorbenzole sind aufgrund ihrer weiten Verbreitung in Industrie und Landwirtschaft und ihrer geringen chemischen Reaktivität sowie guten Fettlöslichkeit ubiquitäre Umweltkontaminanten, die sich in der Nahrungskette anreichern. Eine natürliche, mikrobielle Dechlorierung dieser Verbindungen ist von besonderem Interesse, da die Toxizität chlorierter Benzole mit der Anzahl der Chlorsubstituenten steigt. Im Gegensatz zu Mono- und Dichlorbenzol, die durch aerobe Laborreinkulturen dechlorierbar sind, werden höher chlorierte Benzole bevorzugt unter anaeroben Bedingungen in Mischkulturen mit unbekannter Spezieszusammensetzung dechloriert. Bioreaktoren, in denen solche undefinierten Mischkulturen kontinuierlich kultiviert werden, sind eine vielversprechende Technik bei der Behandlung Chlorbenzol-kontaminierter Abwässer. Aufgrund der unbekannten Zusammensetzung der Population muß die mikrobielle Aktivität jedoch als "black box" betrachtet werden, was eine direkte Steuerung und Optimierung solcher Bioreaktoren erschwert. Zur Bestimmung der mikrobiellen Diversität einer in ihrer Zusammensetzung unbekannten, Trichlorbenzol(TCB)-dechlorierenden Bioreaktorkultur wurde aufgrund der bekannten Limitierungen kulturabhängiger Methoden die kulturunabhängige, vergleichende Sequenzanalyse direkt amplifizierter und klonierter 16S-rDNA gewählt. Die durch eine neuentwickelte Hybridisierungsmethode wesentlich vereinfachte Analyse der 16S-rDNA-Genbibliotheken erlaubte eine phylogenetische Klassifizierung der in der TCB-dechlorierenden Mischkultur abundanten Mikroorganismen (Bakterien und Archaea) und zeigte das Auftreten von 51 bakteriellen 16S-rDNA-Klonfamilien, mit einem weiten phylogenetischen Spektrum und teilweise enge Verwandtschaften zu anaerob dechlorierenden Dehalobacter spp. oder zu unkultivierten Bakterien eines vergleichbaren Biotops. Dieser Diversität stand eine dominierende Methanosaeta-ähnliche Klonfamilie innerhalb der Archaea gegenüber. Die aus der phylogenetischen Klassifizierung abgeleiteten metabolischen Eigenschaften einiger Bakterien und Archaea der TCB-dechlorierenden Mischkultur konnten durch gezielte Anreicherung und in-vitro Kultivierung bzw. die kulturunabhängige Sequenzanalyse funktioneller Gene bestätigt werden. Die dargestellten Ergebnisse lassen eine spezifische Zusammensetzung der TCB-dechlorierenden Mischkultur vermuten und geben Hinweise auf Indikatororganismen, die ein Monitoring und eine damit verbundene Effizienzsteigerung der anaeroben TCB-Dechlorierung im Bioreaktor ermöglichen könnten.
Due to their widespread application in industry and agriculture and their chemical stability chlorobenzenes (CB) are ubiquitous pollutants in soil, sediments, and aquifers. Since toxicity of CBs increases with the number of chlorine substituents, microbial dechlorination of CBs is of major interest. In contrast to di- and monochlorobenzenes (DCB and MCB) higher chlorinated benzenes are more resistant to aerobic dechlorination. However, for these compounds reductive dechlorination by different anaerobic microbial communities is well known. Bioreactors inoculated with complex dechlorinating anaerobic microbiota seem to be promising technologies for bioremediation of CB-contaminated aquifers. Several studies showed the efficiency of such bioreactors in treating chloroaromatic contaminated wastewaters. However, due to the unknown species diversity microbial activity had to be treated as a "black box" and direct optimization was hampered. To determine the microbial diversity of an anaerobic consortium in a fluidized bed reactor used for dechlorination of trichlorobenzene (TCB) I employed comparative sequence analysis of 16S rRNA genes after direct PCR-amplification and cloning from community DNA since culture-based methods have shown to be strongly biased. The application of a new hybridization based screening approach for bacterial 16S rDNA clone libraries drastically simplified the analysis and allowed the phylogenetic classification of the abundant bacteria and archaea. A total of 51 bacterial 16S rDNA clone families were found, which showed a wide distribution among the main bacterial phyla. Several bacterial 16S rDNA clone families were closely related to anaerobic, dechlorinating Dehalobacter spp. and to yet-uncultured bacteria of a similar habitat. In contrast to the high bacterial diversity archaeal 16S rDNA clone libraries were clearly dominated by a Methanosaeta concilii-like clone family. Some yet-uncultured bacteria and archaea of the TCB-dechlorinating consortium were sufficiently closely related to studied organsims that reasonable physiological hypotheses could be formulated. These hypotheses were confirmed by either cultivation of the respective organism or by culture-independent sequence analysis of specific functional genes. The results suggest a specific community structure of the TCB-dechlorinating consortium and give evidence for indiator organisms. Moleculargenetic monitoring of these indicator organisms might allow the optimization of the continous TCB-dechlorination in the fluidized bed reactor.

Das Pflanzenwachstum fördernde Bakterien (PGPB) kommen ubiquitär sowohl an der Wurzel als auch am Spross der Pflanzen vor und sie können über direkte oder indirekte Mechanismen einen bedeutenden Beitrag zur Stickstoffernährung der Pflanzen leisten. Die vorliegende Arbeit umfasst a) die Isolierung von PGPB, welche das Wachstum verschiedener Pflanzenarten fördern und durch Fusarien verursachte Pflanzenkrankheiten bekämpfen, b) die Analyse der Möglichkeiten Probleme der Pflanzenernährung durch den Einsatz von PGPB zu lösen, c) die Entwicklung neuer molekularbiologischer Methoden zur Messung der Diversität und Aktivität der PGPB. Im Rahmen dieser Arbeit wurden Methoden zur Beschreibung der Diversität von rhizosphären PGPB entwickelt und verbessert um Verbindungen zwischen applizierten PGPB und deren Aktivitäten zu prüfen. Die sensitive quantitative real-time-PCR Methode wurde zur Quantifizierung bzw. zum Nachweis der inokulierten PGPB und zum Nachweis des nitrogenase-reduktase-Gens (nifH), des Markergens für potentiell diazotrophe Bakterien. Bakterienartspezifische Primer wurden aus dem Sequenzvergleich der 16S-23S ISR ausgewählter Bakterienstämme selektiert und Protokolle zur Quantifizierung dieser Bakterienarten erarbeitet. Die nifH Gen Quantifizierung an Pflanzen eröffnet die Möglichkeit Schlüsselorganismen in der assoziativen biologischen Luftstickstoffbindung zu identifizieren und kurzfristige Reaktionen der Bakteriengesellschaften auf Umweltveränderungen und Regulationsmechanismen in situ zu analysieren.
Plant growth promoting bacteria (PGPB) are ubiquitous in both plant root and shoot, and are important contributors to the nitrogen-input of plants exerting their positive effects on plant growth directly or indirectly through different mechanisms. The present work focuses on a) the isolation of PGPB, which promotes the growth of different plant cultures and controls plant diseases caused by Fusarium species, b) the prospects of PGPB to solve plant nutritional problems, c) developing new molecular methods for the assessment of their diversity and activity. In the frame of this thesis, the methods for the description of the diversity of root colonizing PGPB have been developed and improved to provide links between introduced PGPB abundance and activities. The approach used was based on the sensitive real – time PCR detection/quantification of introduced PGBP and the nitrogenase reductase gene (nifH), which served as a marker gene for potential diazotrophs. The amplified 16S-23S ISR sequences of studied bacteria were subjected to strain – specific primer design and a highly specific bacteria quantification protocol were developed. The bacteria quantification protocol was based on real – time PCR using strain specific primers in order to evaluate the colonization ability of studied bacteria, which were inoculated to plant roots. The results presented in this thesis have shown that monitoring of nifH amount in plant root is a suitable and promising approach to link inoculated diazotrophic bacteria abundance and its potential activity. The study of nifH gene abundance in plant offers the opportunity to identify key players in asymbiotic nitrogen fixation, to study short-term community responses in changing environments, or to analyze the effect of regulation in situ.

Ecosystem processes drive biogeochemical cycles that influence input and losses of nutrients in the biosphere. Through human activities the environment has been highly enriched with nutrients, especially nitrogen. In most ecosystems, nitrogen availability should be limited, but soils and aquatic ecosystems have been anthropogenically impacted. In streams, availability of nutrients, geochemical characteristics, hydrodynamics, and human activities influence the metabolic activities and structure of microbial communities. The aim of the current study is to contrast gene abundance and metabolic responses of N2-fixing guilds exposed to chronic nitrogen loading in three different types of Kansas prairie streams: urban, agricultural, and pristine. Nitrogen-fixation activity was expected to be negatively correlated to the level of fixed nitrogen, while nifH (nitrogenase gene) abundance would be unchanged. A combination of process-level and molecular techniques were applied to study nitrogen fixation in these small prairie streams. Nitrogen fixation activity was measured with acetylene reduction assays. Rates of acetylene reduction for urban, agricultural, and pristine prairie streams were 1.5 to 93.5, 2.1 to 112.8, and 2.9 to 81.9 fmol N2/g soil/h, respectively. The highest rates were found in leaf litter, sediments and algal bio films. Samples of sediments and leaf litter were field-frozen for molecular analyses of the nitrogen-fixing microbial guild. Direct DNA extracts were examined by SYBR real-time PCR to determine the abundance of nifH, given a detection limit of 2 x 102 nifH gene copies/g sample. The abundance of bacterial 16S rRNA was between 1.0 x 106 to 1.0 x 1012 gene copies per gram to 1.0 x 10gene copies per gram sample. The abundance of nifH genes ranged between 1.0 x 10to 1.0 x 10gene copies per gram in all streams. The assay was quantitative over at least 8 orders of magnitude, from 1 ng to 0.1 pg of nifH target. This study provides a link between the abundance of nifH genes and nitrogen-fixation activity. An understanding of the effects of nitrogen pollution on nitrogen cycling guilds in small streams will increase our ability to overcome the challenges of nutrient pollution. This work was supported by Kansas NSF EPSCoR Ecological Genomics.
Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Biological Sciences

碩士
國立中山大學
海洋資源學系研究所
93
This research investigated the existence of <10 μm nitrogen-fixing unicellular cyanobacteria in the South China Sea. The surveys covered the period from February 2004 to January 2005 and a total of seven cruises. The unicellular cyanobacteria that express orange-yellow from cellular phycoerythrin were observed under a fluorescence microscope. Their expressions in nitrogen-fixation were confirmed by the results from reverse transcription polymerase chain reaction (RT-PCR) and whole cell fluorescence immunolocalization of nitrogenase. The nifH gene sequences of the unicellular cyanobacteria collected from the South China Sea was with >90% identities of their nucleotides similar to the nifH gene sequences of unicellular diazotrophs from ALOHA (Hawaii) as well as Synechocystis sp. WH 8501, Cyanothece sp. ATCC 51142, Cyanothece sp. WH 8902, Cyanothece sp. WH 8904 and Synechococcus sp. RF-1. Positive reactions of fluorescence immunolocalization of nitrogenase were only observed in some, not all, of <10 μm unicellular cyanobacteria, suggesting that cell counting alone can not be used to estimate nitrogen fixation rate. There was great seasonal and spatial variation in the unicellular cyanobacteria cell density. There was, however, no significant relationship between cell density and the investigated environmental factors. Cell density was high when temperature was high or where stratification index of water column was high, such as in summer or in basin in contrast to other seasons or the shelf-slope regions.

博士
國立中興大學
土壤環境科學系所
98
Long-term fertilizer application are known to cause changes in soil physical, chemical properties and microbial population. Biological nitrogen fixation is an important process by soil microorganisms, not only to help plant growth but also maintain the balance of natural nitrogen cycle. Rice is the most important food crop in the world, including Taiwan. Many of nitrogen-fixing bacteria associated with rice rhizosphere by the accumulation of root exudates have been selected and published, but research about nitrogen-fixing bacteria on the rhizosphere under different fertilization management is rare. The purpose of this study was to test the hypothesis that in paddy soil, nitrogen-fixing bacterium population would be changed considerably after a long-term fertilizer application. Unculturable methods was used to study the composition of the nifH gene in rice rhizosphere at different growth stages after long-term (ten years) chemical fertilizer and swine manure compost application, and the potential factors influencing the population was proposed. The results showed that nitrogen-fixing activities of rice rhizosphere under different fertilizer managements were not significant different, but there were significant changes under different growth stages. Restriction enzyme fragment length polymorphism analysis (RFLP) also showed different nifH gene profiles at different growth stages, but not significantly different among fertilization treatments. Cloning technique was thus used to sequence the nifH gene, The results indicated growth stage, significant change of the rhizospheric nifH pool under long-term applications of fertilizers. Several nifH genes disappeared in some fertilization treatments. nifH gene compositions at different growth stages were similar in different fertilization treatments but varied in from dominancy. Although RFLP method could not distinguish the nifH gene profiles under the different fertilizations, cloned nifH gene sequence revealed significantly different among fertilization treatments. These results support the hypothesis that the nitrogen fixing bacterium population changed considerably after long-term fertilizer application. The study also established the first nifH gene database in Taiwan paddy soil, and found that there are a large number of newly discovered nifH gene unknown.