Sinorhizobium meliloti is a model bacterium for the study of symbiotic nitrogen fixation (SNF). It infects the roots of alfalfa as well as some other legumes and differentiates into N2-fixing bacteroids within the plant cells of specialized nodule organs. To understand genes essential for SNF and, in the longer term, to facilitate the manipulation of this SNF process for agricultural purposes, it is highly desirable to construct the minimal genome for SNF in this organism. S. meliloti harbors two replicons required for SNF, a 1.7-Mb chromid (pSymB) and a 1.4-Mb megaplasmid (pSymA). A previous deletion analysis revealed that only four gene regions, accounting for <12% of the total sequences of pSymA and pSymB that, were essential for SNF. In the first part of the thesis, I report the cloning of these two pSymA SNF-essential regions on a plasmid (pTH3255) in Escherichia coli, and the integration of this plasmid into the genome of a ∆pSymA S. meliloti derivative strain (the strain was named as RmP4291 after integration). Plant root dry weight and nitrogenase-catalyzed acetylene reduction assays were carried out on RmP4291 with four host plants, including Medicago sativa, Medicago truncatula, Melilotus alba and Melilotus officinalis. Nodule kinetic assays were also performed on RmP4291 and RmP110(wt). The results showed that the SNF-essential regions from pSymA were sufficient to restore the symbiotic capabilities to the ∆pSymA derivative strain with all the host plants tested, except a significant reduction (~40%) in SNF by RmP4291 was noticed on M. officinalis compared to that by wildtype S. meliloti. A higher alfalfa nodulation efficiency of RmP4291 compared to that of wildtype RmP110 was also discovered. In the second part of the thesis, a histochemical staining method for S. meliloti nodules was developed by integrating the marker genes gusA (β-glucuronidase) and celB (β-glucosidase) into the S. meliloti genome. This staining method was found to be useful in the study of nodule competitiveness. A nodule competition assay was carried out between RmP4291 and RmP110 using the new staining method. RmP4291 was found to be significantly reduced in nodulation competitiveness compared to wildtype S. meliloti. The development of the histochemical staining method for S. meliloti nodules will accelerate the identification of genes required for nodule competitiveness in the organism, which will be of crucial importance to the construction of the minimal genome strains with high SNF efficiency. / Thesis / Master of Science (MSc) / Nitrogen is one of the critical elements for life. Biological nitrogen fixation plays a crucial role in providing fixed nitrogen for the ecosystem on Earth. Our Laboratory has endeavored to establish a minimal symbiotic genome in Sinorhizobium meliloti, a model nitrogen fixing bacterium which forms symbiosis with certain kinds of legumes. Building this minimal symbiotic genome will improve our understanding of the symbiotic nitrogen fixation process in S. meliloti at gene level. It may also help in eventually introducing a nitrogen fixation system into other organisms. In this study, the minimal symbiotic genome of the pSymA replicon in S. meliloti was constructed. In addition, a staining method to detect specific S. meliloti strains in nodules was established. This method is potentially useful in finding genes related to nodule competitiveness, and these are potentially important for augmenting the genes that constitute the minimal symbiotic genome.

Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein's interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997). Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion. Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may "fine-tune" the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates. / Ph. D.

The herbicide simazine (2-chloro-4,6-bis(ethylamino)- 1,3,5-triazine) was found to have an inhibitory effect on nitrification in pure and mixed cultures of the nitrifying bacteria. Simazine concentrations of 6 and 9ppm inhibited the rate of nitrification in soil perfusion units, and an abnormally high level of nitrite nitrogen was observed in the herbicide treatments. The herbicide had no effect on the growth of Nitrosomonas europaea in shake flask culture, but did inhibit the growth of Nitrobacter agilis. The addition of yeast extract to the culture medium did not reverse the inhibition. Simazine did not inhibit the respiration of N. agilis even at a 100ppm concentration. Five organisms were isolated which could utilize simazine as a sole source of carbon and nitrogen. Pure cultures of these organisms were made and several were identified. / Master of Science

The products of nifE and nifN from Azotobacter vinelandii, which are involved in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) co) from nitrogenase, have been analyzed using a variety of mutagenic techniques. NifE was the object of several site-specific, amino acid substitutions that were designed to elicit information regarding metal cluster ligands, subunit-subunit interactions, and the proposed transfer of FeMo-co.from a nifEN-products complex to the apo-MoFe protein. A model of metal cluster binding; regions within the nifEN-products is discussed insofar as it relates to the rationale for the targeting of particular amino acids for-substitution. A translational fusion between nifN and lacZ was constructed and used to study the regulation of nifEN. This gene fusion was regulated in the same manner as wild type nifN and produced a fusion protein which was enzymatically active with respect to substrates of β-galactosidase. Results from mutant strains which carry lesions in nifH or nifA in addition to the nifN / Master of Science

Previous studies revealed that α-275-Cys provides an essential ligand to one of the Fe atoms on the FeMo-cofactor, and its substitutions resulted in inactive nitrogenase. In order to study the structural-functional relationship of the protein environment in this region with respect to the FeMo-cofactor, subtle changes were introduced through substitutions using a site-directed mutagenesis and gene-replacement method at α-274-His, α-276-Tyr and α-277-Arg in Azotobacter vinelandii nitrogenase. Characterization of mutants strains resulting from amino acid substitutions at residues, α-274-His, α-276-Tyr or α-277-Arg, using activity assays, resulted in mixed Nif phenotypes. Therefore, none of these residues is absolutely required for nitrogenase activity. However, the changed EPR spectra of the altered MoFe proteins from some strains with substitutions at either α-276-Tyr or α-277-Arg indicated that the FeMo-cofactor environment had been perturbed by these substitutions. Together with its changed EPR spectrum, substituting α-277-Arg with His showed some extraordinary catalytic features, such as its inability to reduce N₂ while retaining respectable C₂H₂- and H⁺-reduction activities. It was also found that this altered protein used a higher percentage of total electron flux for H₂ evolution under an C₂H₂/Ar atmosphere than did wild type. Further characterization of the purified α-277^his MoFe protein in parallel with its wild type counterpart revealed that the alteration in the α-277^his MoFe protein caused a lower affinity for C₂H₂ binding, whereas it did not affect the CO binding. Interestingly, CO-induced cooperativity during C₂H₂ reduction was observed in this altered MoFe protein clearly indicating two sites for C₂H₄ evolution, one of which might be in the vicinity of this residue. Furthermore, the α-277^his MoFe protein does not bind or reduce N₂ leading to the proposal of a nonexistent E₄ redox state in the MoFe protein catalytic cycle which was supported by stopped-flow spectrophotometric evidence. This altered α-277^his MoFe protein showed comparable physical stabilities to that of the wild-type protein, and its ATP hydrolysis rates remained constant under a number of substrates assayed. Therefore, the substitution has not affected the overall protein structure, rather, it has changed the local FeMo-cofactor environment. When we studied the purified α-276^his and α-274^gin/α-276^his MoFe proteins and compared the results with the data from the α-277^his and wild-type MoFe protein, we found that there is no direct correlation between the additional set of EPR signals observed in these altered MoFe proteins and their catalytic activities. The current understanding concerning the functionality of these residues is that they are involved in maintaining a proper environment for FeMoco to bind and in stabilizing the different redox states of the enzyme during catalysis. / Ph. D.

Throughout the African highlands forage legumes are relied on to add soil N, support increasing livestock populations, and reduce soil erosion. This research addresses a constraint designated by the International Livestock Center for Africa (ILCA) to identify African clover (Trifolium) germplasm and Rhizobium trifolii strain combinations with high productivity potential for the African highlands. A Vertisol and an Eutric Nitosol (Paleudalf), and seed from Trifolium decorum, T. quartinianum, T. rueppellianum, T. steudneri, and T. tembense were obtained from ILCA in Addis Ababa, Ethiopia. A commercial collection and R. trifolii strains isolated from the Ethiopian soils were evaluated for symbiotic effectiveness with these clovers. Effective combinations were evaluated for growth and biological nitrogen fixation (BNF) in a greenhouse on both soils with limited, adequate, and excessive soil moisture. Rhizobia were also evaluated for survival in desiccated soil (12.5 and 17.5 g H₂O g⁻¹ soil) and for competitive nodule forming ability. Effective strains were found among soil isolates but not in the commercial collection. Highest dry matter yields and total BNF accumulation were obtained from T. tembense on all soil and moisture treatments followed by T. decorum and T. quartinianum. Trifolium rueppellianum and T. steudneri had low yields and BNF capacities. Plants receiving adequate and excessive moisture had higher yields than moisture stressed plants. The highest levels of cumulative BNF were obtained on the nitosol soil with either adequate or limited moisture. The moisture limited vertisol supported the lowest BNF levels. Rhizobial strains survived desiccation only in the vertisol at 17.5 g H₂O g⁻¹ soil. All strains could compete with background rhizobia populations to nodulate host plants. Nodule occupancy rates of 20 to 30% were required for high yields. Trifolium tembense, T. decorum, and T. quartinianum are adapted to soils with adequate or excessive moisture, T. rueppellianum and T. steudneri are suited to moisture limited conditions or short growing seasons. Effective rhizobia inoculants and selected clovers have the potential for increasing forage productivity in highland areas. / Ph. D.

Submitted by RODRIGO ALBERTO REPKE null (rodrigorepke@hotmail.com) on 2016-10-27T12:42:23Z No. of bitstreams: 1 Tese FINAL (Corrigida).pdf: 1048387 bytes, checksum: 7e4212c9a987cc6df565f0d8dc5423a4 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-11-01T16:01:05Z (GMT) No. of bitstreams: 1 repke_ra_dr_bot.pdf: 1048387 bytes, checksum: 7e4212c9a987cc6df565f0d8dc5423a4 (MD5) / Made available in DSpace on 2016-11-01T16:01:05Z (GMT). No. of bitstreams: 1 repke_ra_dr_bot.pdf: 1048387 bytes, checksum: 7e4212c9a987cc6df565f0d8dc5423a4 (MD5) Previous issue date: 2016-09-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O triticum aestivum é uma espécie de ciclo anual, pertencente à família Poaceae. Cultivado em regiões de climas subtropical e temperado, ocupa a segunda maior área plantada no mundo. Para obtenção de altas produtividade dentre outros cuidados, é essencial o fornecimento de nutrientes na quantidade demandada pelas cultivares de alto potencial produtivo, com destaque para o nitrogênio (N). A fixação biológica de nitrogênio é realizada por microrganismos simbióticos associados com raízes das plantas. Entre os microrganismos simbióticos que fixam nitrogênio associados com raízes de plantas, destacam-se os do gênero Azospirillum brasilense. O objetivo no presente trabalho foi avaliar a eficiência do uso da inoculação Azospirillum brasilense no desenvolvimento e componentes produtivos da cultura do trigo. O estudo foi dividido em dois experimentos sendo o primeiro em ambiente protegido e o segundo em ambiente não protegido, ambos na Faculdade de Ciências Agronômicas - Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Botucatu, SP, no ano agrícola 2013. O delineamento utilizado foi o de blocos inteiramente casualizados para ambiente protegido e blocos casualizados para ambiente não protegido, com 10 tratamentos em esquema fatorial 3x3+1. Sendo os tratamentos compostos pelo uso de duas estirpes (Ab-V5 e Ab-V6) isoladamente e em combinação das mesmas (Ab-V5+Ab-V6), todos aplicados em três concentrações 65, 130 e 195 milhões de unidades formadoras de colônia (UFC) de bactérias. Mais a testemunha, sem inoculação de bactérias. Para o experimento em ambiente protegido, cada parcela experimental foi constituída por plantas cultivadas em um recipiente de amianto com capacidade para 0,462 m3 de solo. Foram 7 linhas de 0,88 m cada, espaçadas em 0,15 m, com 40 sementes por metro. Já no experimento em ambiente não protegido, a parcela experimental foi composta por 10 linhas de 2,0 m cada, espaçadas em 0,17 m, e 45 sementes por metro, onde ambos os experimentos teve uma densidade de semeadura em 266 sementes m2 almejando uma densidade de 250 plantas m2. Para atender a necessidade de água da cultura durante todo o ciclo, adotou-se o sistema de irrigação localizada por gotejamento, monitorada por tensiômetros de mercúrio. Após a emergência das plântulas até a colheita, foram realizadas avaliações de crescimento em diversos componentes morfológicos e de produtividade das plantas de trigo. Os dados obtidos foram submetidas à análise de variância pelo teste F a 5% de probabilidade, sendo os dados quantitativos submetidos a análise de regressão em função das doses de A.brasiliense os dados qualitativos em função das estirpes pelo teste de Tukey. Em ambiente protegido, a inoculação das sementes com Azospirillum brasilense promove maior crescimento das plantas de trigo, sem influencia na produção de grãos. Em condições de ambiente não protegido o crescimento das plantas é influenciado pela inoculação de Azospirillum brasilense. O uso de bactérias promotoras de crescimento em plantas aumentam o desenvolvimento das plantas de trigo, o que representa uma estratégia viável, além dos benefícios ambientais associados à redução no uso de fertilizantes nitrogenados. / Triticum aestivum is a yearly crop species belonging to the Poaceae family. It is cultivated in regions with subtropical and temperate climate, and takes up the second largest planted area in the world. Nutrient provision in the demanded amount by high productive potential cultivars, mainly nitrogen (N), is essential to obtain high productivity. The biological fixation of nitrogen is done by symbiotic microorganisms associated with plan roots. Azospirillum brasilense is one of the genera in which the symbiotic microorganisms fixate nitrogen associated with plant roots. This study aimed to evaluate the agronomical efficiency of Azospirillum brasilense inoculation on the growth and productive components of wheat cultivation. The study was divided into two experiments: the fist protected environment, and the second non-protected environment conditions, both in the School of Agriculture - Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus of Botucatu, SP, Brazil, during 2013 crop year. The experiment had completely randomized block design for the fist experiment in protected environment, and random blocks for the second experiment in non-protected environment, with 10 treatments in 3x3+1 factorial scheme. The treatments consisted of two separate (V5 and V6) and combined (V5+V6) stocks and three concentrations (65, 130 and 195 million UFC of bacteria) were applied. The control treatment had no bacterial inoculation. For the protected environment, each experimental plot consisted of plants cultivated in an asbestos recipient with the following dimensions: 0.50 m of height, 0.88 m of width and 1.05 m of length, and 0.462 m3 of soil. Seven 0.88-m rows with 0.15 m spacing and 40 seeds per meter were utilized. In the field experiment, the experimental plot consisted of ten 2.0-m rows with 0.17 m spacing and 45 seeds per meter. For both experiments, the seeding density was 266 seeds m2, aiming a density of 250 plants m2. To meet water cultivation requirements throughout the cycle, dripping irrigation was used and monitored by mercury tensiometers. After seedling emergence until harvest, growth evaluations of several morphological and productivity components of wheat plants were carried out. The obtained data were submitted to analysis of variance by F test at 5% probability and quantitative data were submitted to regression analysis in function of A.brasiliense doses whereas qualitative data in function of stocks were submitted to Tukey’s test. In protected environment, the inoculation of Azospirillum brasilense seeds provides greater growth of wheat plants, without influencing grain production. Under non-protected environment conditions, the plant growth is influenced by the inoculation of Azospirillum brasilense. The use of growth-promoting bacteria in plants increase the development of wheat plants, which represents a viable strategy, besides the environmental benefits related to the reduced utilization of nitrogen fertilizers.

No description available.

Thesis (PhD (Plant biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In soils, the concentration of available phosphate (P) for plants is normally very low (ca. 1 µM in the soil solution), because most of the P combines with iron, aluminium and calcium to form relatively insoluble compounds. Inorganic P (Pi)-deficiency is thought to be one of the limiting factors of nitrogen fixation due to the high energy requirement for nitrogenase function of plants taking part in nitrogen fixation. Pideficiency has important implications for the metabolic Pi and adenylate pools of plants, which influence respiration and nitrogen fixation. An alternative route of pyruvate supply during Pi stress has been proposed involving the combined activities of phosphoenolpyruvate carboxylase (PEPc), malate dehydrogenase (MDH) and NAD-malic enzyme (ME) supplying pyruvate to the mitochondrion during Pi stress. Previously, three isoforms of PEPc were isolated from lupin nodules and roots, with two forms being nodule specific. The aim of this project was to determine the effect of Pi stress on these PEPc isoforms in Lupinus luteus at transcript and protein expression level with a view to produce genetically modified crops for nutrient-poor soils. Cytosolic P levels were measured over a time course to give an indication of temporal development of P stress in nodules. The changes in enzyme activities of PEPc, MDH and PK (pyruvate kinase) under P stress were measured and the downstream effect on amino and organic acid pools were analysed. Two novel PEPc isoforms, LUP1 (AM235211) and LUP2 (AM237200) were isolated from nodules, followed by transcriptional and protein expression analyses. Nodules under P stress had lower amounts of metabolically available Pi and as P stressed developed, the amount of Pi decreased. This decline in Pi levels was associated with lower growth, but higher biological nitrogen fixation (BNF). A greater proportion of root-nodule respiration was devoted to nutrient acquisition than to new growth. A typical P-stress response is higher anaplerotic carbon fixation via PEPc. However, in this study, no significant differences were found for PEPc, MDH or PK in P-stressed plants compared to P-sufficient plants which would lead to an increase in organic acids. An increase in key amino acids was reported along with unchanged levels of organic acids. These levels of organic and amino acid are in congruence with the increases in BNF under P-starvation. No significant differences were found in expression of PEPC1 or PEPC2 at 12 and 20 days for both P-sufficient and P-stressed plants which further supported the lack of engagement of the PEPc-MDH-ME bypass. PEPc activity appeared not to be regulated by gene expression or phosphorylation indicating that other posttranslational modifications such as a decrease in protein degradation may be of importance. / AFRIKAANSE OPSOMMING: Die konsentrasie van fosfaat (P) beskikbaar vir opname deur plante vanuit die grond is gewoonlik baie laag (in die omgewing van 1 µM) aangesien die P onoplosbare komplekse vorm met katione soos yster, aluminium en kalsium. ‘n Tekort aan anorganiese P (Pi) word gereken as een van die beperkende faktore van stikstofbinding as gevolg van die hoë energie behoefte wat nitrogenase plaas op plante wat van gefikseerde stikstof gebruik maak. Hierdie P-tekort het ook belangrike betrekking op die metaboliese fosfaat- en adenilaatpoele wat weer op hul beurt respirasie en stikstofbinding beÏnvloed. ‘n Alternatiewe roete van pirovaatvoorsiening aan mitochondria tydens fosfaatstres is voorgestel wat bestaan uit die aktiwiteite van fosfoenolpirovaat karboksilase (PEPc), malaat dehidrogenase en NAD-malaat ensiem. Vantevore is drie isovorme van PEPc uit Lupinus luteus wortelknoppies en wortels geïsoleer, met twee van die isovorme wat wortelknoppie-spesifiek was. The doel van hierdie projek was om die invloed van P-tekort op die transkripsie en proteien uitdrukkingsvlak van hierdie PEPc isovorme te bepaal met die doel van gemodifiseerde gewasse vir arm gronde ingedagte. Sitoplasmiese P konsentrasies is gemeet oor tyd om ‘n aanduiding te gee van die ontwikkeling van P-tekort oor tyd. Veranderinge in ensiemaktiwiteite van PEPc, MDH en pirovaatkinase (PK) is gemeet gedurende P-tekort as ook die moontlike effek van hierdie ensiemaktiwiteite op aminosuur en organiese suur poele. Twee nuwe PEPc isovorme, LUP1 (AM235211) en LUP2 (AM237200) is uit wortelknoppies geïsoleer en gekarakteriseer. Transkripsie en proteïenuitdrukking is geanaliseer. Wortelknoppies wat P-tekort behandeling ontvang het, het laer vlakke van metabolise beskikbare Pi gehad en soos die P-tekort ontwikkel het oor tyd, het die Pi vlakke gedaal. Hierdie afname in vlakke van Pi was geassosieer met laer groei, maar met ‘n toename in biologiese stikstofbinding. ‘n Groter proporsie van respirasie is toegestaan aan minerale opname as aan nuwe groei. ‘n Tipiese reaksie op P-tekort is hoër anaplerotiese koolstofbinding via PEPc. Alhoewel, in hierdie studie is geen gevind betekenisvolle verandering gevind in die aktiwiteite van PEPc, MDH en PK nie in plante wat P-tekort ervaar het nie. Verhoogde aktiwiteit van hierdie ensieme sou verhoogde organise suur konsentrasies tot gevolg hê. ‘n Toename in aminosuur konsentrasies is gevind tesame met onveranderde vlakke van organiese sure. Hierdie toename in aminosure word onderskryf deur die verhoogde biologiese stikstofbinding tydens P-tekort. Geen betekenisvolle verskille is gevind in die geenuitdrukking van pepc1 en pepc2 by beide 12 en 20 dae van P-tekort nie, wat verder die afwesigheid van die PEPc- MDH-ME alternatiewe roete beaam het. Dit blyk dat PEPc aktiwiteit nie deur geenuitdrukking of proteïenfosforilering beheer word nie, maar eerder dat ander posttranslasie modifikasies soos ‘n verlaagde afbraak van proteïen ‘n rol speel.

Inoculation with diazotrophic bacteria is well documented as a means to enhance growth and increase yields of various crops, especially when used as an alternative or a supplement to the use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. Nitrogen is the most limiting nutrient for increasing crop productivity, and the use of chemical sources of N fertilizers is expensive, and may contribute to environmental pollution. Therefore, there is a need to identify diazotrophic inoculants as an alternative or supplement to N-fertilizers for sustainable agriculture. The search for effective diazotrophic bacterial strains for formulation as biofertilizers has been going on for over 40 years and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 195 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within the KwaZulu-Natal Province, Republic of South Africa. Ninety five of the isolates were selected for further screening because they were able to grow on N-free media using different carbon sources. Isolates that were very slow to grow on N-free media were discarded. Of these, 95 isolates were screened in vitro for growth promotion traits tests including tests for ammonia production and acetylene reduction. The best 20 isolates that were also able to reduce acetylene into ethylene were selected for growth-promotion trials on maize under greenhouse conditions. Of the 20 isolates, ten isolates enhanced (P = 0.001) growth of maize above the Un-inoculated Control. Molecular tests were conducted to identify the ten most promising isolates selected in the in vitro study. In the greenhouse study, these diazotrophic isolates were screened for their ability to enhance various growth parameters of maize (Zea mays L.), following various inoculation techniques (drenching, seed treatment, foliar spray and combination of these). Inoculations with the five best diazotrophic isolates by various methods of application increased dry weight and leaf chlorophyll content (P < 0.001, P = 0.001), respectively, compared to the Untreated Control. Although, all methods of application of diazotrophic inoculants used in this study resulted in increased dry weight and leaf chlorophyll content, combined methods of application (seed treatment + drenching) and sole application (seed treatment) were significantly more (P < 0.05) efficient. The best five most promising isolates were identified for growth promotion of maize under greenhouse conditions. They were also assessed for their effects on germination of wheat in vitro and were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of wheat (Triticum aestivum L.). These five isolates were also investigated for their potential to enhance growth and yields of maize and wheat crops in field trials, when combined with a low dose of nitrogenous fertilizer. These isolates were further studied for their contribution for enhancing plant growth through nitrogen fixation by predicting N content in leaves using a chlorophyll content meter (CCM-200) and correlated to extractable chlorophyll level at R2 = 0.96. In this study, relative to the Un-inoculated Control, the best five isolates enhanced growth of maize and wheat when combined with a 33% N-fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry weight of maize and wheat; and enhanced root and shoot development of these crops in both greenhouse and field conditions. The best contributions of diazotrophic bacteria was achieved by Isolate LB5 + 0% NPK (41%), V9 + 65% NPK (28.9%), Isolate L1 + 50% NPK (25%), Isolate L1 + 25%NPK (22%) and LB5 + 75% NPK (15%) undergreenhouse conditions. At 30 or 60 DAP, isolates with 33%N-fertilizer caused relatively higher dry weight than the 100%NPK. Inoculation of Isolate StB5 without 33N% fertilizer cuased significant (P<0.005) increases in stover dry weight. In field studies, inoculation of diazotrophic bacteria alone or with 33%N-fertilizer resulted in relatively greater increases of dry weight, stover dry weight, number of spikes and yield at different growth stages higher than the Un-inoculated or Unfertilized Control. However, the increases were not statistically significant. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising yields. The isolates obtained in this study can effectively fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture. / Ph.D. University of KwaZulu-Natal, Pietermaritzburg 2013.