DNA homology within the Rhizobiaceae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

The relationship of rhizobia that nodulate Galega officinalis to the known species of Rhizobium and Bradyrhizobium was investigated. Similarly, the recently discovered fast­ growing soybean nodulating group of rhizobia was studied. Both groups were investigated using DNA:DNA hybridization as well as nodulation on legumes and phage-typing. The Galega nodulating rhizobia were found to form a distinct DNA homology group. The mean relative homology of 11 strains of Galega nodulating rhizobia with the reference strains gal 1 and gal NW 3, which effectively nodulate Galega officinalis, was significantly higher than the mean relative homology of other groups of rhizobia. The Galega rhizobia only nodulated Galega officinalis and formed a distinct phage-typing group in agreement with the DNA homology results. These rhizobia therefore appear to form a unique taxonomic group within the genus Rhizobium. The fast-growing soybean nodulating rhizobia formed a distinct DNA homology group with at least two subgroups. The mean relative homology of 11 of these strains with the reference strains USDA 208 and USDA 191 which nodulate Glycine max, was significantly higher than the mean relative homology of other groups of rhizobia. Low DNA homologies were found between the fast-growing soybean strains and Bradyrhizobium japonicum ATCC 10324. The fast-growing soybean nodulating rhizobia nodulated glycine max and formed ineffective nodules on Lotus pedunculatus. None of these strains were lysed by the bacteriophages used in the study, but as yet, no bacteriophage specific for this group of rhizobia has been isolated. The fast­ growing soybean nodulating rhizobia were concluded to be taxonomically distinct from other species of Rhizobium. The thermal stability of reassociated DNA duplexes was examined for both the Galega and fast-growing soybean rhizobia and further indicated the uniqueness of both groups. The use of colony hybridization as a means of identifying different strains of Rhizoiium was investigated and was found to be useful in distinguishing between genetically distinct rhizobia and to identify rhizobia within root nodules

Plant Hormones Differentially Control the Sub-Cellular Localization of Plasma Membrane Microdomains during the Early Stage of Soybean Nodulation

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and GmFWL1 or GmFLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are suffcient to trigger or inhibit the translocation of GmFWL1 and GmFLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of GmFWL1 and GmFLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation

Explaining coexistence of nitrogen fixing and non-fixing rhizobia in legume-rhizobia mutualism using mathematical modeling

In the mutualism established between legumes and soil bacteria known as rhizobia, bacteria from soil infect plants roots and reproduce inside root nodules where they fix atmospheric N2 for plant nutrition, receiving carbohydrates in exchange. Host-plant sanctions against non N2 fixing, cheating bacterial symbionts have been proposed to act in the legume-Rhizobium symbiosis, to preserve the mutualistic relationship. Sanctions include decreased rhizobial survival in nodules occupied by cheating rhizobia. Previously, a simple population model experimentally based showed that the coexistence of fixing and cheating rhizobia strains commonly found in field conditions is possible, and that the inclusion of sanctions leads to the extinction of cheating strains in soil. Here, we extend the previous model to include other factors that could complicate the sanction scenario, like horizontal transmission of symbiotic plasmids, turning non-nodulating strains into nodulating rhizobia, and competition between fixing and cheating strains for nodulation. In agreement with previous results, we show that plant populations persist even in the presence of cheating rhizobia without incorporating any sanction against the cheater populations in the model, under the realistic assumption that plants can at least get some amount of fixed N2 from the effectively mutualistic rhizobia occupying some nodules. Inclusion of plant sanctions leads to the unrealistic extinction of cheater strains in soil. Our results agree with increasing experimental evidence and theoretical work showing that mutualisms can persist in presence of cheating partners.Fil: Moyano, Gabriel Eduardo. Universidad Nacional de Córdoba. Facultad de Matemática, Astronomía y Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marco, Diana Elizabeth. Universidad Nacional de Córdoba. Facultad de Cs.exactas Físicas y Naturales. Departamento de Matemáticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Knopoff, Damián Alejandro. Universidad Nacional de Córdoba. Facultad de Matemática, Astronomia y Física. Sección Matemática; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigación y Estudios de Matemática. Universidad Nacional de Córdoba. Centro de Investigación y Estudios de Matemática; ArgentinaFil: Torres, German Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Modelado e Innovación Tecnológica. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Modelado e Innovación Tecnológica; ArgentinaFil: Turner, Cristina Vilma. Universidad Nacional de Córdoba. Facultad de Matemática, Astronomía y Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigación y Estudios de Matemática. Universidad Nacional de Córdoba. Centro de Investigación y Estudios de Matemática; Argentin

An experimental and modelling exploration of the host-sanction hypothesis in legume-rhizobia mutualism

Despite the importance of mutualism as a key ecological process, its persistence in nature is difficult to explain since the existence of exploitative, 'cheating' partners that could erode the interaction is common. By analogy with the proposed policing strategy stabilizing intraspecific cooperation, host sanctions against non N2 fixing, cheating symbionts have been proposed as a force stabilizing mutualism in legume-Rhizobium symbiosis. Following this proposal, penalizations would include decreased nodular rhizobial viability and/or early nodule senescence in nodules occupied by cheating rhizobia. In this work, we analyze the stability of Rhizobium-legume symbiosis when "cheating" strains are present, using an experimental and modelling approach. We used split-root experiments with soybean plants inoculated with two rhizobial strains, a cooperative, normal N2 fixing strain and an isogenic non-fixing, “perfect” cheating mutant derivative that lacks nitrogenase activity but has the same nodulation abilities inoculated to split-root plants. We found no experimental evidence of functioning plant host sanctions to cheater rhizobia based on nodular rhizobia viability and nodule senescence and maturity molecular markers. Based on these experiments, we developed a population dynamic model with and without the inclusion of plant host sanctions. We show that plant populations persist in spite of the presence of cheating rhizobia without the need of incorporating any sanction against the cheater populations in the model, under the realistic assumption that plants can at least get some amount of fixed N2 from the effectively mutualistic rhizobia occupying some nodules. Inclusion of plant sanctions merely reduces the time needed for reaching plant population equilibrium and leads to the unrealistic effect of ultimate extinction of cheater strains in soil. Our simulation results are in agreement with increasing experimental evidence and theoretical work showing that mutualisms can persist or even improve in presence of cheating partners

Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia

The current classification of the rhizobia (root-nodule symbionts) assigns them to six genera. It is strongly influenced by the small subunit (16S, SSU) rRNA molecular phylogeny, but such single-gene phylogenies may not reflect the evolution of the genome as a whole. To test this, parts of the atpD and recA genes have been sequenced for 25 type strains within the alpha -Proteobacteria, representing species in Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Agrobacterium, Phyllobacterium, Mycoplana and Brevundimonas. The current genera Sinorhizobium and Mesorhizobium are well supported by these genes, each forming a distinct phylogenetic clade with unequivocal bootstrap support. There is good support for a Rhizobium clade that includes Agrobacterium tumefaciens, and the very close relationship between Agrobacterium rhizogenes and Rhizobium tropici is confirmed. There is evidence for recombination within the genera Mesorhizobium and Sinorhizobium, but the congruence of the phylogenies at higher levels indicates that the genera are genetically isolated. rRNA provides a reliable distinction between genera, but genetic relationships within a genus may be disturbed by recombination

Test of Host Sanction Hypothesis in Soybean Plants Co-inoculated with Nitrogen Fixing and Non-fixing Bradyrhizobium japonicum

Aims: We tested the proposed mechanism for potential sanctions, that the plant would reduce viability of non-fixing rhizobia inside nodules, performing viable Bradyrhizobium japonicum counts from co-occupied and single-occupied nodules in co-inoculated soybean plants. Study Design: Plants were co-inoculated with two strains of B. japonicum, a highly efficient nitrogen fixing wild-type strain BJD321, and the non-fixing, nifH mutant derivative A3, to produce co-occupied nodules as well as single-occupied nodules. Strain A3 lacks nitrogenase activity but shows similar infection and nodule formation levels respect to the wild-type. As the strains used are equivalent in competitive and nodulation abilities and only differ in the nitrogen fixation ability (by nitrogenase inactivation), and share the same plant, root and even nodule, we can assert that themechanism being tested is plant host sanction, and no other proposed mechanisms like partner choice. Place and Duration of Study: Nitrogen Metabolism Lab, Department of Soil Microbiology and Symbiotic Systems at Zaidín Experimental Station (CSIC State Agency, Granada, Spain). 2010- 2011. Methodology: Axenic seedlings of soybean (Glycine max) cultivar Williams were inoculated with 2 ml of bacterial suspension of BJD321 or A3 strains, alone or in 1:1 mixture and supplied with sterilized N free nutrient solution. Four weeks after inoculation plants of each inoculation treatment (BJD321 + A3, BJD321 only, A3 only) were harvested, nodules were counted and weighed and plated to determine rhizobial strain occupation and population. In the aerial part of plants, determinations of weight, N and C content were done. Results: Co-inoculated plants and plants only inoculated with the BJD321 strain showed a similar nitrogen fixation since they did not differ in dry weight, total N content and total C content. Plants with different inoculation treatments (BJD321 + A3, BJD321 only and A3 only) did not differ in nodule number. In co-inoculated plants, nodule occupation did not differ from the expected among strains (about 33% BJD321 + A3, BJD321 only and A3 only), and the weight of nodules occupied by both strains, BJD321 or A3 alone did not differ. In co-inoculated plants rhizobial viability did not differ between BJD321 and A3 strains, either comparing co-occupied nodules or single-occupied nodules. Nodule size and CFU of rhizobia inside nodules were not correlated, either in coinoculated plants or plants inoculated with BJD321 strain alone. Conclusion: We can conclude that in the soybean-B. japonicum system, plants facing the presence of fixing and non-fixing rhizobial strains do not sanction cheating and can perform as well as plants inoculated with the fixing strain alone.Fil: Marco, Diana Elizabeth. Universidad Nacional de Córdoba. Facultad de Cs.agropecuarias. Area de Producción Organica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Talbi, Chouhra. Universidad Nacional Autónoma de México; MéxicoFil: Bedmar, Eulogio. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; Españ

Multi-purpose cowpea inoculation for improved yields in small holder farms in Kenya

Introduction In Kenya, cowpea is the most important pulse crop in the dry lands of Eastern and Coastal regions where it is commonly inter cropped with maize and sorghum. The poor yields obtained in small holder farms in Kenya (150 kg ha-1) can in part be attributed to the use of poor yielding varieties, low soil fertility (mainly N and P deficiency) low presence of effective indigenous rhizobia and high occurrence of highly competitive but inefficient indigenous rhizobia strains. Biological nitrogen fixation (BNF) through exploitation of the rhizobia-legume symbiosis and use of inoculants coupled with soil amendments such as Phosphorus offers in part a means to improve cowpea yield, nutrition and soil fertility

Denitrification by rhizobia: A possible factor contributing to nitrogen losses from soils

The intensive pastoral farming system on which New Zealand animal production is based is almost completely dependent upon the rhizobium-legurne symbiosis for the fixed nitrogen required for pasture production. The average annual fixation has been measured as 184 kg nitrogen/ha in developed lowland pastures Hoglund et cii., 1979 and about 13 kg nitrogen/ha in poorly developed bill country pastures (Grant and Lambert, 1979). From these figures it can be estimated that rhizobia in New Zealand pastures fix in excess of one million tonnes of nitrogen an nually. The current annual application of fertilizer nitrogen to pastures is about 12 500 tonnes (O'Connor, 1979)