Photosynthetic Bradyrhizobia from Aeschynomene spp. Are Specific to Stem-Nodulated Species and Form a Separate 16S Ribosomal DNA Restriction Fragment Length Polymorphism Group

We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene species A. elaphroxylon, A. uniflora, or A. schimperi and 69 root or stem nodule isolates from the stem-nodulated Aeschynomene species A. afraspera, A. ciliata, A. indica, A. nilotica, A. sensitiva, and A. tambacoundensis from various places in Senegal. These isolates, together with 45 previous isolates from various Aeschynomene species, were studied for host-specific nodulation within the genus Aeschynomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732–734, 1985). The whole collection of Aeschynomene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of puf genes in photosynthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 Aeschynomene isolates, including strain BTAi1, and 19 reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii, and other Bradyrhizobium sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomene nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicum and B. elkanii.     

Rhizobium–legume symbiosis in the absence of Nod factors: two possible scenarios with or without the T3SS

The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NF-independent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all non-photosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism.     

Phylogenetic Analysis of Bradyrhizobium japonicum and Photosynthetic Stem-Nodulating Bacteria from Aeschynomene Species Grown in Separated Geographical Regions

Nearly complete and short partial 16S rRNA sequences were derived from PCR-amplified ribosomal DNAs of Bradyrhizobium japonicum USDA 136 and USDA 110 and five strains of bacteriochlorophyll-synthesizing bacteria isolated from stem nodules of Aeschynomene indica and other Aeschynomene species growing in different geographic regions, including India, The Philippines and North America. We confirmed that the five stem-nodulating strains examined synthesize bacteriochlorophyll a, and the absorption spectra of methanol-extracted cells contained a major absorbance peak at 770 nm. Strains isolated on different continents and from different Aeschynomene species were found to be phylogenetically homogeneous and exhibited levels of sequence similarity of more than 99%. The bacteriochlorophyll-synthesizing rhizobia, Bradyrhizobium japonicum, Blastobacter denitrificans, Afipia felis, and Rhodopseudomonas palustris exhibited levels of sequence similarity of 97% or greater and belong to a distinct line of descent within the alpha-2 subdivision of the Proteobacteria. Variable regions between positions 995 and 1045 provide potential target sites for design of a probe that is able to distinguish the photosynthetic rhizobia from closely related taxa.     

Photosynthetic Bradyrhizobia Are Natural Endophytes of the African Wild Rice Oryza breviligulata

We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulata could significantly enhance cultivated rice production.     

The LPS O-Antigen in Photosynthetic Bradyrhizobium Strains Is Dispensable for the Establishment of a Successful Symbiosis with Aeschynomene Legumes

The photosynthetic bradyrhizobia are able to use a Nod-factor independent process to induce nitrogen-fixing nodules on some semi-aquatic Aeschynomene species. These bacteria display a unique LPS O-antigen composed of a new sugar, the bradyrhizose that is regarded as a key symbiotic factor due to its non-immunogenic character. In this study, to check this hypothesis, we isolated mutants affected in the O-antigen synthesis by screening a transposon mutant library of the ORS285 strain for clones altered in colony morphology. Over the 10,000 mutants screened, five were selected and found to be mutated in two genes, rfaL, encoding for a putative O-antigen ligase and gdh encoding for a putative dTDP-glucose 4,6-dehydratase. Biochemical analysis confirmed that the LPS of these mutants completely lack the O-antigen region. However, no effect of the mutations could be detected on the symbiotic properties of the mutants indicating that the O-antigen region of photosynthetic Bradyrhizobium strains is not required for the establishment of symbiosis with Aeschynomene.     

Floral ontogeny of Aeschynomene falcata and A. sensitiva (Leguminosae: Papilionoideae) supports molecular phylogenetic data

Floral ontogeny and morphology of the Leguminosae are of interest because of their potential to provide characteristics useful for phylogeny. To determine if these features corroborate the phylogenetic segregation of the section Ochopodium from Aeschynomene, this study used comparative analysis between Aeschynomene falcata and A. sensitiva, which are within the sections Ochopodium and Aeschynomene, respectively. Flower buds were analysed by use of scanning electron microscopy. Aeschynomene falcata has a unidirectional initiation of sepals from the abaxial side, and a tendency toward whorled initiation for petals and stamens. At maturity, it has a calyx tube with five lobes, a pubescent standard petal, keel petals with coherent (but not fused) margins above and below the stamens, and a carpel with a long hairy stipe. Aeschynomene sensitiva has a distinct initiation pattern of petals (1st abaxial, 2nd adaxial, and 3rd lateral) and a tendency toward whorled initiation of sepals and stamens. Overlap between sepals, petals, and antesepalous stamens initiation was observed. At maturity, A. sensitiva has a glabrous bilobed calyx and a glabrous standard petal, keel petals postgenitally fused above the stamens, and a carpel with a short and glabrous stipe. Floral ontogeny and morphology of A. falcata are very similar to those of Machaerium and Dalbergia species so far studied, corroborating the phylogenetic proximity of section Ochopodium to these genera. Important features of the floral ontogeny of A. sensitiva seem to be related to the origin of the bilobed calyx, which is shared with the rest of Aeschynomeninae except section Ochopodium, suggesting they are synapomorphies for those species.     

The nodulation of micro-propagated plants of Parasponia andersonii by tropical legume rhizobia

A simple clonal micro-propagation system for Parasponia andersoniiwas employed to study the nodulation response of this non-legumeto inoculation by the broad host range Rhizobium sp. NGR234,isolated from Lablab purpureus, and also to tropical legumerhizobia isolated from Aeschynomene species. Partially effectivenodules, assayed by acetylene reduction and ¹⁵N dilution procedures,were induced with strain NGR234 and its spontaneous streptomycinresistantmutant ANU240. Effective nodules were produced by one of theAeschynomene strains (ORS302) tested, with rates of acetylenereduction comparable to those of root nodules produced by Bradyrhizobiumstrain CP279, originally isolated from P. andersonii. Lightand transmission electron microscopy showed that there was acorrelation between the nitrogen fixing capability of the symbiosisbetween NGR234 and Parasponia and the number of persistent infection(fixation) threads within the nodule cells. Key words: Parasponia, Bradyrhizobium, Rhizobium, Aeschynomene, micro-propagation, root nodules, nitrogen fixation     

Multilocus sequence analysis of bradyrhizobia isolated from Aeschynomene species in Senegal

This study reports the multilocus sequence analysis (MLSA) of nine house-keeping gene fragments (atpD, dnaK, glnA, glnB, gltA, gyrB, recA, rpoB and thrC) on a collection of 38 Bradyrhizobium isolated from Aeschynomene species in Senegal, which had previously been characterised by several phenotypic and genotypic techniques, allowing a comparative analysis of MLSA resolution power for species delineation in this genus. The nifH locus was also studied to compare house-keeping and symbiotic gene phylogenies and obtain insights into the unusual symbiotic properties of these Aeschynomene symbionts. Phylogenetic analyses (maximum likelihood, Bayesian) of concatenated nine loci produced a well-resolved phylogeny of the strain collection separating photosynthetic bradyrhizobial strains (PB) from non-photosynthetic bradyrhizobial (NPB) ones. The PB clade was interpreted as the remains an expanding ancient species that presently shows high diversification, giving rise to potential new species. B. denitrificans LMG8443 and BTAi1 strains formed a sub-clade that was identified as recently emerging new species. Congruence analyses (by Shimodaira–Hasegawa (S–H) tests) identified three gene-fragments (dnaK, glnB and recA) that should be preferred for MLSA analyses in Bradyrhizobium genus. The nine loci or nifH phylogenies were not correlated with the unusual symbiotic properties of PB (nod-dependent/nod-independent). Advantages and drawbacks of MLSA for species delineation in Bradyrhizobium are discussed.     

Symbiotic properties of a chimeric Nod-independent photosynthetic Bradyrhizobium strain obtained by conjugative transfer of a symbiotic plasmid

The lateral transfer of symbiotic genes converting a predisposed soil bacteria into a legume symbiont has occurred repeatedly and independently during the evolution of rhizobia. We experimented the transfer of a symbiotic plasmid between Bradyrhizobium strains. The originality of the DOA9 donor is that it harbours a symbiotic mega-plasmid (pDOA9) containing nod, nif and T3SS genes while the ORS278 recipient has the unique property of inducing nodules on some Aeschynomene species in the absence of Nod factors (NFs). We observed that the chimeric strain ORS278-pDOA9* lost its ability to develop a functional symbiosis with Aeschynomene. indica and Aeschynomene evenia. The mutation of rhcN and nodB led to partial restoration of nodule efficiency, indicating that T3SS effectors and NFs block the establishment of the NF-independent symbiosis. Conversely, ORS278-pDOA9* strain acquired the ability to form nodules on Crotalaria juncea and Macroptillium artropurpureum but not on NF-dependent Aeschynomene (A. afraspera and A. americana), suggesting that the ORS278 strain also harbours incompatible factors that block the interaction with these species. These data indicate that the symbiotic properties of a chimeric rhizobia cannot be anticipated due to new combination of symbiotic and non-symbiotic determinants that may interfere during the interaction with the host plant.     

The Aquatic Budding Bacterium Blastobacter denitrificans Is a Nitrogen-Fixing Symbiont of Aeschynomene indica

Blastobacter spp. are freshwater bacteria that form rosette structures by cellular attachment to a common base. Comparative analyses of ribosomal 16S rRNA gene and internally transcribed spacer region sequences indicated that B. denitrificans is a member of the α-subdivision of Proteobacteria. Among the α-Proteobacteria, B. denitrificans was related to a cluster of genera, including Rhodopseudomonas palustris, Afipia felis, Nitrobacter hamburgensis, and Bradyrhizobium spp. Although the precise phylogenetic relationships among these genera could not be established with a high degree of confidence, the sequences of B. denitrificans and several bradyrhizobial isolates from nodules of Aeschynomene indica were almost identical. Bradyrhizobia are bacteria that form nitrogen-fixing symbioses with legumes, including soybeans (Glycine max) and members of the genus Aeschynomene. From symbiotic infectiveness tests we demonstrated that the type strain for B. denitrificans, IFAM 1005, was capable of forming an effective nitrogen-fixing symbiosis with A. indica. Not only do these results reveal a previously unknown ecological adaptation of a relatively obscure aquatic bacterium, but they also demonstrate how evidence gathered from molecular systematic analyses can sometimes provide clues for predicting ecological behavior.     

Nod factor-independent ‘crack-entry’ symbiosis in dalbergoid legume Arachis hypogaea

Dalbergoids are typified by crack-entry symbiosis which is evidenced to be Nod Factor (NF)-independent in several Aeschynomene legumes. Natural symbionts of the dalbergoid legume Arachis hypogaea are always NF-producing, prompting us to check whether symbiosis in this legume could also be NF-independent. For this, we followed the symbiosis with two NF-containing bradyrhizobial strains – SEMIA6144, a natural symbiont of Arachis and ORS285, a versatile nodulator of Aeschynomene legumes, along with their corresponding nodulation (nod) mutants. Additionally, we investigated NF-deficient bradyrhizobia like BTAi1, a natural symbiont of Aeschynomene indica and the WBOS strains that were natural endophytes of Oryza sativa, collected from an Arachis-Oryza intercropped field. While SEMIA6144ΔnodC was non-nodulating, both ORS285 and ORS285ΔnodB could induce functional nodulation, although with lower efficiency than SEMIA6144. On the other hand, all the NF-deficient strains – BTAi1, WBOS2 and WBOS4 showed comparable nodulation with ORS285 indicating Arachis to harbour an NF-independent mechanism of symbiosis. Intriguingly, symbiosis in Arachis, irrespective of whether it was NF-dependent or independent, was always associated with the curling or branching of the rosette root hairs at the lateral root bases. Thus, despite being predominantly described as an NF-dependent legume, Arachis does retain a vestigial, less-efficient form of NF-independent symbiosis.     

Nitrogen-fixing symbiosis between photosynthetic bacteria and legumes

Rhizobia having photosynthetic systems form nitrogen-fixing nodules on the stem and/or root of some species of the legumes Aeschynomene and Lotononis. This review is focused on the recent knowledge about the physiology, genetics and role of the photosystem in these bacteria. Photosynthetic electron transport seems to involve reaction centers, soluble cytochrome c2 and cytochrome bc1. Anaerobically, the electron transport system becomes over-reduced. The photosynthesis genes have been partially characterized; their organization is classical but their regulation is unusual as it is activated by far-red light via a bacteriophytochrome. This original mechanism of regulation seems well adapted to promote photosynthesis during stem symbiosis. Photosynthesis plays a major role in the efficiency of stem nodulation. It is also observed that infrared light stimulates nitrogen fixation in nodules containing photosynthetic bacteroids, suggesting that photosynthesis may additionally provides energy for nitrogen fixation, allowing for more efficient plant growth. Other aspects of these bacteria are discussed, in particular their taxonomic position and nodulation ability, the role of carotenoids and the potential for application of photosynthetic rhizobia in rice culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

N2 fixation in phototrophs: adaptation to a specialized way of life

Phototrophic diazotrophs include the photosynthetic green and purple bacteria, the heliobacteria, many cyanobacteria and the unusual chlorophyll-containing rhizobia that are found in the stem nodules of Aeschynomene spp. In this review, which concentrates on cyanobacteria, the interrelations between photosynthesis and N₂ fixation are discussed. Photosynthesis can, in theory, directly provide the ATP and reductant needed to support N₂ fixation but the link between these two processes is usually indirect, mediated through accumulated carbon reserves. In cyanobacteria, which possess an oxygenic photosynthesis, this serves to separate the O₂ that is produced by photosynthesis from the O₂-sensitive nitrogenase. However, in certain circumstances, oxygenic photosynthesis and N₂ fixation coexist. Under these conditions, respiratory consumption of photosynthetically generated O₂ may have an important role in minimizing O₂-damage to nitrogenase.     

Hard ticks (Acari,Ixodidae) of Croatia

The present paper is based on original and literature data. In Croatia the first studies on the occurrence of ixodid species were made about 80 years ago. The number of tick species recorded in Croatia considerably increased during the 1950s, 60s, 70s and 80s of the past century. A total of 21 species of hard tick belonging to 5 genera have been recorded in Croatia. Ixodes is the best represented genus, with seven species recorded. Haemaphysalis is represented by six species, followed by Rhipicephalus with four species. Dermacentor and Hyalomma are represented by two species each. The ticks were collected on 47 different host species. Eleven tick species were collected on Bos taurus and Ovis aries, followed by Capra hircus and Equus caballus with 8 species and Canis lupus familiaris with 6 species. On the remaining 42 host species one, two or three tick species were collected. The most widespread tick is Ixodes ricinus which was found on 25 different host species.     

Taxonomic survey of fossil isocrinids with a list of the species found in the USSR

The taxonomic scheme of the two families of the order Isocrinida (Isocrinidae and Pentacrinidae) is given. The first family is divided at five subfamilies: Balanocrininae, Isocrininae, Metacrininae, Diplocrininae and Isselicrininae. Six genera are included in subfamily Balanocrininae: Balanocrinus (four species found in USSR), Laevigatocrinus (USSR: 3 species), Margocrinus (USSR: 4 species), Percevalicrinus (USSR: 5 species), Singularocrinus nov. gen. (monotypic) and Terocrinus nov. gen. (USSR: 1 species). Five fossil genera are included in subfamily Isocrininae: Chariocrinus (USSR: 1 species), Chladocrinus (USSR: 4 species), Isocrinus (USSR: 6 species), Raymondicrinus nov. gen. (Oligocene of USA : 2 species) and Tyrolecrinus nov. gen. (6 triassic species). Fossil representatives of three genera, namely, Metacrinus (Miocene-Recent), Nielsenicrinus (USSR: 4 species) and Cainocrinus (USSR: 1 species) are noted in subfamily Metacrininae. Five genera are included in subfamily Isselicrininae: Austinocrinus (USSR: 5 species), Buchicrinus (USSR: 5 species), Doreckicrinus (USSR: 1 species?), Isselicrinus (USSR: 4 species) and Praeisselicrinus (USSR: 1 species). Two genera are included in family Pentacrinidae: Pentacrinus (USSR: 1 species) and Seirocrinus (USSR: 4 species). Besides, the localities of 24 isocrinid species, systematic position of which is unknown, are listed (from Triassic upon Cretaceous). Three erroneous attributions to Isocrinida in USSR are pointed out. In the conclusion an outline of the phylogeny of the Isocrinida is discussed. For a majority of the wide-spread species in USSR figures are given.     

Thysanoptera of Bulgaria

The present checklist includes data on the species composition, geographic distribution and feeding preferences of thrips species in Bulgaria. In total, 155 species in 48 genera are listed. Of these, 125 species belong to suborder Terebrantia and include 103 species of 33 genera in family Thripidae, 14 species of two genera in Aeolothripidae, seven species of two genera in Melanthripidae and one species in Fauriellidae. In suborder Tubulifera, 30 species of 10 genera in the single family Phlaeothripidae are listed. Of the 155 Bulgarian thrips species, 87.7% are phytophagous, 4.5% are obligate predators, 5.8% are mycophagous and 1.9% are with unknown feeding preferences. Fourteen pest species are listed for Bulgaria, of which Frankliniella occidentalis, Thrips tabaci and Haplothrips tritici are of economic importance. The list provides detailed information on the horizontal and vertical distribution of Thysanoptera in 5 regions and 45 subregions of Bulgaria. The present paper also includes an evaluation of the biodiversity of Thysanoptera and the extent to which each region of the country has been studied.     

History and current taxonomic status of genus Agrobacterium

The genus Agrobacterium was created a century ago by Conn who included it in the family Rhizobiaceae together with the genus Rhizobium. Initially, the genus Agrobacterium contained the non-pathogenic species Agrobacterium radiobacter and the plant pathogenic species Agrobacterium tumefaciens and Agrobacterium rhizogenes. At the end of the past century two new pathogenic species, Agrobacterium rubi and Agrobacterium vitis, were added to the genus. Already in the present century these species plus Agrobacterium larrymoorei were reclassified into genus Rhizobium. This reclassification was controversial and for a time both genus names were used when new species were described. Few years ago, after a taxonomic revision based on genomic data, the old species A. rhizogenes was maintained in the genus Rhizobium, the old species A. vitis was transferred to the genus Allorhizobium and several Rhizobium species were transferred to the genus Agrobacterium, which currently contains 14 species including the old species A. radiobacter, A. tumefaciens, A. rubi and A. larrymoorei. Most of these species are able to produce tumours in different plants, nevertheless the genus Agrobacterium also encompasses non-pathogenic species, one species able to nodulate legumes and one human pathogenic species. Taking into account that the species affiliations to five Agrobacterium genomospecies have not been determined yet, an increase in the number of species within this genus is expected in the near future.