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

Olive Mill Wastewater (OMW), by-product of oil industry, is a dark liquid with a characteristic fetid smell, bitter taste and bright appearance / having a high pollution potential, creating serious problems in countries producing olive oil. Azotobacter chroococcum as a Nitrogen-fixing bacteria can bioremediate OMW, by degrading its toxic constituents. With the help of this detoxification process OMW can be used as biofertilizer. In this study, the dynamics of growth and nitrogen fixation at different physiological conditions and nutrient requirements of A. chroococcum in chemically defined N-free medium was determined. These parameters were cultivation conditions such as pH, temperature and aeration and some additives such as inorganic salts, boric acid and nitrogen. Consequently, the maximum cell concentration were obtained when A. chroococcum was grown at neutral pH, 35&amp / #61616 / C, 150 rpm and in medium supplemented with manganese salt at 0.01% concentration. The maximum nitrogen fixation products were attained when A. chroococcum was grown under the same conditions except at pH 8. Further, bioremediation of OMW by A. chroococcum was examined. When A. chroococcum was cultivated in OMW containing basal medium at 10% OMW concentration, a cell density 12 times higher than in the OMW free medium was achieved. Also, it was found to have maximum increase in extracellular protein concentration (112 mg/l) at 10% OMW containing medium and maximum increase in ammonia concentration (9.05 mg/l) at 5% OMW containing medium.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 130-136). / Microfiche. / xi, 136 leaves, bound 29 cm

Forage growth in the subarctic is sub-optimal due to low soil nutrient levels. Forage crops in the Yukon Territory consistently require nitrogen (N) and phosphorus fertilization to meet plant requirements. Fertilization is expensive due to transportation costs and potentially harmful to the environment so alternative, more sustainable, sources of nutrients are being sought. Alfalfa is an alternative, but there is limited knowledge in the Yukon of the benefits and management of this crop as a replacement for fertilizer N. Experiments were carried out in south central Yukon during the 2005 and 2006 field seasons to examine the potential of co-inoculation of alfalfa with N-fixing Ensifer meliloti and phosphate-solubilizing Penicillium bilaii to increase the dry matter yield and N fixation of monoculture alfalfa (Medicago sativa) cv Peace and binary mixed alfalfa with smooth bromegrass (Bromus inermis) cv Carlton or timothy (Phleum pratense) cv Climax forage swards. Interactions between alfalfa inoculation and N fertilization and late season harvest treatments were assessed. The TagTeam® inoculant from Philom Bios was used as the rhizobium source which contains both Ensifer meliloti isolate NRG-34 and Penicillium bilaii isolate PB-50. Nitrogen fixation was determined using the total plant N difference method. Alfalfa growth and nodulation was successful in the trials. Inoculation had a positive impact on N fixation, whereas urea fertilizer at 25 kg N/ha had a negative impact in most cases. In the mixed alfalfa and smooth bromegrass stand there was a positive contribution from the alfalfa in both the establishment and second year with N fixation rates of up to 14 kg/ha. In the mixed timothy and alfalfa stand the N fixation reached 35 kg/ha in the establishment year and 102 kg/ha in the second year. In the establishment year the dry matter yield and N fixation of the TagTeam® inoculated, monoculture alfalfa plots were 3.1 t/ha and 77 kg N/ha. In the second year, the unharvested inoculated alfalfa treatment yielded 3.4 t/ha with N fixation of 66 kg/ha compared to the late harvest treatment which yielded only 1.5 t/ha and an N fixation rate of 20 kg/ha. The effects of the late season harvest are startling and reflect the importance of removing grazing animals during the fall to allow plant energy reserves to accumulate in the roots. Fertilizer N replacement is possible with the seeding of alfalfa into existing hay stands or in monoculture.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23956号 / 農博第2505号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5391(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 准教授 黒田 浩一 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Trifolium ambiguum (Caucasian clover) is being released in New Zealand for use in areas where growth of T. repens (white clover) is marginal. Although closely related to T. repens, T. ambiguum has unique and highly specific nodulation requirements and as rhizobial strains capable of effectively nodulating T. ambiguum are not naturally found in New Zealand soils, they must be introduced with the seed. Rhizobium leguminosarum bv. trifolii strains such as ICC105 form effective nodules on T. ambiguum but ineffective (Fix⁻) nodules on T. repens. The T. repens nodules nevertheless develop normally and contain bacteroids. R. l. bv. trifolii strains that are effective on T. repens such as NZP561, fail to nodulate T. ambiguum. As the host-specific nitrogen fixation defect of Caucasian clover rhizobia on T. repens has potentially adverse agronomic implications, the genetic basis for this Fix⁻ phenotype was investigated. Rhizobium leguminosarum bv. trifolii strain ICC105 was converted to Fix⁺ on T. repens by the introduction of an 18-kb fragment of DNA from a white clover rhizobial strain (NZP514) symbiotic plasmid. This fragment contained several nif and fix genes, including nifHDKEN, fixABCX, nifA, nifB, fdxN and fixU. Tn5 mutation of these white clover rhizobial genes demonstrated that most were required to impart the Fix⁺ phenotype on T. repens to ICC105, with the exception of nifA. Mutagenesis of the ICC105 nifA gene and subsequent complementation with various combinations of the white clover rhizobia nif/fix genes as well as transcriptional lacZ fusion studies of the ICC105 nifA and nifH genes demonstrated that ICC105 nifA is expressed and functional during the ineffective nodulation of T. repens and able to activate expression of nifHDKEN and fixABCX operons derived from white clover rhizobium but not from ICC105. Sequence analysis and comparison of the intergenic region between the divergently transcribed nif/fix operons revealed a conserved 111-bp region found between the nifH/fixA promoters of Caucasian clover rhizobia, but not in white clover rhizobia. Attempts to modify this region in ICC105 failed in creating a strain which was Fix⁺ on T. repens; however recombination of the nifHD/fixAB region from a white clover rhizobium into the ICC105 genome produced several strains with a �swapped� nitrogen fixation phenotype (i.e. Fix⁺ on T. repens and Fix⁻ on T. ambiguum). A hypothesis was therefore proposed by which differences in the nifH/fixA promoter regions of Caucasian clover rhizobia and white clover rhizobia modulate the expression of the upstream genes in response to the particular plant host they are nodulating. The incompatibility between the symbiotic plasmid of R. l. bv. trifolii ICC105 and the white clover rhizobium symbiotic plasmid cointegrate, pPN1, was also investigated and potential regions of each plasmid involved in this incompatibility were identified. The research presented in this thesis has contributed to the genetic knowledge of the nitrogen fixation genes, and regulation of these genes in R. l. bv. trifolii. It has also provided progress towards the goal of creating a suitable inoculant strain for T. ambiguum that is able to fix nitrogen in symbiosis with both T. repens and T. ambiguum.

Bibliography: leaves 209-254. / xv, 254 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Experiments detailed in this thesis, evaluate the impact of various herbicides and herbicide mixtures on the growth, nodulation and nitrogen fixation of annual pasture legumes, the efficacy of the herbicides for weed control, and the potential role of mechanical defoliation to replace herbicides, leading to lower cost and more sustainable farming systems. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1996

Bibliography: last 24 unnumbered leaves / 161, [32] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1985

[Truncated abstract] Some plants, such as legumes, are able to use atmospheric nitrogen as a nitrogen source due to the nitrogen-fixing bacteria residing in specialised root structures called nodules. The exchange of carbon and nitrogen between the host plant (legume) and the nitrogen- fixing micro-symbiont is vital for biological nitrogen fixation. In particular, transport of C4-dicarboxylates, mainly malate, from the plant to the micro-symbiont, and the reverse transport of fixed nitrogen in the form of ammonium are essential for symbiotic nitrogen fixation. In the legume nodule, the symbiosome membrane (SM) surrounds the bacteroid and all exchanges of metabolites and nutrients that occur between the plant and the micro-symbiont must cross this membrane. Recently it has been established that cycling of amino acids across the SM is also critical for optimal symbiotic nitrogen fixation. Therefore to fully understand this agriculturally significant phenomenon, the mechanisms facilitating these exchanges need to be investigated. The major aim of this study was to increase the understanding of nutrient exchange within the nodule at the molecular level by isolating and characterising genes encoding transporters responsible for malate and amino acids transport in soybean (Glycine max, L.), with particular interest in genes significantly or highly expressed in nodules. A combination of molecular and biochemical techniques was used to achieve this. ... Southern blot analysis showed that a small gene family of up to five members encodes these proteins in soybean. A full-length cDNA, designated GmAAP5, was isolated that encodes a novel, putative amino acid transporter. Molecular characterisation of this cDNA and that of GmAAP1 (GenBank Accession no: AY029352), a previously identified putative amino acid transporter gene, was done. Expression analyses showed relatively high expression of GmAAP5 in soybean nodules compared to that in leaf and root tissues, while GmAAP1 showed uniformly high expression in root, leaf and nodule tissues. Phylogenetic analysis of the deduced amino acid sequences of known functional AAPs from dicotyledonous plants revealed that GmAAP1 is most closely related to AAP2 from V. faba, while GmAAP5 is more closely related to AAPs from non-leguminous plants than from leguminous plants. Based on the functional characterisation of the AAPs with which GmAAP1 and GmAAP5 cluster, it is likely that both transporters are neutral and acidic amino acid transporters within the AAP subfamily.

Biogeochemical processes in the marine biosphere are important in global element cycling and greatly influence the gas composition of the Earth’s atmosphere. The nitrogen cycle is a key component of marine biogeochemical cycles. Nitrogen is an essential constituent of living organisms, but bioavailable nitrogen is often short in supply thus limiting primary production. The largest input of nitrogen to the marine environment is by N2-fixation, the transformation of inert N2 gas into bioavailable ammonium by a distinct group of microbes. Hence, N2-fixation bypasses nitrogen limitation and stimulates productivity in oligotrophic regions of the marine biosphere. Extensive blooms of N2-fixing cyanobacteria occur regularly during summer in the Baltic Sea. N2-fixation during these blooms adds several hundred kilotons of new nitrogen into the Baltic Proper, which is similar in magnitude to the annual nitrogen load by riverine discharge and more than twice the atmospheric nitrogen deposition in this area. N2-fixing cyanobacteria are therefore a critical constituent of nitrogen cycling in the Baltic Sea. In this thesis N2 fixation of common cyanobacteria in the Baltic Sea and the direct fate of newly fixed nitrogen in otherwise nitrogen-impoverished waters were investigated. Initially, the commonly used 15N-stable isotope assay for N2-fixation measurements was evaluated and optimized in terms of reliability and practicality (Paper I), and later applied for N2-fixation assessments (Paper II–IV). N2 fixation in surface waters of the Baltic Sea was restricted to large filamentous heterocystous cyanobacteria (Aphanizomenon sp., Nodularia spumigena, Dolichospermum spp.) and absent in smaller filamentous cyanobacteria such as Pseudanabaena sp., and unicellular and colonial picocyanobacteria (Paper II-III). Most of the N2-fixation in the Northern Baltic Proper was contributed by Aphanizomenon sp. due to its high abundance throughout the summer and similar rates of specific N2-fixation as Dolichospermum spp. and N. spumigena. Specific N2 fixation was substantially higher near the coast than in an offshore region (Paper II). Half of the fixed nitrogen was released as ammonium at the site near the coast and taken up by non-N2-fixing organisms including phototrophic and heterotrophic, prokaryotic and eukaryotic planktonic organisms. Newly fixed nitrogen was thereby rapidly turned-over in the nitrogen-depleted waters (Paper III). In colonies of N. spumigena even the potential for a complete nitrogen cycle condensed to a microcosm of a few millimeters could be demonstrated (Paper IV). Cyanobacterial colonies can therefore be hot-spots of nitrogen transformation processes potentially including nitrogen gain, recycling and loss processes. In conclusion, blooms of cyanobacteria are instrumental for productivity and CO2 sequestration in the Baltic Sea. These findings advance our understanding of biogeochemical cycles and ecosystem functioning in relation to cyanobacterial blooms in the Baltic Sea with relevance for both ecosystem-based management in the Baltic Sea, and N2-fixation and nitrogen cycling in the global ocean. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>