No description available.

The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number of chaperone proteins were highly expressed in the nip mutant, indicating high stress in the mutant prior to infection by rhizobia. Additionally, a database containing the information regarding the nip expression profile at both 0 days post inoculation (dpi) and 10 dpi were created for screening of candidate genes as predicted from sequence in the genomic region containing NIP.

Thesis (MTech (Horticulture Science))--Cape Peninsula University of Technology, 2009 / The study used common bean (P. vulgaris L. variety Provider) in a spilt-split-plot design involving 2 levels of Rhizobium inoculation (with and without rhizobia), 3 levels of lime (0, 2 and 3 t.ha-1) and 3 levels of molybdenum (0, 6 and 12 g.kg-1 of seeds) in a glasshouse experiment. The glasshouse experiment was then verified in the field during 2008 and 2009 cropping seasons. The aim was to assess the effects of Rhizobium inoculation, molybdenum and lime supply on: i) yield and yield components of the P. vulgaris L. ii) Changes in soil pH and the concentrations of selected plant-available nutrients in the rhizosphere, iii) photosynthesis and chlorophyll formation in P. vulgaris L. and (iv) plant growth and N2-fixation in P. vulgaris L. The results showed that Rhizobium inoculation had significant effects in increasing yield components and ultimately the final seed yield. Rhizobial inoculation also significantly increased the levels of chlorophyll content in leaves, improved all photosynthetic parameters, increased dry matter yield of different organs and decreased 15N values in all organs assessed. As a result, % nitrogen derived from atmosphere (%Ndfa) in all organs as well as the amount of N derived from fixation was improved. In the field, the whole plant level of N-fixation of P. vulgaris L. from Rhizobium inoculation accounted for approximately 33 kg N.ha-1. Furthermore, soil pH and the concentration of mineral nutrients (P, K, Ca, Mg, Na, Fe, Cu, Zn and Mn) in the rhizosphere were significantly increased with Rhizobium inoculation when compared with the control.

A utilização das técnicas de inoculação e coinoculação de rizobactérias promotoras do crescimento de plantas (RPCPs) pode ser uma alternativa com grande potencial para o mercado agrícola, visando uma maior eficiência no uso de fertilizantes e consequentemente redução do impacto ambiental. Os rizóbios também apresentam capacidade para a promoção do crescimento de poáceas, grupo de plantas no qual essas bactérias não realizam a fixação simbiótica de nitrogênio. Nesse contexto, a principal hipótese do trabalho é que o desenvolvimento de poáceas é beneficiado quando as plantas são inoculadas com RPCPs. O objetivo geral deste trabalho foi verificar a capacidade de rizóbios em promover o crescimento dos cereais: arroz, milho e trigo, inoculados isoladamente e coinoculados com Azospirillum brasilense. Os experimentos foram realizados a campo e em casa de vegetação, com plantas de arroz, milho e trigo, inoculadas e coinoculadas com os rizóbios UFRGS Vp16 e UFRGS Lc348, e A. brasilense, e sob níveis de doses de nitrogênio recomendadas para cada espécie vegetal. A inoculação de rizóbios, isoladamente ou em coinoculação com A. brasilense promove aumentos de produção de grãos de milho híbrido (Morgan 30A77PW) em manejo sequeiro e irrigado. Em áreas de pousio a coinoculação do rizóbio UFRGS Lc348+A. brasilense mantem o rendimento de grãos de arroz (cultivar IRGA 424), com 60% da dose nitrogênio recomendada. Já em safras com inoculações sucessivas na área de plantio, todos os tratamentos com RPCPs e 60% da dose de nitrogênio, mantém o rendimento de grãos. Em plantas de trigo (cultivares TBIO Sossego e BRS Parrudo), a inoculação e coinoculação aumentam o rendimento de grãos. A cinética de absorção de nitrogênio em plantas de milho híbrido (Morgan 30A77PW) é modificada pela inoculação com rizóbios UFRGS Vp16 e UFRGS Lc348 tanto de forma isolada, ou coinoculada com A. brasilense. / The use of inoculation and coinoculation techniques of plant growth promoting rhizobacteria (PGPR) can be an alternative with great potential for the agricultural market, aiming at a greater efficiency in the use of fertilizers and consequently reduction of environmental impact. Rhizobia also have the capacity to promote the growth of grasses, a group of plants in which these bacteria do not perform symbiotic nitrogen fixation. In this context, the main hypothesis of the work is that the development of grasses benefits when the plants are inoculated with PGPR. The general objective of this work was to verify the rhizobia of capacity to promote cereal growth: rice, maize and wheat, inoculated in isolation and coinoculated with Azospirillum brasilense. The experiments were carried out in the field and in a greenhouse with rice, maize and wheat plants, inoculated and coinoculated with the rhizobia UFRGS Vp16 and UFRGS Lc348, and A. brasilense, and under levels of nitrogen rates recommended for each plant species. The inoculation of rhizobia, alone or in combination with A. brasilense, promotes hybrid maize grain yield increases (Morgan 30A77PW) in dry and irrigated management. In fallow areas the coinoculation of the rhizobia UFRGS Lc348 + A. brasilense maintains the yield of rice grains (cultivar IRGA 424), with 60% of the recommended nitrogen dose. In crops with successive inoculations in the plantation area, all the treatments with PGPR and 60% of the nitrogen dose, maintain the yield of grains. In wheat plants (cultivars TBIO Sossego and BRS Parrudo), inoculation and coinoculation increase the yield of grains. Nitrogen uptake kinetics in hybrid maize plants (Morgan 30A77PW) is modified by inoculation with rhizobia UFRGS Vp16 and UFRGS Lc348 either in an isolated form, or coinoculated with A. brasilense.

Espécies com potencial forrageiro adaptadas aos solos do Rio Grande do Sul, como L.corniculatus, L.glaber, L.subbiflorus e L.uliginosus, se destacam pela capacidade de fixação simbiótica de nitrogênio, boa produção e tolerância à acidez e baixa fertilidade dos solos. A seleção de rizóbios para Lotus, resistentes a baixo pH, a alumínio tóxico e à salinidade é fundamental para a implantação e aumento da produção destas leguminosas no estado. Foram avaliados 52 rizóbios da coleção de culturas da UFRGS quanto à resistência a pH 4,2 e a 50μM de alumínio tóxico em meio mínimo de Wood e Cooper (MWC) modificado e quanto à resistência a diferentes concentrações de NaCl e KCl em meio Levedura-Manitol (LM) a pH 6,8. A diversidade genética de rizóbios de Lotus spp foi avaliada usando-se PCR com os oligonucleotídeos iniciadores BOX A1-R, ERIC e RISA, sendo o perfil eletroforético dos produtos da amplificação do DNA genômico de cada rizóbio analisado e construído um dendrograma de similaridade. Entre os isolados resistentes a pH 4,2 e a 50μM de Al3+, sete foram avaliados quanto à eficiência simbiótica com plantas em casa de vegetação. Entre os rizóbios estudados, 16 foram resistentes a pH 4,2 e a 50μM de alumínio, 11 foram resistentes até concentrações de 12gL-1 de NaCl e seis foram resistentes até a 20gL-1 de KCl. Foram selecionados rizóbios para Lotus spp resistentes à acidez e ao estresse salino. Observou-se alta diversidade genética, indicando que os rizóbios estudados não são reisolamentos de estirpes utilizadas em inoculantes para as espécies de Lotus estudadas. / Species with forage potential adapted to the soils from the state of Rio Grande do Sul, such as L.corniculatus, L.glaber, L.subbiflorus and L.uliginosus, stand out for their capacity of symbiotic nitrogen fixation, good production and tolerance to acid and low-fertility soils. Selection of rhizobia that are resistant a low pH, aluminum toxicity and salinity, is fundamental to establishing and increasing the production of to Lotus legumes in this state. In the present research, 52 rhizobia, from the culture collection of UFRGS, were assessed for resistance to pH 4.2 and 50μM of aluminum in modified minimal Wood and Cooper medium (MWC), and for resistance to different concentrations of NaCl and KCl in yeast mannitol medium (LM) at pH 6.8. The genetic diversity of Lotus spp rhizobia was assessed by using PCR with the primers BOX-A1 R, ERIC and RISA. The electrophoretic profile of the PCR products from each rhizobium was analyzed and a dendrogram of similarity was built. Among the isolates that were resistant to pH 4.2 and 50μM of Al3+, seven were assessed for symbiotic efficiency with plants in the greenhouse. Among the studied rhizobia, 16 were resistant to pH 4.2 and 50μM of aluminum, 11 were resistant to concentrations of 12gL-1 NaCl and six were resistant to the 20gL-1 KCl. It was possible to select Lotus spp rhizobia resistant to acidity and salinity stress. A high genetic diversity was observed, indicating that the studied rhizobia were not reisolations of strains used in inoculants for Lotus species.

Twenty-nine Rhizobium leguminosarum biovar trifolii strains were tested for acidity tolerance in acidified liquid medium. Only 41\ of the strains grew at pH 4.1. One acid-tolerant strain, USDA 2160, and one acid-sensitive strain, 162-X-103 from Nitragin Co., were inoculated on seeds of 'Nungarin', 'Seaton Park' and 'Clare' subclover cultivars. The inoculated and pelleted seeds were sown in potted Cluff soil with pH 5. 7. Three desiccation levels were imposed by delaying watering for 0, 15 or 30 days. Four gravimetric soil water contents (6.0, 6.6, 10.5 and 12.5\) were maintained under a greenhouse line-source sprinkler system for 7 weeks. The desiccation treatments were more detrimental to the survival of the acid-tolerant Rhizobium strain (USDA 2160) than they were for the acid sensitive strain (162-X-103). Symbiotic effectiveness, measured as shoot dry weight, was higher with strain 162-X- 103 than with strain USDA 2160 and was comparable to the Nfertilized control at the highest water level (12.5 %). At the lower water levels (6.0, 6.6 %) symbiotic N2-fixation was more affected than N-uptake. The Rhizobium strains were able to survive and grow even at the lowest soil water level. The number of rhizobia in the soil and nodulation of the subclover plants had a correlation of 0.56.

No description available.

Genome plasticity in soil bacteria is predicted to be evolutionarily advantageous, allowing bacteria to sample genetic variation for adaptation to local soil ecology. In the field population of mesorhizobia where the symbiosis island (ICEMlSym[R7A]; an I̲ntegrative C̲onjugative E̲lement) was first identified, individual members were found to have significant chromosomal variation downstream of the phe-tRNA gene or phe-tRNA integrated ICEMlSym[R7A]. However, the nature of this genetic variation and whether it contributed to the adaptation of the indigenous mesorhizobia to their field environment were unknown. This work focused on a nodule isolate, Mesorhizobium sp. strain R88B, a member of the indigenous mesorhizobial population that received ICEMlSym[R7A] from strain R7A. The region downstream of ICEMlSym[R7A] was sequenced, revealing three distinct regions of non-conserved DNA, totalling 34.5 kb. Integrated directly downstream of ICEMlSym[R7A] was IMEMlAdh[R88B], a 24.3-kb novel I̲ntegrative M̲obilisable E̲lement. Using a PCR-based assay, it was shown that the IMEMlAdh[R88B] integrase could excise not only IMEMlAdh[R88B], but also a dual-IMEMlAdh[R88B]/ICEMlSym[R7A] hybrid, indicating the potential mobility of IMEMlAdh[R88B], and a likely evolutionary intermediate of a novel ICE. However, a functional role for MadA, (a putative adhesin and the sole adaptive trait encoded on IMEMlAdh[R88B]) was not discovered. Southern hybridisations with the mesorhizobial population provided evidence for the existence of a novel family of IMEs in the mesorhizobia, which, by diversifying their internal sequences, provide allele-specific variation to the population. The two other regions downstream of IMEMlAdh[R88B] possessed no obvious mobile genetic element structures, and only the region adjacent to the core-chromosome encoded ORFs with putative functions. Mutation of two of these ORFs, fhuD1 and fhuB1, identified their function as two of the four components of a ferrichrome ABC-uptake (Fhu) system. Using genetic screens, the remaining components of this transporter were mapped to two separate loci. Thus, the functional transporter in R88B was a composite of at least two independently-acquired Fhu systems. The genetic screens also revealed that ferrichrome utilisation was dependent on a TonB energy-transduction system encoded downstream of the Fhu ATPase gene, fhuC. Expression studies on the three fhu loci demonstrated that, despite their separate acquisition, their expression was coordinately up-regulated in response to low-iron conditions. Bioinformatics on the predicted promoter regions of the fhu genes identified the binding site of the rhizobial Fur analogue, RirA, which is likely to be responsible for this expression profile. Southern hybridisations of DNA isolated from members of the mesorhizobial population revealed the three fhu loci were not conserved in the mesorhizobial population. The presence of FhuA was the best predictive marker for the trait. It is proposed that multiple rounds of acquisitions and recombinations, both illegitimate and legitimate, formed this transporter, with the constant need for iron offset by the negative selection pressure of FhuA being a target for phage. None of the Fhu-specific genes was present in the sequenced M. loti strain MAFF303099 though flanking sequences were, further emphasizing the role of genome microevolution in forming the Fhu phenotype.

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 189-210 / xxxiv, 210 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, Dept. of Agricultural Biochemistry, 1987