No description available.

No description available.

Soybean has a strong demand for nitrogen that can be acquired from atmosphere for vegetative growth and seed production through the symbiosis with the soil bacterium Bradyrhizobium japonicum (B. japonicum). However, the native soil bradyrhizobia may be ineffective in nitrogen fixation and the greatest limiting factor in increasing symbiotic nitrogen fixation is the inability to influence the infection of soybean roots by a desired strain of B. japonicum due to competition from the native bradyrhizobia. Previous studies have demonstrated the efficacy of a co-inoculum seed treatment on the symbiotic competency of the soybean cultivar LS90-1920 in greenhouse and field trials. The co-inoculation by the soil bacterium Streptomyces kanamyceticus (S. kanamyceticus) strain ATCC 12853 and strains of B. japonicum more efficient in nitrogen fixation and resistant to the antibiotics kanamycin and neomycin may have an advantage over the native bradyrhizobia regarding soybean root infection (Gregor et al., 2003). However, inconsistent inoculation responses in field trials and low efficacy in nodule competency by selected Bradyrhizobium japonicum (B. japonicum) co-inocula were observed under greenhouse conditions. These results were attributed to insufficient population size or growth of viable co-inocula associated with the seed treatments. This recent study showed that the nodulation response of LS90-1920 to B. japonicum strains KNI-1 and KNI-3 is independent of the inoculum dose and age of the broth culture. Iron supplement to the inoculum nutrient solution significantly increased the total biomass of nodules formed by strain KNI-1 but not by strain KNI-3 on a per plant basis and had no effect on the nodule number regardless of B. japonicum strain. In the glass bead viability study, the effect of inoculum nutrient solution concentration on the viability of bacterial co-inocula is species-specific and influenced by seed coating material. The growth of Pseudomonas putida strains displayed a dependency on the concentration of the inoculum nutrient solution with graphite or vermicompost as the seed coating material treatment or with activated charcoal treatment associated with 0.1% or 1.0 % inoculum nutrient solution. The seed coating material treatments of vermicompost and graphite promote stronger growth of S. kanamyceticus strain ATCC 12853 than the activated charcoal treatment. After a six-day incubation at 28oC, a 1.0 % inoculum nutrient solution maintained the highest viable populations of co-inocula with activated charcoal and a 0.1% inoculum nutrient solution was most effective in the maintenance of the co-inocula population when graphite or vermicompost was employed as the seed coating material. By applying the appropriate level of inoculum nutrient solution, the viability of a selected B. japonicum KNI strain and co-inocula remained stable for six days in activated charcoal and graphite treatment regardless of the number of applied co-inocula. However, the vermicompost treatment did not maintain the viable populations of the B. japonicum KNI strains and P. putida strain G11-32 but support the vigorous growth of S. kanamyceticus strain ATCC 12853 and P. putida strain 17-29. Greenhouse studies employing sterilized vermiculite as a soybean growth medium showed no significant differences in nodule competency by the inoculum/seed coating treatments associated with B. japonicum strain KNI-1. However, the co-inoculum treatments significantly increased either the total nitrogenase activity (B. japonicum strain KNI-3 with S. kanamyceticus strain ATCC 12853) or the nodule number (B. japonicum strain KNI-3 with S. kanamyceticus strain ATCC 12853 and P. putida strain 17-29) versus the singular inoculum treatment of strain KNI-3. Soil-pot studies under the same greenhouse conditions showed no significant differences in the nodule competency between the inoculum treatment of B. japonicum strain KNI-3, the co-inoculum treatment of strain KNI-3 and S. kanamyceticus, and the non-inoculated control regardless of seed coating material. However, co-inoculation of emergence-promoting rhizobacteria (Pseudomonas putida strain 17-29 and G11-32) with strain KNI-3 and S. kanamyceticus strain ATCC 12853 may improve the total nitrogenase activity and specific nitrogenase activity, depending on the seed coating material and soil type. The treatment with activated charcoal employed as a seed coating material and the co-inocula of strain KNI-3, S. kanamyceticus strain ATCC 12853 and P. putida strains 17-29 or G11-32 showed significantly higher total nitrogenase activity (Stoy silt loam) and specific nitrogenase activity (Drummer silty clay loam) versus the non-inoculated control. For the Bethalto silty clay loam, the same co-inoculum treatment associated with graphite and vermicompost as the seed coating material significantly increased the total nitrogenase activity. The seed coating treatment by activated charcoal enhanced nodulation competency for both the 2010 and 2011 field trials resulting in higher grain yield, seed nitrogen content, and seed protein content versus the seed coating treatment by graphite. No significant differences by the inoculum treatments were determined.

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Nous nous sommes intéressés à la compréhension des mécanismes régissant la symbiose Rhizobium-Acacia dans les conditions de stress salin. Les lipopolysaccharides jouent un rôle important dans les étapes de cette symbiose. Le but était de mettre en évidence les modifications pariétales de la bactérie en réponse au stress salin par l’étude de la structure des lipopolysaccharides des souches isolées du désert marocain tolérant NaCl 7%. Ainsi, une nouvelle méthode d’hydrolyse des lipopolysaccharides sensible, non destructive et compatible avec la spectrométrie de masse a été développée. En présence de stress salin, nous avons montré que la membrane externe devenait plus hydrophobe en augmentant l’acylation de la région lipidique ainsi qu’en réduisant la présence des molécules de LPSs à longues chaînes de sucres.Des essais d’évaluation de l’efficience et de l’infectivité des Rhizobia étudiés ont été mis en œuvre pour déterminer l’impact de ces modifications des LPSs sur la symbiose sous stress salin. / We were interested in the understanding of the mechanisms governing Rhizobium-Acacia symbiosis in salt stress conditions. Lipopolysaccharides play an important role in the stages of this symbiosis. The aim of this work was to highlight the changes occurring in the bacterial membrane in response to salt stress by studying the structure of the lipopolysaccharides isolated from Moroccan desert strains tolerating 7% NaCl. Thus, a new method of hydrolysis of the lipopolysaccharide - sensitive, non-destructive and compatible with mass spectrometry- was developed. We studied the LPSs strains grown with or without salt stress and we showed that in salt stress conditions, the outer membrane becomes more hydrophobic by increasing acylation of the lipid region and reducing the number of long sugar chains in LPSs. Tests for evaluating the efficiency and infectivity of the studied rhizobia were carried out to determine the impact of these LPS modifications on symbiosis under salt stress.

La Tara (Caesalpinia spinosa) est une espèce forestière d’une grande importance en raison d’une forte demande sur le marché international pour les tanins présents dans ses gousses, et pour les gommes provenant de ses graines. Malgré son importance économique pour le Pérou, la majeure partie de la production provient de forêts naturelles non aménagées. Ces forêts présentent des problèmes de sol (érosion, faible fertilité, présence d’agents pathogènes, manque d'irrigation), qui conduisent à des rendements faibles. C’est pourquoi dans le présent travail, nous étudions les composantes microbiologiques du sol associé à cette culture, telles que les mycorhizes et les bactéries dont l’utilisation, selon de nombreuses études, s’est révélée être une alternative à l'utilisation d'engrais chimiques. Pour cela, nous avons procédé à l'analyse moléculaire de la diversité des champignons mycorhiziens arbusculaires par la technique de Miseq Illumina, ce qui nous a permis de mettre en évidence une prépondérance de Gloméracées parmi lesquelles les Rhizophagus spp. étaient retrouvés dans 70% des séquences. En outre, la dépendance de la Tara vis-à-vis de la mycorhization a été démontrée car, après avoir testé la mycorhization contrôlée de la Tara par Rhizophagus irregularis, il a été constaté que la croissance de Caesalpinia spinosa était considérablement améliorée, ainsi que l'absorption d'éléments nutritifs tels que l'azote (N) et le phosphore (P). Pour vérifier la capacité à noduler de la Tara, différents milieux de culture ont été utilisés ainsi que différentes conditions de croissance, en serre et in vitro. Ces expérimentations ont toutes montré que les racines de Tara ne présentaient pas de nodules, confirmant que cette légumineuse de la sous-famille des Caesalpinioideae est non nodulante. Par conséquent et afin d’étudier la diversité des rhizobia présents dans le sol de la plantation de Tara, nous avons utilisé en serre une plante-piège, le pois (Pisum sativum) car c’est une légumineuse nodulante et de plus est traditionnellement associée à la culture de Tara. Les rhizobia identifiés moléculairement se sont révélés très spécifiques et différents des rhizobia présents dans les sols extérieurs à la plantation de Tara. Plus particulièrement, ces rhizobia se sont révélés être phylogénétiquement proches de R. etli, R. phaseoli, R. pisi et R. leguminosarum. Enfin, un test d'inoculation contrôlée (in vitro) a été réalisé sur des plantules de pois, avec ces bactéries préalablement piégées et isolées du pois. Il a été observé que les rhizobia piégés à partir des sols collectés entre deux lignes de Tara et sur la ligne de plantation de Tara, ont stimulé la croissance du pois par rapport aux rhizobia présents dans les sols collectés à l'extérieur de la plantation. / The Tara (Caesalpinia spinosa) is a forest species of great importance due to its high demand in the international market for the tannins present in its pods and its seeds’ gum. Despite its great importance for Peru, most of the production comes from unmanaged natural forests. These forests present soil problems (e.g., erosion, low fertility, pathogens, lack of irrigation), which cause low yields. Therefore, in the present work we seek to study the soil components associated with Tara plantation , such as mycorrhizae and bacteria that have proved to be an alternative for reducing the use of chemical fertilizers in similar context (Aboubacar et al., Flores Chavez 20015, E and Saif 1987, Dia et al. 2010; Bilgo et al., 2013) . We used molecular analysis of the arbuscular diversity by the Miseq Illumina technique that allowed to verify the arbuscular diversity with a preponderance of Glomeraceae among which the Rhizophagus spp were found to be present in 70% of the sequences. In addition, the dependence of the Tara on obligatory mycorrhization was demonstrated, after testing the controlled mycorrhization of the Tara by the Rhizophagus irregularis. We found that the growth of this crop was significantly improved, as well as the absorption of nutrients such as nitrogen (N) and phosphorus (P).To check the nodulation of the Tara, different culture media were used (JenSen, sand mixture with Tara plantation soil, attapulgite mixture with Tara plantation soils) in greenhouse and in vitro condition. We did not manage to find rhizobial nodules in the roots which let us think that Tara is a non-nodular legume. Therefore, we used Pisum sativum as a trap plant to study the diversity of rhizobia present in the soil of the Tara plantation since this legume is often associated with Tara crop. The rhizobia found in the trap plant were very specific and different from the rhizobia present in soils outside the Tara plantation. Likewise, these rhizobia found to be phylogenetically close to R. etli, R. phaseoli, R. pisi and R. leguminosarum. Finally, we inoculated the trapped bacterias (in vitro) in Pisum sativum with the bacterias previously trapped and isolated from the pea (which grew in the green house); where it was observed that the rhizospheric bacteria of the zones IL (soil collected between two lines) and L (soil collected from the same line) from the plantation of Tara stimulated the growth of this crop with respect to the bacteria present in soils collected outside of the plantation (OP zone).

Cette étude a pour principal objectif de mettre en évidence le rôle des bactéries solubilisatrices de phosphate (rhizobia) dans une meilleure gestion des systèmes de cultures associant la fève (légumineuse) et le blé (céréale) en monoculture et en cultures mixtes sous des conditions de déficience en P. Cent neuf souches de rhizobia ont été isolées à partir des nodules racinaires des plantes de V. faba dans la région de Marrakech. Les analyses phylogénétiques basées sur les gènes 16S rDNA, recA et nodD montrent que les souches étudiées sont proches phylogénétiquement de R. leguminosarum biovar viciae, de R. laguerreae et de Ensifer meliloti. Nous avons également décelé la présence du gène pqqC qui est impliqué dans la solubilisation du phosphate (pyrroloquinoline quinone synthase C) chez 15 souches de rhizobia. 22% de ces souches ont été identifiées comme étant capables de solubiliser le phosphate minéral. Ensuite, nous avons montré que l’inoculation avec certaines souches de rhizobia ayant la capacité de solubiliser le P in vitro améliore la croissance des plants de fève et de blé en serre (en monoculture et culture mixte). Nous avons aussi noté des réponses différentes à l’effet de l’inoculation selon les combinaisons symbiotiques étudiées fève-rhizobia. Nous avons également montré que les souches de rhizobia peuvent stimuler le transfert d'azote fixé de la fève au blé, ce qui pourrait contribuer de manière significative au processus de facilitation plante-plante dans les cultures en association dans des conditions de sols carencés en phosphore. En plus de ces avantages de l’association, cette pratique culturale n’est pas limitée à un effet trophique (amélioration de la nutrition azotée des plantes), mais pourrait résulter d'interactions microbiennes qui peuvent agir directement sur la croissance du blé (effet PGPR). Les résultats des expérimentations menées en serre et au champ montrent, en effet, que les cultures mixtes stimulent la croissance du blé ainsi que sa nutrition minérale (N et P).Ces résultats soulignent l'utilité de développer ce type de pratiques culturales associant les légumineuses et les céréales et d'utiliser les ressources microbiennes locales dans les agro-écosystèmes afin d’améliorer la production agricole, en réduisant l’utilisation des intrants chimiques coûteux et néfastes pour l'environnement. / The main purpose of this study is to evaluate the role of phosphate solubilizing rhizobial strains in promoting faba bean (legume) and wheat (cereal) growth in mono and mixed cultures under phosphorus deficiency conditions. 109 rhizobial strains have been isolated from nodules of V. faba plants in Marrakech region. Phylogenetic analysis based on 16S rDNA, recA and nodD genes showed that the studied strains are close to Rhizobium leguminosarum biovar viciae, R. laguerreae and Ensifer meliloti. We detected the presence of pqqC gene which is implicated on phosphate solubilization (Pyrroloquinoline-quinone synthase C) in 15 rhizobia strains. 22 % of isolated strains were found to be able to solubilize mineral phosphate. We have demonstrated that the inoculation with some of rhizobial strains with PSolubilization capacity in vitro, improved the growth of fava bean and wheat plants in greenhouse (both in mono and mixed cultures). We noticed different reactions to the inoculation's effect, depending on the symbiotic combinations faba bean-rhizobia studied. We also showed that rhizobial strains can stimulate the transfer of fixed nitrogen from the bean to the wheat, which could contribute significantly to the plant-plant facilitation process in associated cultures with phosphorus deficiency conditions. Besides this association advantages, this cultural practice is not limited to a trophic effect (improvement of the nitrogenous nutrition of plants), but could engender microbial interactions which can directly enhance the wheat growth (PGPR effect). The results of the experiments conducted in greenhouse and on field show that mixed cultures stimulate wheat growth and its mineral nutrition (N and P). These results underline the utility of developing this kind of cultural practices associating legumes and cereals, and to use the local microbial resources in the agro-ecosystems to improve the agricultural production by reducing the use of agricultural improvers among others expensive artificial fertilizers which represent a threat to the environment.

Chemotaxis is the process by which motile bacteria navigate chemical gradients in order to position themselves in optimum environments for growth and metabolism. Sensory input from both the external environment and the internal cellular environment are sensed by chemotaxis transducers and transduced to a two-component system whose output interacts with the flagellum thereby regulating motility. Chemotaxis has been implicated in establishing the endosymbiotic relationship between the motile alpha-proteobacterium Rhizobium leguminosarum biovar viciae and its host Pisum sativa, the pea plant. An approach combing bioinformatical sequence analysis, molecular genetics, and behavioral analysis was used to characterize the chemotaxis system of R. leguminosarum and determine its contribution to this bacterium s lifestyle. A genome search revealed the presence of two chemotaxis gene clusters, che1 and che2. Homologs of each che cluster are major chemotaxis operons controlling flagellar motility in other bacterial species. For this reason we sought to determine the contribution of each che cluster to chemotaxis in R. leguminosarum. We found that while both che clusters contribute to the regulation of motility, che1 is the major che cluster controlling chemotaxis. Using competitive nodulation assays we determined that che1, but not che2, is essential for competitive nodulation. The major che cluster, che1, encodes a chemotaxis transducer, IcpA-Rl, with a globin coupled sensor domain. Chemotaxis transducers with a globin coupled sensor domain comprise a large class of proteins found in bacteria and archaea. These proteins have been shown to bind heme and sense oxygen and are therefore termed HemATs for heme-binding aerotaxis transducers. However, sequence analysis of IcpA-Rl reveals that it lacks the requisite amino acid residues for heme-binding and is therefore unlikely to sense oxygen. We present evidence that IcpA-Rl is likely an energy transducer and represents a novel function of the globin coupled sensor domain in sensing energy related parameters.

Biserrula pelecinus L. has been introduced to Australia from the Mediterranean region, in the last decade due to many attractive agronomic features. This deep rooted, hard seeded, acid tolerant and insect resistant legume species provides high quality food for cattle and sheep, and grows well under the harsh edaphic and environmental conditions of Australia. In 1994, B. pelecinus was introduced to a site in Northam, Western Australia where there were no native rhizobia capable of nodulating this legume. The introduced plants were inoculated with a single inoculant strain of Mesorhizobium sp., WSM1271. This study investigated whether a diversity of rhizobia emerged over time. A second objective was to investigate the possible mechanisms involved in the diversification of rhizobia able to nodulate B. pelecinus. Eighty eight isolates of rhizobia were obtained from nodules on B. pelecinus growing at the Northam site in August 2000, six years after introduction. These plants were self-regenerating offspring from the original seeds sown. Molecular fingerprinting PCR with RPO1 and ERIC primers revealed that seven strains (novel isolates) had banding patterns distinct from WSM1271 while 81 strains had similar banding patterns to WSM1271. A 1400 bp internal fragment of the 16S rRNA gene was amplified and sequenced for four of the novel isolates (N17, N18, N45 and N87) and WSM1271. The phylogenetic tree developed using these sequences clustered the novel isolates in Mesorhizobium. There were >6 nucleotide mismatches between three of the novel isolates (N17, N18, N87) and WSM1271 while there were 23 nucleotide mismatches between N45 and WSM1271. When B. pelecinus cv. Casbah was inoculated with the novel isolates, five (N17, N18, N39, N46 and N87) yielded <40% of the shoot dry weight of the plants inoculated with the original inoculant (WSM1271). Novel isolates N15 and N45 were completely ineffective on B. pelecinus cv. Casbah. Physiological experiments to test the ability of the novel isolates and WSM1271 to grow on 14 different carbon sources (N acetyl glucosamine, arabinose, arbutine, dulcitol, β-gentiobiose, lactose, maltose, melibiose, D-raffinose, saccharose, L-sorbose, D-tagatose, trehalose and D-turanose) as the sole source of carbon, intrinsic resistance to eight different antibiotics (ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, spectinomycin, streptomycin and tetracycline) and pH tolerance (pH 4.5, 5.0, 7.0, 9.0) revealed that the novel isolates had significantly different carbon source utilization patterns to WSM1271. However, pH tolerance and intrinsic resistance to antibiotics were similar between the novel isolates and WSM1271 except for streptomycin (100 μg/ml). Novel isolates N17, N18, N46 and N87 were susceptible for this antibiotic while the other novel isolates and WSM1271 were resistant. Host range experiments were performed for the novel isolates N17, N18, N45, N87, WSM1271 and two other root nodule bacteria (RNB) previously isolated from B. pelecinus growing in the Mediterranean region (WSM1284 and WSM1497) for twenty one legumes (Amorpha fruticosa, Astragalus adsurgens, Astragalus membranaceus, Astragalus sinicus, Biserrula pelecinus cv Casbah, Dorycnium hirsutum, Dorycnium rectum, Glycyrrhiza uralensis, Hedysarum spinosissimum, Leucaena leucocephala, Lotus corniculatus, Lotus edulis, Lotus glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus parviflorus, Lotus pedunculatus, Lotus peregrinus, Lotus subbiflorus, Macroptilium atropurpureum, and Ornithopus sativus). Only isolate N17 have the same host range as WSM1271 in that they both nodulated B. pelecinus and A. membranaceus, while the other three novel isolates, WSM1284 and WSM1497 had a broader host range than WSM1271. Three isolates N18, N45 and N87 formed small white nodules on M. atropurpureum, in addition to nodulating the above hosts. Isolates N18 and N45 also nodulated A. adsurgens while N45 was the only isolate to nodulate L. edulis. Isolate N87 was the only isolate to nodulate A. fruticosa. WSM1497 nodulated A. adsurgens, A. membranaceus, B. pelecinus and L. corniculatus while WSM1284 was a promiscuous strain that nodulated 16 host species out of the 21 tested. A 710 bp internal region of nifH, a 567 bp internal region of nodA and a 1044 bp internal region of intS were sequenced for N17, N18, N45, N87 and WSM1271. The sequence comparison showed that the sequences of the above three genes of the four novel isolates were identical to that of WSM1271. Eckhardt gel electrophoresis revealed that WSM1271, three other RNB isolates from B. pelecinus from the Mediterranean region and isolate N18 each have a plasmid of approximately 500 kb while N17, N45 and N87 are plasmid free. Probing of the plasmid DNA from the Eckhardt gel with nifH and nodA probes indicated that these two genes were not located on the plasmid. Furthermore, the results of this study demonstrated that 92% of the nodules on B. pelecinus growing in the Northam site six years after the introduction of this plant were occupied by the inoculant strain and the N2 fixation efficiency of the progeny strains of WSM1271 remain similar to the mother culture. This study also showed that the carbon source utilization pattern, intrinsic antibiotic resistance and pH range of the progeny strains of WSM1271 remain relatively similar, except for few variations in carbon source utilization patterns. This thesis clearly demonstrated that phenotypicaly, genetically and phylogenetically diverse strains capable nodulating B. pelecinus evolved through symbiotic gene transfer from the inoculant strain to other soil bacteria within six years. The presence of intS, and the evidence of gene transfer between these Mesorhizobium strains indicates that transfer of symbiotic genes may have occurred via a symbiosis island present in WSM1271.

Mycorrhizal associations are very important to plant growth. This is mainly due to the hyphal stands that are able to utilize concentrations of nutrients that would normally be considered unavailable for plant growth, especially phosphorus. These associations are of great importance in soils that have been disturbed by mineral extraction and are consequently low in available nutrients. The ability of mycorrhizal fungi to form associations on disturbed sites may be dictated by a number of factors: moisture, soil pH, soil fertility (including heavy metal ions), salinity, and plant response. Two factorial experiments were conducted with the legume Hedysarum boreale cv utahensis and the mycorrhizal fungus Glomus mosseae. The first to observe the effects of three factors: fungi level (mycorrhizal or non-mycorrhizal), type of phosphate (P) (monocalcium or superphosphate), and irrigation intervals (2, 4, or 8 day) on root and shoot dry weights. The second experiment was conducted to observe the effects of three factors: fungi level, type of P and level of P on root and shoot dry weights, root and shoot P concentration, nodulation and nitrogen fixation. In the first experiment, the combination of fungi level and type of P had a significant effect on root /shoot ratio (weight ). In the second experiment, the interaction of fungi level, type of P and level of P had a significant effect on nodulation. The interaction of P type and P level had a significant effect on the amount of phosphorus found in plant roots and shoots. The factor, fungi level, had a significant effect on shoot dry weight, with mycorrhizal plant's shoots having a significantly greater dry weight than non-mycorrhizal shoots. Mycorrhizal infection may have been greatly reduced by the adverse soil conditions found in the mine spoil (i.e. low pH, high salinity , and high heavy metal concentration). What infection did occur may have been great enough to produce changes in the shoot dry weight, but not great enough to significantly enhance plant P uptake. The reintroduction of mycorrhizae into disturbed areas for reclamation purposes may be important for rapid establishment of plant cover and to speed up succession. Before mycorrhizae can be reestablished in a disturbed area, many potential stress factors need to be addressed and, if possible, overcome.