Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

Virulence factors from Aeromonas species

Isolates (116) of Aeromonas were obtained from various sources and subjected to tests to establish their virulence factors. A high number of the isolates (69.8%) were found to be enterotoxigenic. The isolates from snails had more enterotoxigenic strains (73.3%), while those from cattle faeces had the lowest (33.3%). Haemolysin production was found to be high (60.3%) amongst the isolates, and human isolates gave the highest number of haemolysin producing strains (70.6%), while the least number (33.3%) was obtained from cattle strains. About 50% of the strains produced both enterotoxin and haemolysin. The enzyme profile of the isolates included amylase, lecithinase, lipase and protease. There was no definite pattern in the elaboration of these enzymes and the production of haemolysin and enterotoxin, thus inferring that the production of these factors is not specific to the source. Two isolates were seen to produce none of these enzymes, and one was positive for enterotoxin and haemolysin production, leaving only one isolate which yielded none of these factors. The work adds more support to the pathogenicity of Aeromonas species, and indicates the existence of non-pathogenic strains.     

Burkholderia cepacia complex: distribution of genomovars among isolates from the maize rhizosphere in Italy

Burkholderia cepacia is a 'complex' in which seven genomic species or genomovars have so far been identified. It appears that all seven B. cepacia genomovars are capable of causing infections in vulnerable persons; in particular, the importance of Burkholderia multivorans (genomovar II) and B. cepacia genomovar III among cystic fibrosis isolates, especially epidemic ones, has been emphasized. In order to acquire a better comprehension of the genomovar composition of environmental populations of B. cepacia, 120 strains were isolated from the rhizosphere of maize plants cultivated in fields located in northern, central and southern Italy. The identification of the different genomovars was accomplished by a combination of molecular polymerase chain reaction (PCR)-based techniques, such as restriction fragment length polymorphism (RFLP) analysis of 16S rDNA (ARDRA), genomovar-specific PCR tests and RFLP analyses based on polymorphisms in the recA gene whole-cell protein electrophoresis. ARDRA analysis allowed us to distinguish between all B. cepacia genomovars except B. cepacia genomovar I, B. cepacia genomovar III and Burkholderia ambifaria (genomovar VII). The latter genomovars were differentiated by means of recA PCR tests and RFLP analyses. Among the rhizospheric isolates of B. cepacia, we found only B. cepacia genomovar I, B. cepacia genomovar III, Burkholderia vietnamiensis (genomovar V) and B. ambifaria. B. cepacia genomovars I and III and B. ambifaria were recovered from all three fields, whereas B. vietnamiensis was detected only in the population isolated from the field located in central Italy. Among strains isolated from northern and southern Italy, the most abundant genomovars were B. ambifaria and B. cepacia genomovar III respectively; in contrast, the population isolated in central Italy showed an even distribution of strains among genomovars. These results indicate that it is not possible to differentiate clinical and environmental strains, or pathogenic and non-pathogenic strains, of the B. cepacia complex simply on the basis of genomovar status, and that the environment may serve as a reservoir for B. cepacia genomovar III infections in vulnerable humans.     

Forward from the crossroads of ecology and evolution

Community genetics is a synthesis of community ecology and evolutionary biology. It examines how genetic variation within a species affects interactions among species to change ecological community structure and diversity. The use of community genetics approaches has greatly expanded in recent years and the evidence for ecological effects of genetic diversity is growing. The goal of current community genetics research is to determine the circumstances in which, and the mechanisms by which community genetic effects occur and is the focus of the papers in this special issue. We bring a new group of researchers into the community genetics fold. Using a mixture of empirical research, literature reviews and theoretical development, we introduce novel concepts and methods that we hope will enable us to develop community genetics into the future.     

Application of a recA gene-based identification approach to the maize rhizosphere reveals novel diversity in Burkholderia species

Burkholderia species are widely distributed in the natural environment. We evaluated the use of the recA gene in a cultivation-independent approach to examine the Burkholderia diversity associated with the maize rhizosphere. Two types of recA gene library were constructed, one with broad-specificity recA primers (BUR1 and BUR2) and a second from the products of nested PCRs using Burkholderia-specific primers (BUR3 and BUR4). The broad-specificity primer set provided near full-length recA sequences (869 bp) suitable for the creation of robust environmental sequence data sets; however, the nested PCR approach demonstrated the greatest specificity (84%) for detection of Burkholderia species recA genes. In addition, the screening approach was able to identify recA phylotypes matching Burkholderia cepacia complex species previously cultivated from the maize samples and discriminate these from other Burkholderia. The ecological benefit of Burkholderia species cultivated from maize rhizosphere is well documented, however, the fact that the majority of Burkholderia recA genes detected in this study (90%) were suggestive of novel taxa indicates that a wealth of potentially important interactions with uncultivated Burkholderia species remain unstudied in this habitat.     

Very early evolution from the perspective of microbial ecology

The universal ancestor at the root of the species tree of life depicts a population of organisms with a surprising degree of complexity, posessing genomes and translation systems much like that of microbial life today. As the first life forms were most likely to have been simple replicators, considerable evolutionary change must have taken place prior to the last universal common ancestor. It is often assumed that the lack of earlier branches on the tree of life is due to a prevalence of random horizontal gene transfer that obscured the delineations between lineages and hindered their divergence. Therefore, principles of microbial evolution and ecology may give us some insight into these early stages in the history of life. Here, we synthesize the current understanding of organismal and genome evolution from the perspective of microbial ecology and apply these evolutionary principles to the earliest stages of life on Earth. We focus especially on broad evolutionary modes pertaining to horizontal gene transfer, pangenome structure, and microbial mat communities.     

Identification and onion pathogenicity of Burkholderia cepacia complex isolates from the onion rhizosphere and onion field soil

Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source of B. cenocepacia.     

Current status of the tardigrada: evolution and ecology

The Tardigrada are bilaterally symmetrical micrometazoans withfour pairs of lobopod legs terminating in claws or sucking disks.They occupy a diversity of niches in marine, freshwater, andterrestrial environments throughout the world. Some have a cosmopolitandistribution, while others are endemic. About 900 species havebeen described thus far, but many more species are expectedas additional habitats are investigated. Most are less than1 mm in body length and are opaque or translucent, exhibitingcolors such as brown, green, orange, yellow, red, or pink inthe cuticle and/or gut. Marine species are more variable inbody shape and overall appearance and generally exhibit lowpopulation density with high species diversity. Reproductivemodes include sexual reproduction and parthenogenesis, but muchremains to be known about development. Tardigrades have a hemocoel-typeof fluid-filled body cavity, a complete digestive tract, anda lobed dorsal brain with a ventral nerve cord with fused ganglia.Recent molecular analyses and additional morphological studiesof the nervous system have confirmed the phylogenetic positionof tardigrades as a sister group of the arthropods. The abilityof tardigrades to undergo cryptobiosis has long intrigued scientists.Although tardigrades are active only when surrounded by a filmof water, they can enter latent states in response to desiccation(anhydrobiosis), temperature (cryobiosis), low oxygen (anoxybiosis),and salinity changes (osmobiosis). Cryptobiotic states aid indispersal.     

Oxidative stress in ecology and evolution: lessons from avian studies

Although oxidative stress is a central topic in biochemical and medical research, the number of reports on its relevance in life‐history studies of non‐human animals is still low. Information about oxidative stress in wild birds may help describe functional interactions among the components of life‐history traits. Currently available evidence suggests that oxidative stress may impart an important physiological cost on longevity, reproduction, immune response or intense physical activity. Given the gaps in our present knowledge, it is still premature to attempt to draw definitive conclusions and basic questions (e.g. how is oxidative stress generated and how do organisms cope with it?) have yet to be fully explored under natural conditions. Therefore, caution is needed in developing hypotheses or drawing general conclusions until additional data become available to perform more rigorous comparative analyses.     

High allozyme variation in populations of three spiny rat species from Upper Amazonia: association with ecology and evolution]

The allozyme variation in three spiny rat species of the genus Proechimys from Upper Amazonia was studied in relation to their ecology and evolution. The ecological environmental factors and biotopic distribution of species were analyzed. The unusually high allozyme variation was found in P. simonsi and P. sp. (2n = 34) inhabiting native forest biotopes. A relatively low allozyme variation in P. brevicauda was assumed to be associated with eurybiotic properties and the ability of this species to adapt to anthropogenic biotopes. Data on chromosome homeology and reconstruction of chromosome rearrangements in six spiny rat species were correlated with allozyme variation. The results suggested that chromosome rearrangements played the major role in evolution of the spiny rat species, and that the reorganization of the P. brevicauda genome was not random. P. simonsi and P. sp. (2n = 34), which live in native forest biotopes and carry an excessive genomik "informational load", were assumed to be highly susceptible to any novel external factors. These species are potentially able to produce new chromosome forms and are most significantly affected by deforestation.     

The ecology and evolution of the monito del monte,a relict species from the southern South America temperate forests

The arboreal marsupial monito del monte (genus Dromiciops, with two recognized species) is a paradigmatic mammal. It is the sole living representative of the order Microbiotheria, the ancestor lineage of Australian marsupials. Also, this marsupial is the unique frugivorous mammal in the temperate rainforest, being the main seed disperser of several endemic plants of this ecosystem, thus acting as keystone species. Dromiciops is also one of the few hibernating mammals in South America, spending half of the year in a physiological dormancy where metabolism is reduced to 10% of normal levels. This capacity to reduce energy expenditure in winter contrasts with the enormous energy turnover rate they experience in spring and summer. The unique life history strategies of this living Microbiotheria, characterized by an alternation of life in the slow and fast lanes, putatively represent ancestral traits that permitted these cold‐adapted mammals to survive in this environment. Here, we describe the ecological role of this emblematic marsupial, summarizing the ecophysiology of hibernation and sociality, updated phylogeographic relationships, reproductive cycle, trophic relationships, mutualisms, conservation, and threats. This marsupial shows high densities, despite presenting slow reproductive rates, a paradox explained by the unique characteristics of its three‐dimensional habitat. We finally suggest immediate actions to protect these species that may be threatened in the near future due to habitat destruction and climate change.     

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.     

Network properties, species abundance and evolution in a model of evolutionary ecology

We study the evolution of the network properties of a populated network embedded in a genotype space characterized by either a low or a high number of potential links, with particular emphasis on the connectivity and clustering. Evolution produces two distinct types of network. When a specific genotype is only able to influence a few other genotypes, the ecosystem consists of separate non-interacting clusters (i.e. isolated compartments) in genotype space. When different types may influence a large number of other sites, the network becomes one large interconnected cluster. The distribution of interaction strengths--but not the number of connections--changes significantly with time. We find that the species abundance is only realistic for a high level of species connectivity. This suggests that real ecosystems form one interconnected whole in which selection leads to stronger interactions between the different types. Analogies with niche and neutral theory and assembly models are also considered.     

Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.     

Diversity and distribution of Burkholderia cepacia complex in the rhizosphere of rice and maize

A survey of Burkholderia cepacia complex (Bcc) species was conducted in agricultural fields within Hangzhou, China. Out of the 251 bacterial isolates recovered on the selective media from the rhizosphere of rice and maize, 112 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the majority belong to B. cepacia, Burkholderia cenocepacia recA lineage IIIB, Burkholderia vietnamiensis and Burkholderia pyrrocinia. Burkholderia cenocepacia and B. vietnamiensis dominated the rhizosphere of maize and rice, respectively, indicating that species composition and abundance of Bcc may vary dramatically in different crop rhizospheres. In addition, one isolate (R456) formed a single discrete cluster within the phylogenetic analysis of the Bcc recA gene, and it may belong to a new genomovar.