Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere.     

The diversity of Rhizobia, Sinorhizobia and novel non-Rhizobial Paenibacillus nodulating wild herbaceous legumes

The objective of the present study was to isolate and characterize nodulating bacteria associated with wild legumes. For this purpose, we recovered twenty isolates from root nodules of five wild legume species: Melilotus alles, Melilotus officinalis, Trifolium pratense, Trifolium repens and Medicago sp. Most of the isolates were morphologically analogous with only few exceptions in colony shape, appearance and incubation time. All isolates were Gram negative except T.P2-4. Random amplification of polymorphic DNA showed genetic variation among isolates. The 16S rRNA sequence analysis revealed these isolates as Rhizobium, Sinorhizobium and Paenibacillus. Each of these was also screened for nod D and nod F genes with marked variation at these loci; however, the nucleotide sequence analysis confirmed the presence of nod genes. The assignment of strains to their hosts revealed a unique symbiotic association of Paenibacillus sp. nodulating T .pratense which is being reported here for the first time.     

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.     

Molecular characterization of novel Bradyrhizobium strains nodulating Eriosema chinense and Flemingia vestita,important unexplored native legumes of the sub-Himalayan region (Meghalaya) of India

Root nodule bacterial strains were isolated from the little-studied legumes Eriosema chinense and Flemingia vestita (both in tribe Phaseoleae, Papilionoideae) growing in acidic soil of the sub-Himalayan region of the Indian state of Meghalaya (ME), and were identified as novel strains of Bradyrhizobium on the basis of their 16S rRNA sequences. Seven isolates selected on the basis of phenotypic characters and assessment of ARDRA and RAPD patterns were subjected to multilocus sequence analysis (MLSA) using four protein-coding housekeeping genes (glnII, recA, dnaK and gyrB). On the basis of 16S rRNA phylogeny as well as a concatenated MLSA five strains clustered in a single separate clade and two strains formed novel lineages within the genus Bradyrhizobium. The phylogenies of the symbiotic genes (nodA and nifH) were in agreement with the core gene phylogenies. It appears that genetically diverse Bradyrhizobium strains are the principal microsymbionts of these two important native legumes. The novel genotypes of Bradyrhizobium strains isolated in the present study efficiently nodulate the Phaseoloid crop species Glycine max, Vigna radiata and Vigna umbellata. These strains are genetically different from strains of Bradyrhizobium isolated earlier from a different agro-climatic region of India suggesting that the acidic nature of the soil, high precipitation and other local environmental conditions are responsible for the evolution of these newly-described Bradyrhizobium strains. In global terms, the sub-Himalayan region of India is geographically and climatically distinct and the Bradyrhizobium strains nodulating its legumes appear to be novel and potentially unique to the region.     

Diversity of sporadic symbionts and nonsymbiotic endophytic bacteria isolated from nodules of woody, shrub, and food legumes in Ethiopia

Fifty-five bacterial isolates were obtained from surface-sterilized nodules of woody and shrub legumes growing in Ethiopia: Crotalaria spp., Indigofera spp., and Erythrina brucei, and the food legumes soybean and common bean. Based on partial 16S rRNA gene sequence analysis, the majority of the isolates were identified as Gram-negative bacteria belonging to the genera Achromobacter, Agrobacterium, Burkholderia, Cronobacter, Enterobacter, Mesorhizobium, Novosphingobium, Pantoea, Pseudomonas, Rahnella, Rhizobium, Serratia, and Variovorax. Seven isolates were Gram-positive bacteria belonging to the genera Bacillus, Paenibacillus, Planomicrobium, and Rhodococcus. Amplified fragment length polymorphism (AFLP) fingerprinting showed that each strain was genetically distinct. According to phylogenetic analysis of recA, glnII, rpoB, and 16S rRNA gene sequences, Rhizobium, Mesorhizobium, and Agrobacterium were further classified into six different genospecies: Agrobacterium spp., Agrobacterium radiobacter, Rhizobium sp., Rhizobium phaseoli, Mesorhizobium sp., and putative new Rhizobium species. The strains from R. phaseoli, Rhizobium sp. IAR30, and Mesorhizobium sp. ERR6 induced nodules on their host plants. The other strains did not form nodules on their original host. Nine endophytic bacterial strains representing seven genera, Agrobacterium, Burkholderia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium, and Serratia, were found to colonize nodules of Crotalaria incana and common bean on co-inoculation with symbiotic rhizobia. Four endophytic Rhizobium and two Agrobacterium strains had identical nifH gene sequences with symbiotic Rhizobium strains, suggesting horizontal gene transfer. Most symbiotic and nonsymbiotic endophytic bacteria showed plant growth-promoting properties in vitro, which indicate their potential role in the promotion of plant growth when colonizing plant roots and the rhizosphere.     

Evidence of Environmental and Vertical Transmission of Burkholderia Symbionts in the Oriental Chinch Bug,Cavelerius saccharivorus (Heteroptera: Blissidae)

The vertical transmission of symbiotic microorganisms is omnipresent in insects, while the evolutionary process remains totally unclear. The oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae), is a serious sugarcane pest, in which symbiotic bacteria densely populate the lumen of the numerous tubule-like midgut crypts that the chinch bug develops. Cloning and sequence analyses of the 16S rRNA genes revealed that the crypts were dominated by a specific group of bacteria belonging to the genus Burkholderia of the Betaproteobacteria. The Burkholderia sequences were distributed into three distinct clades: the Burkholderia cepacia complex (BCC), the plant-associated beneficial and environmental (PBE) group, and the stinkbug-associated beneficial and environmental group (SBE). Diagnostic PCR revealed that only one of the three groups of Burkholderia was present in ∼89% of the chinch bug field populations tested, while infections with multiple Burkholderia groups within one insect were observed in only ∼10%. Deep sequencing of the 16S rRNA gene confirmed that the Burkholderia bacteria specifically colonized the crypts and were dominated by one of three Burkholderia groups. The lack of phylogenetic congruence between the symbiont and the host population strongly suggested host-symbiont promiscuity, which is probably caused by environmental acquisition of the symbionts by some hosts. Meanwhile, inspections of eggs and hatchlings by diagnostic PCR and egg surface sterilization demonstrated that almost 30% of the hatchlings vertically acquire symbiotic Burkholderia via symbiont-contaminated egg surfaces. The mixed strategy of symbiont transmission found in the oriental chinch bug might be an intermediate stage in evolution from environmental acquisition to strict vertical transmission in insects.     

Profiling a besieged flora: endemic and threatened plants of the Cape Peninsula, South Africa

The Cape Peninsula (area: 471 km²), situated at the south-western extremity of the Cape Floristic Region, has exceptionally high plant species richness (2285 species and infraspecific taxa) and numbers of endemic (90; 88 species and two infraspecific) and threatened (141; 138 species and three infraspecific) taxa (termed species from here on). This biodiversity is threatened by urban development and the spread of invasive alien plants. Peninsula endemics are concentrated in a few, predominantly species-rich families and these correspond well with endemic-rich families in other areas of the Cape Floristic Region. A high level of similarity exists between families with threatened and families with endemic species. A frequency analysis of the biological traits of both endemic and threatened species shows that low growing, ant-dispersed shrubs are over-represented in both groups. Endemics are most likely to be non-sprouters, but threatened plants do not have a specific post-fire regeneration strategy. Threatened species have higher frequencies of geophytes, sprouters and wind-dispersed species compared to endemic species. Numbers of endemic and threatened species are not randomly distributed with regard to occurrence in vegetation types and patterns are similar for both groups. The habitat and biological profiles of both endemic and threatened species suggest that they are highly vulnerable to extinction as a result of increasing rates of alien plant infestation, urbanization and inappropriate fire regimes.     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariae-josephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain

The genomic diversity of a collection of 103 indigenous rhizobia isolates from Lupinus mariae-josephae (Lmj), a recently described Lupinus species endemic to alkaline-limed soils from a restricted habitat in Eastern Spain, was investigated by molecular methods. Isolates were obtained from soils of four geographic locations in the Valencia province that harbored the known Lmj plant populations. Using an M13 RAPD fingerprinting technique, 19 distinct RAPD profiles were identified. Phylogenetic analysis based on 16S rDNA and the housekeeping genes glnII, recA and atpD showed a high diversity of native Bradyrhizobium strains that were able to establish symbiosis with Lmj. All the strains grouped in a clade unrelated to strains of the B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. The phylogenetic tree based on concatenated glnII, recA and atpD gene sequences grouped the Lmj isolates in six different operational taxonomic units (OTUs) at the 93% similarity level. These OTUs were not associated to any specific geographical location, and their observed divergence predicted the existence of different Bradyrhizobium genomic species. In contrast, phylogenetic analysis of symbiotic genes based on nodC and nodA gene sequences, defined only two distinct clusters among the Lmj strains. These two Lmj nod gene types were largely distinct from nod genes of bradyrhizobia nodulating other Old World lupine species. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host.     

Recent changes to the classification of symbiotic,nitrogen-fixing,legume-associating bacteria: a review

The Rhizobia are collectively comprised of gram negative soil bacteria that have the ability to form symbiotic nitrogen-fixing root and/or stem nodules in association with leguminous plants. The taxonomy of these bacteria is continually in a state of flux, in large part due to rapid development of refined molecular biology techniques. The isolation and characterization of new, and often different, legumes-nodulating bacteria on a variety of plant hosts has resulted in the naming of many new rhizobial species. Here we update the taxonomy of the legume-nodulating bacteria and describe newly identified rhizobia capable of nodulating edible legumes and legume trees. In 1990, there was only one bacterial species that was known to nodulate common bean worldwide (Rhizobium leguminosarum sv. phaseoli), one species that nodulated faba bean (Rhizobium leguminosarum sv. viciae), and two species that nodulated soybean (Bradyrhizobium japonicum and Rhizobium fredii). Today, nearly 14, 11, 6, 5, 5, 4, 3 and 2 species have been defined that are capable of nodulating common bean, soybean, cowpea, chickpea, peanut, lentils, faba bean and pea, respectively. The recent use of whole genome based taxonomy (genomotaxonomy) will surely change how we define this important group of bacteria. The identification of several rhizobial species that are able to nodulate and fix nitrogen with edible legumes may enhance the production of these crops and can compensate for worldwide deficiencies in human nutritional needs in the future.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. Nodule Bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both β-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

Diverse Mesorhizobium bacteria nodulate native Astragalus and Oxytropis in arctic and subarctic areas in Eurasia

Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. ¹⁵N analysis suggested that the legumes in association with these isolates were actively fixing nitrogen.     

Genotypic and symbiotic diversity of Rhizobium populations associated with cultivated lentil and pea in sub-humid and semi-arid regions of Eastern Algeria

The genetic structure of rhizobia nodulating pea and lentil in Algeria, Northern Africa was determined. A total of 237 isolates were obtained from root nodules collected on lentil (Lens culinaris), proteaginous and forage pea (Pisum sativum) growing in two eco-climatic zones, sub-humid and semi-arid, in Eastern Algeria. They were characterised by PCR-restriction fragment length polymorphism (RFLP) of the 16S–23S rRNA intergenic region (IGS), and the nodD-F symbiotic region. The combination of these haplotypes allowed the isolates to be clustered into 26 distinct genotypes, and all isolates were classified as Rhizobium leguminosarum. Symbiotic marker variation (nodD-F) was low but with the predominance of one nod haplotype (g), which had been recovered previously at a high frequency in Europe. Sequence analysis of the IGS further confirmed its high variability in the studied strains. An AMOVA analysis showed highly significant differentiation in the IGS haplotype distribution between populations from both eco-climatic zones. This differentiation was reflected by differences in dominant genotype frequencies. Conversely, no host plant effect was detected. The nodD gene sequence-based phylogeny suggested that symbiotic gene diversity in pea and lentil nodulating rhizobial populations in Algeria was low compared to that reported elsewhere in the world.     

Characterization of bacteria associated with nodules of two endemic legumes of Algeria,Hedysarum naudinianum and H. perrauderianum

The root nodules of two wild legume species endemic to Algeria, Hedysarum naudinianum and He. perrauderianum, were investigated with regard to their anatomy and histology, and the identity of the associated bacteria. Both plants were found to form root nodules with regular features and well infected by rod-shaped bacteria. The culturable fraction of bacteria that could be obtained from surface-sterilized nodules included a prevailing presence of Enterobacteriaceae having 100 % 16S rDNA sequence identity with both Enterobacter cloacae and E. ludwigii. In H. perrauderianum, this taxon was the sole cultured isolate, while from H. naudinianum we also found Bacillus, Lactobacillus, Staphylococcus, Rothia, and isolates that were 100 % identical to Corynebacterium pseudodiphthericum, which is known to be an agent of respiratory and cardiac infections in humans. Whereas no culturable rhizobia and alike could be obtained on plates, PCR-based culture-independent approaches revealed in both plants the presence of a Mesorhizobium sp., which in H. perrauderianum was identical to isolates nodulating other legumes from Africa, European Mediterranean countries, and Asia, while in H. naudinianum it bore a single nucleotide polymorphism which is so far unique for any observed mesorhizobia. Data from the microsymbionts appear to suggest interesting clues to interpret the evolutionary ecology of their host plants.     

Plant-Associated Symbiotic Burkholderia Species Lack Hallmark Strategies Required in Mammalian Pathogenesis

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.     

Molecular diversity and phylogeny of indigenous <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> strains associated with <Emphasis Type="Italic">Trifolium repens</Emphasis> plants in Romania

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.     

Gazania lanata and G. splendidissima: Two new species of Asteraceae (tribe Arctotideae) from the Greater Capensis, with an updated key for the genus

Two new species of Gazania Gaert. (Asteraceae, tribe Arctotideae) from the Greater Cape Floristic Region of South Africa are described. Gazania lanata Magee & Boatwr., a local endemic of dolomite lenses near Robertson, is distinguished by the spatulate to sublyrate, simple or weakly pinnatilobed leaves, the bristle-like hairs along the inflorescence and the densely lanate vestiture of the young growth and involucre. Gazania splendidissima Mucina, Magee & Boatwr. from the arid Namaqualand coast between Port Nolloth and Hondeklipbaai, is distinguished by the suffrutescent habit, semi-succulent leaves with a densely mealy uppermost surface, and prominently maculate involucre. An updated key to the now 18 recognised Gazania species is provided to facilitate identification of these and existing taxa in the genus.