An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host–symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host–symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host–symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed.     

Molecular Method To Assess the Diversity of Burkholderia Species in Environmental Samples

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.     

Multivariate Analyses of Burkholderia Species in Soil: Effect of Crop and Land Use History

The assessment of Burkholderia diversity in agricultural areas is important considering the potential use of this genus for agronomic and environmental applications. Therefore, the aim of this work was to ascertain how plant species and land use management drive the diversity of the genus Burkholderia. In a greenhouse experiment, different crops, i.e., maize, oat, barley, and grass, were planted in pots containing soils with different land use histories, i.e., maize monoculture, crop rotation, and permanent grassland, for three consecutive growth cycles. The diversity of Burkholderia spp. in the rhizosphere soil was assessed by genus-specific PCR-denaturing gradient gel electrophoresis (DGGE) and analyzed by canonical correspondence analysis (CCA). CCA ordination plots showed that previous land use was the main factor affecting the composition of the Burkholderia community. Although most variation in the Burkholderia community structure was observed between the permanent grassland and agricultural areas, differences between the crop rotation and maize monoculture groups were also observed. Plant species affected Burkholderia community structure to a lesser extent than did land use history. Similarities were observed between Burkholderia populations associated with maize and grass, on the one hand, and between those associated with barley and oat, on the other hand. Additionally, CCA ordination plots demonstrated that these two groups (maize/grass versus barley/oat) had a negative correlation. The identification of bands from the DGGE patterns demonstrated that the species correlated with the environmental variables were mainly affiliated with Burkholderia species that are commonly isolated from soil, in particular Burkholderia glathei, B. caledonica, B. hospita, and B. caribiensis.     

Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis.     

Characterization of Burkholderia bacteria clade compositions in soil and Riptortus pedestris (Hemiptera: Alydidae) in South Korea

Riptortus pedestris (Hemiptera: Alydidae) is known to acquire the genus Burkholderia, symbiotic bacteria, from soil. Therefore, symbiont acquisition of R. pedestris would be directly affected by bacterial diversity in soil. Soil typically harbors diverse microbes including different Burkholderia clades such as SBE (stinkbug-associated beneficial and environmental), PBE (plant-associated beneficial and environmental), and BCC (Burkholderia cepacia and complex). Nevertheless, little is known about Burkholderia acquisition patterns of R. pedestris in nature, especially in the context of bacteria clade compositions in soil. Therefore, based on diagnostic PCR analysis, we investigated Burkholderia clade compositions in field-collected soil itself and R. pedestris when the insects were provided with the soil. Also, wild R. pedestris were surveyed to characterize their Burkholderia compositions. First, 88.44% of soil samples were detected with the genus Burkholderia, and triple clade (SBE + PBE + BCC) was most frequently detected. Second, R. pedestris nymphs readily acquired Burkholderia bacteria from field-collected soil where 91.25% of the reared insects harbored the bacteria in their midguts. In contrast to soil, the detection of single BCC clade was the most dominant among the three identified Burkholderia clades. Third, from wild R. pedestris, 80.62% of the insects were found to harbor the genus Burkholderia, and single BCC clade was most frequently detected. Finally, 29.13% and 47.06% of the reared and wild R. pedestris were detected with unidentified Burkholderia clade, which does not belong to any of the three identified clades. Our findings provide baseline information to better understand ecological associations between R. pedestris and Burkholderia bacteria in different clades.     

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.     

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.     

Genome characterization of a novel Burkholderia cepacia complex genomovar isolated from dieback affected mango orchards

We characterized the genome of the antibiotic resistant, caseinolytic and non-hemolytic Burkholderia sp. strain TJI49, isolated from mango trees (Mangifera indica L.) with dieback disease. This isolate produced severe disease symptoms on the indicator plants. Next generation DNA sequencing and short-read assembly generated the 60X deep 7,631,934 nucleotide draft genome of Burkholderia sp. TJI49 which comprised three chromosomes and at least one mega plasmid. Genome annotation studies revealed a total 8,992 genes, out of which 8,940 were protein coding genes. Comparative genomics and phylogenetics identified Burkholderia sp. TJI49 as a distinct species of Burkholderia cepacia complex (BCC), closely related to B. multivorans ATCC17616. Genome-wide sequence alignment of this isolate with replicons of BCC members showed conservation of core function genes but considerable variations in accessory genes. Subsystem-based gene annotation identified the active presence of wide spread colonization island and type VI secretion system in Burkholderia sp. TJI49. Sequence comparisons revealed (a) 28 novel ORFs that have no database matches and (b) 23 ORFs with orthologues in species other than Burkholderia, indicating horizontal gene transfer events. Fold recognition of novel ORFs identified genes encoding pertactin autotransporter-like proteins (a constituent of type V secretion system) and Hap adhesion-like proteins (involved in cell–cell adhesion) in the genome of Burkholderia sp. TJI49. The genomic characterization of this isolate provided additional information related to the ‘pan-genome’ of Burkholderia species.     

Invasion of Spores of the Arbuscular Mycorrhizal Fungus Gigaspora decipiens by Burkholderia spp.

Burkholderia species are bacterial soil inhabitants that are capable of interacting with a variety of eukaryotes, in some cases occupying intracellular habitats. Pathogenic and nonpathogenic Burkholderia spp., including B. vietnamiensis, B. cepacia, and B. pseudomallei, were grown on germinating spores of the arbuscular mycorrhizal fungus Gigaspora decipiens. Spore lysis assays revealed that all Burkholderia spp. tested were able to colonize the interior of G. decipiens spores. Amplification of specific DNA sequences and transmission electron microscopy confirmed the intracellular presence of B. vietnamiensis. Twelve percent of all spores were invaded by B. vietnamiensis, with an average of 1.5 × 10⁶ CFU recovered from individual infected spores. Of those spores inoculated with B. pseudomallei, 7% were invaded, with an average of 5.5 × 10⁵ CFU recovered from individual infected spores. Scanning electron and fluorescence microscopy provided insights into the morphology of surfaces of spores and hyphae of G. decipiens and the attachment of bacteria. Burkholderia spp. colonized both hyphae and spores, attaching to surfaces in either an end-on or side-on fashion. Adherence of Burkholderia spp. to eukaryotic surfaces also involved the formation of numerous fibrillar structures.     

Molecular mechanisms underlying the close association between soil Burkholderia and fungi

Bacterial species belonging to the genus Burkholderia have been repeatedly reported to be associated with fungi but the extent and specificity of these associations in soils remain undetermined. To assess whether associations between Burkholderia and fungi are widespread in soils, we performed a co-occurrence analysis in an intercontinental soil sample collection. This revealed that Burkholderia significantly co-occurred with a wide range of fungi. To analyse the molecular basis of the interaction, we selected two model fungi frequently co-occurring with Burkholderia, Alternaria alternata and Fusarium solani, and analysed the proteome changes caused by cultivation with either fungus in the widespread soil inhabitant B. glathei, whose genome we sequenced. Co-cultivation with both fungi led to very similar changes in the B. glathei proteome. Our results indicate that B. glathei significantly benefits from the interaction, which is exemplified by a lower abundance of several starvation factors that were highly expressed in pure culture. However, co-cultivation also gave rise to stress factors, as indicated by the increased expression of multidrug efflux pumps and proteins involved in oxidative stress response. Our data suggest that the ability of Burkholderia to establish a close association with fungi mainly lies in the capacities to utilize fungal-secreted metabolites and to overcome fungal defense mechanisms. This work indicates that beneficial interactions with fungi might contribute to the survival strategy of Burkholderia species in environments with sub-optimal conditions, including acidic soils.     

A computational approach to identify CRISPR-Cas loci in the complete genomes of the lichen-associated Burkholderia sp. PAMC28687 and PAMC26561

The genus Burkholderia and its strains PAMC28687 and PAMC26561 are lichen-associated bacteria isolated from the Antarctic region. Our study is the first to provide the genome sequence of the Burkholderia sp. PAMC26561 strain. The genus Burkholderia includes bacteria that are pathogenic to plants, animals, and humans. Computational analysis of complete genomes of strains from the uncategorized Burkholderia group was performed using the NCBI databank and PATRIC (for genome sequence information) and CRISPRCasFinder (online and offline versions) software in order to predict the CRISPR loci and Cas genes. The RNAfold Webserver online software was used to predict RNA secondary structures. Our study showed that strain MSMB0852 (plasmid) possesses CRISPR-Cas system Class 2, and two lichen-associated strains, PAMC28687 (chromosome I) and PAMC26561 (chromosome I), possess CRISPR-Cas system Class 1. Additionally, only the two lichen-associated strains possess a variety of Cas genes.     

The Tomato Rhizosphere, an Environment Rich in Nitrogen-Fixing Burkholderia Species with Capabilities of Interest for Agriculture and Bioremediation

Burkholderia strains are promising candidates for biotechnological applications. Unfortunately, most of these strains belong to species of the Burkholderia cepacia complex (Bcc) involved in human infections, hampering potential applications. Novel diazotrophic Burkholderia species, phylogenetically distant from the Bcc species, have been discovered recently, but their environmental distribution and relevant features for agro-biotechnological applications are little known. In this work, the occurrence of N₂-fixing Burkholderia species in the rhizospheres and rhizoplanes of tomato plants field grown in Mexico was assessed. The results revealed a high level of diversity of diazotrophic Burkholderia species, including B. unamae, B. xenovorans, B. tropica, and two other unknown species, one of them phylogenetically closely related to B. kururiensis. These N₂-fixing Burkholderia species exhibited activities involved in bioremediation, plant growth promotion, or biological control in vitro. Remarkably, B. unamae and B. kururiensis grew with aromatic compounds (phenol and benzene) as carbon sources, and the presence of aromatic oxygenase genes was confirmed in both species. The rhizospheric and endophyte nature of B. unamae and its ability to degrade aromatic compounds suggest that it could be used in rhizoremediation and for improvement of phytoremediation. B. kururiensis and other Burkholderia sp. strains grew with toluene. B. unamae and B. xenovorans exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity, and the occurrence of acdS genes encoding ACC deaminase was confirmed. Mineral phosphate solubilization through organic acid production appears to be the mechanism used by most diazotrophic Burkholderia species, but in B. tropica, there presumably exists an additional unknown mechanism. Most of the diazotrophic Burkholderia species produced hydroxamate-type siderophores. Certainly, the N₂-fixing Burkholderia species associated with plants have great potential for agro-biotechnological applications.     

Development of a recA Gene-Based Identification Approach for the Entire Burkholderia Genus

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.     

Rapid identification of nitrogen-fixing and legume-nodulating Burkholderia species based on PCR 16S rRNA species-specific oligonucleotides

Several novel N₂-fixing Burkholderia species associated with plants, including legume-nodulating species, have recently been discovered. Presently, considerable interest exists in studying the diazotrophic Burkholderia species, both for their ecology and their great potential for agro-biotechnological applications. However, the available methods used in the identification of these Burkholderia species are time-consuming and expensive. In this study, PCR species-specific primers based on the 16S rRNA gene were designed, which allowed rapid, easy, and correct identification of most known N₂-fixing Burkholderia. With this approach, type and reference strains of Burkholderia kururiensis, B. unamae, B. xenovorans, B. tropica, and B. silvatlantica, as well as the legume-nodulating B. phymatum, B. tuberum, B. mimosarum, and B. nodosa, were unambiguously identified. In addition, the PCR species-specific primers allowed the diversity of the diazotrophic Burkholderia associated with field-grown tomato and sorghum plants to be determined. B. tropica and B. xenovorans were the predominant species found in association with tomato, but the occurrence of B. tropica with sorghum plants was practically exclusive. The efficiency of the species-specific primers was validated with the detection of B. tropica and B. xenovorans from DNA directly recovered from tomato rhizosphere soil samples. Additionally, using PCR species-specific primers, all of the legume-nodulating Burkholderia were correctly identified, even from single nodules collected from inoculated common bean plants. These primers could contribute to rapid identification of the diazotrophic and nodulating Burkholderia species associated with important crop plants and legumes, as well as revealing their environmental distribution.     

Comparative genome analysis of rice-pathogenic Burkholderia provides insight into capacity to adapt to different environments and hosts

Background

In addition to human and animal diseases, bacteria of the genus Burkholderia can cause plant diseases. The representative species of rice-pathogenic Burkholderia are Burkholderia glumae, B. gladioli, and B. plantarii, which primarily cause grain rot, sheath rot, and seedling blight, respectively, resulting in severe reductions in rice production. Though Burkholderia rice pathogens cause problems in rice-growing countries, comprehensive studies of these rice-pathogenic species aiming to control Burkholderia-mediated diseases are only in the early stages.

Results

We first sequenced the complete genome of B. plantarii ATCC 43733^T. Second, we conducted comparative analysis of the newly sequenced B. plantarii ATCC 43733^T genome with eleven complete or draft genomes of B. glumae and B. gladioli strains. Furthermore, we compared the genome of three rice Burkholderia pathogens with those of other Burkholderia species such as those found in environmental habitats and those known as animal/human pathogens. These B. glumae, B. gladioli, and B. plantarii strains have unique genes involved in toxoflavin or tropolone toxin production and the clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bacterial immune system. Although the genome of B. plantarii ATCC 43733^T has many common features with those of B. glumae and B. gladioli, this B. plantarii strain has several unique features, including quorum sensing and CRISPR/CRISPR-associated protein (Cas) systems.

Conclusions

The complete genome sequence of B. plantarii ATCC 43733^T and publicly available genomes of B. glumae BGR1 and B. gladioli BSR3 enabled comprehensive comparative genome analyses among three rice-pathogenic Burkholderia species responsible for tissue rotting and seedling blight. Our results suggest that B. glumae has evolved rapidly, or has undergone rapid genome rearrangements or deletions, in response to the hosts. It also, clarifies the unique features of rice pathogenic Burkholderia species relative to other animal and human Burkholderia species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1558-5) contains supplementary material, which is available to authorized users.     

High diversity of culturable Burkholderia species associated with sugarcane

Sugarcane is an important crop around the world. Burkholderia genus has emerged as an important plant associated bacteria in the last years. In this study, the occurrence of Burkholderia species associated with two sugarcane varieties cultivated in Mexico was assessed. Burkholderia species were isolated with and without diazotrophs enrichment from sugarcane. Burkholderia strains were identified using a semi-selective set of primers and clustered by restriction analysis of 16S rRNA. The isolates were characterized by 16S rRNA, recA and nifH sequence analysis, whole-cell protein patterns, and plant-growth promotion (PGP) characteristics. Diazotrophic B. unamae and B. tropica were predominant using diazotroph enrichment method. Non-diazotrophic B. cepacia complex (Bcc) species were predominant without enrichment. Among non-diazotrophs, B. tropica was identified. The diazotrophic Burkholderia species exhibit in vitro PGP activities: biosynthesis of indolic compounds, phosphate solubilization, siderophores production and acdS gene presence, which encodes the enzyme ACC (1-aminocyclopropane-1-carboxylate) deaminase. The present study confirms the broad environmental and geographic distribution of diazotrophic B. unamae and B. tropica, and reveals the riches of Bcc and other Burkholderia species associated with sugarcane field-grown in Mexico. This work also shows the potential activities in PGP.     

Complementary cooperation between two syntrophic bacteria in pesticide degradation

Interactions between microbial species, including competition and mutualism, influence the abundance and distribution of the related species. For example, metabolic cooperation among multiple bacteria plays a major role in the maintenance of consortia. This study aims to clarify how two bacterial species coexist in a syntrophic association involving the degradation of the pesticide fenitrothion. To elucidate essential mechanisms for maintaining a syntrophic association, we employed a mathematical model based on an experimental study, because experiment cannot elucidate various conditions for two bacterial coexistence. We isolated fenitrothion-degrading Sphingomonas sp. TFEE and its metabolite of 3-methyl-4-nitrophenol (3M4N)-degrading Burkholderia sp. MN1 from a fenitrothion-treated soil microcosm. Neither bacterium can completely degrade fenitrothion alone, but they can utilize the second intermediate, methylhydroquinone (MHQ). Burkholderia sp. MN1 excretes a portion of MHQ during the degradation of 3M4N, from which Sphingomonas sp. TFEE carries out degradation to obtain carbon and energy. Based on experimental findings, we developed mathematical models that represent the syntrophic association involving the two bacteria. We found that the two bacteria are characterized by the mutualistic degradation of fenitrothion. Dynamics of two bacteria are determined by the degree of cooperation between two bacteria (i.e., supply of 3M4N by Sphingomonas sp. TFEE and excretion of MHQ by Burkholderia sp. MN1) and the initial population sizes. The syntrophic association mediates the coexistence of the two bacteria under the possibility of resource competition for MHQ, and robustly facilitates the maintenance of ecosystem function in terms of degrading xenobiotics. Thus, the mathematical analysis and numerical computations based on the experiment indicate the key mechanisms for coexistence of Sphingomonas sp. TFEE and Burkholderia sp. MN1 in syntrophic association involving fenitrothion degradation.     

Phylogenetic Analysis of Burkholderia Species by Multilocus Sequence Analysis

Burkholderia comprises more than 60 species of environmental, clinical, and agro-biotechnological relevance. Previous phylogenetic analyses of 16S rRNA, recA, gyrB, rpoB, and acdS gene sequences as well as genome sequence comparisons of different Burkholderia species have revealed two major species clusters. In this study, we undertook a multilocus sequence analysis of 77 type and reference strains of Burkholderia using atpD, gltB, lepA, and recA genes in combination with the 16S rRNA gene sequence and employed maximum likelihood and neighbor-joining criteria to test this further. The phylogenetic analysis revealed, with high supporting values, distinct lineages within the genus Burkholderia. The two large groups were named A and B, whereas the B. rhizoxinica/B. endofungorum, and B. andropogonis groups consisted of two and one species, respectively. The group A encompasses several plant-associated and saprophytic bacterial species. The group B comprises the B. cepacia complex (opportunistic human pathogens), the B. pseudomallei subgroup, which includes both human and animal pathogens, and an assemblage of plant pathogenic species. The distinct lineages present in Burkholderia suggest that each group might represent a different genus. However, it will be necessary to analyze the full set of Burkholderia species and explore whether enough phenotypic features exist among the different clusters to propose that these groups should be considered separate genera.     

Screening of plant growth promoting Rhizobacteria isolated from sunflower (Helianthus annuus L.)

Background and Aims

This study was aimed at assessing the diversity of putatively diazotrophic rhizobacteria associated with sunflower (Helianthus annuus L.) cropped in the south of Brazil, and to examine key plant growth promotion (PGP) characteristics of the isolates for the purposes of increasing plant productivity.

Methods

299 strains were isolated from the roots and rhizosphere of sunflower cultivated in five different areas using N-free media. 16S rDNA PCR-RFLP and 16S rRNA partial sequencing were used for identification and the Shannon index was used to evaluate bacterial diversity. Production of siderophores and indolic compounds (ICs), as well phosphate solubilization activities of each isolate were also evaluated in vitro. On the basis of multiple PGP activities, eight isolates were selected and tested for their N-fixation ability, and their capacity as potential PGPR on sunflower plants was also assessed.

Results

All except three Gram-positive strains (phylum Actinobacteria) belonged to the Gram-negative Proteobacteria subgroups [Gamma (167), Beta (78), and Alpha (50)] and the family Flavobacteriaceae (1)]. Shannon indexes ranged from 0.96 to 2.13 between the five sampling sites. Enterobacter and Burkholderia were the predominant genera isolated from roots and rhizosphere, respectively. Producers of siderophores and ICs were widely found amongst the isolates, but only 19.8% of them solubilized phosphate. About 8% of the isolates exhibited all three PGP traits, and these mostly belonged to the genus Burkholderia. Four isolates were able to stimulate the growth of sunflower plants under gnotobiotic conditions.

Conclusions

Enterobacter and Burkholderia were the dominant rhizospheric bacterial genera associated with sunflower plants. Inoculation with isolates belonging to the genera Achromobacter, Chryseobacterium, Azospirillum, and Burkholderia had a stimulatory effect on plant growth.     

Toluene removal efficiency, process robustness, and bacterial diversity of a biotrickling filter inoculated with Burkholderia sp. Strain T3

Microorganisms determine the overall biofilter performance under specific operating conditions. The toluene removal and process robustness of a laboratoryscale, ceramisite-based biotrickling filter inoculated with Burkholderia sp. strain T3 (BTFb) were compared with those of another biotrickling filter inoculated with activated sludge (BTFa) for 3 months under various operating conditions. Denaturing gradient gel electrophoresis was applied to visualise the bacterial community of the BTFa and BTFb. Real-time polymerase chain reaction was performed to determine the genes coding for toluenedegrading enzymes. Burkholderia sp. strain T3, which possesses the major toluene-degrading genes in BTFb, was traced in the BTFb bacterial community. The strain was found to stabilize the relative quantity steadily at higher than 60% during toluene biofiltration. Thus, BTFb performed more efficiently than BTFa as evidenced by achieving 98.86% toluene removal efficiency (RE) on 3 day, critical elimination capacity (EC) of 234.23 ± 10.54 g/m3/h, and rapid restoration of the initial RE and EC levels within 3 day of reoperation, even after 1 month of shutdown. The efficiency of BTFb is also evident by the stabilised RE and EC levels within a wide temperature range and a gradually decreasing system pH. Maintaining the pressure drop levels below 150 Pa during prolonged operation also contributed to the efficiency of BTFb. Thus, based on the study results, we propose that Burkholderia sp. strain T3 is a highly efficient and applicable inoculum for toluene biofiltration.