Model plants for studying the interaction between Methylobacterium mesophilicum and Xylella fastidiosa

Over the last few years, endophytic bacterial communities associated with citrus have been studied as key components interacting with Xylella fastidiosa. In this study, we investigated the possible interaction between the citrus endophyte Methylobacterium mesophilicum SR1.6/6 and X. fastidiosa in model plants such as Catharanthus roseus (Madagaskar periwinkle) and Nicotiana clevelandii (Clevelands tobacco). The aim of this study was to establish the fate of M. mesophilicum SR1.6/6 after inoculation of C. roseus and N. clevelandii plants, using PCR-DGGE (polymerase chain reaction--denaturing gradient gel electrophoresis) and plating techniques. Shifts in the indigenous endophytic bacterial communities were observed in plants inoculated with strain SR1.6/6, using specific primers targeting alpha- and beta-Proteobacteria. Cells of strain SR1.6/6 were observed in a biofilm structure on the root and hypocotyl surfaces of in vitro seedlings inoculated with M. mesophilicum SR1.6/6. This emphasizes the importance of these tissues as main points of entrance for this organism. The results showed that C. roseus and N. clevelandii could be used as model plants to study the interaction between M. mesophilicum and X. fastidiosa.     

Endophytic Colonization of Potato (Solanum tuberosum L.) by a Novel Competent Bacterial Endophyte,Pseudomonas putida Strain P9, and Its Effect on Associated Bacterial Communities

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.The colonization of plant tissue is of paramount importance for successful application of plant-growth-promoting bacteria. For instance, efficient root colonization was shown to be important for the control of Fusarium oxysporum in tomato by a phenazine-1-carboxamide-producing Pseudomonas chlororaphis strain (6). The migratory response of bacterial inoculants to compounds released by plant roots is often the first step required for establishment of the bacteria in the rhizosphere and rhizoplane (29, 48, 50). Following the initial colonization by introduced bacteria, these organisms may spread further to the aerial parts of the plant (35). The degree of root colonization by bacterial inoculants depends on, in addition to the mode of application (35), factors intrinsic to the organism used, like the presence of flagella (11) and/or the presence of particular outer membrane lipopolysaccharides (13). Such features may differ from strain to strain. Once attached to plant roots, the inoculant bacteria may evoke “protective” responses in the plants, enabling them to resist phytopathogen attack (4).Endophytic colonization, characterized by colonization of internal plant tissues concomitant with growth and systemic spread, can be an important factor for plant growth (23, 24). For instance, cells of Rhizobium etli G12 (used as a biocontrol agent) marked with a green fluorescent protein were visible in root hairs, around epidermal cells, and within the vascular tissue of Arabidopsis thaliana plants (22), and these plants exhibited maximum control of the nematode Meleidogyne incognita. Moreover, cells of the green fluorescent protein-labeled plant-growth-promoting bacterial strain Burkholderia phytofirmans PsJN were present in xylem vessels and different plant organs, including inflorescences, of grape plants (7). Endophytic colonization was also observed for the nitrogen-fixing bacteria Acetobacter diazotrophicus in sugarcane (12) and Serratia marcescens in rice (20). Hence, different types of bacteria apparently have the capacity to colonize the internal compartments of plants and eventually interact with their hosts, thus occupying niches inside plants where they may evoke responses that are important for plant health maintenance and nutrient acquisition.Introduced bacteria can communicate with each other using a range of signaling systems, including quorum sensing, and most likely also with (related) bacteria indigenous to plants and even with their host plant (41). We hypothesize that introduced strains also impact plant-associated indigenous microbial communities by cross talk with members of these communities, by competition for nutrients or space, or by production of antibiotics. Shifts in endophytic bacterial communities are therefore expected to occur after bacterial inoculation. This is a phenomenon that has been observed after inoculation of Madagascar periwinkle (Catharanthus roseus) and Cleveland tobacco (Nicotiana clevelandii) plants with Methylobacterium mesophilicum (1).In situ microscopic detection of endophytes allows determination of the preferred colonization site. However, it is hard to detect cells (e.g., cells marked with gfp) at levels below certain threshold levels in plants, especially when they are grown under nonsterile conditions. For this reason it is not possible to precisely determine in situ interactions between inoculants and indigenous bacterial populations. Molecular fingerprinting techniques, like PCR-denaturing gradient gel electrophoresis (DGGE), are suitable for studying microbial communities in the rhizospheres (14, 26, 44, 51), rhizoplanes (48), and endospheres (18, 38, 42, 51) of different plant species. The use of bacterial group-specific primer systems has been proposed for studies of different taxonomic groups in the plant endosphere (52). The impact of factors like crop history, plant growth stage, and cultivar (genotype) on plant-associated microbial populations can be established by using multivariate statistical analyses (17, 40, 51). The combination of molecular fingerprinting techniques and multivariate analyses enabled us to show that the plant growth stage and cultivar contributed strongly and significantly to the composition of plant-associated microbial communities (37, 51).The aim of this study was to establish the fate and impact of strain P9 during endophytic colonization of potato plants. The polyphasic approach used allowed us to investigate the presence of strain P9 at culture-dependent and -independent levels in plants.     

Bacterial soil community in a Brazilian sugarcane field

The assessment of bacterial communities in soil gives insight into microbial behavior under prevailing environmental conditions. In this context, we assessed the composition of soil bacterial communities in a Brazilian sugarcane experimental field. The experimental design encompassed plots containing common sugarcane (variety SP80-1842) and its transgenic form (IMI-1 — imazapyr herbicide resistant). Plants were grown in such field plots in a completely randomized design with three treatments, which addressed the factors transgene and imazapyr herbicide application. Soil samples were taken at three developmental stages during plant growth and analyzed using 16S ribosomal RNA (rRNA)-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries. PCR-DGGE fingerprints obtained for the total bacterial community and specific bacterial groups — Actinobacteria, Alphaproteobacteria and Betaproteobacteria — revealed that the structure of these assemblages did not differ over time and among treatments. Nevertheless, slight differences among 16S rRNA gene clone libraries constructed from each treatment could be observed at particular cut-off levels. Altogether, the libraries encompassed a total of eleven bacterial phyla and the candidate divisions TM7 and OP10. Clone sequences affiliated with the Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria were, in this order, most abundant. Accurate phylogenetic analyses were performed for the phyla Acidobacteria and Verrucomicrobia, revealing the structures of these groups, which are still poorly understood as to their importance for soil functioning and sustainability under agricultural practices.     

Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry.     

Properties of microtubule sliding disintegration in isolated tetrahymena cilia

Properties of the sliding disintegration response of demembranated tetrahymena cilia have been studied by measuring the spectrophotomeric response or turbidity of cilia suspensions at a wavelength of 350 nm relative to changes in the dynein substrate (MgATP(2-)) concentration. The maximum decrease in turbidity occurs in 20 muM ATP, and 90 percent of the decrease occurs in approximately 5.9 s. At lower ATP concentrations (1-20 muM), both the velocity and magnitude of the turbidity decreases are proportional to ATP concentration. The velocity data for 20 muM ATP permit construction of a reaction velocity curve suggesting that changes in turbidity are directly proportional to the extent and velocity of disintegration. At ATP concentrations more than 20 muM (50muM to 5mM), both velocity and magnitude of the turbidimetric response are reduced by approximately 50 percent. This apparent inhibition results in a biphasic response curve that may be related to activation of residual shear resistance or regulatory components at the higher ATP concentrations. The inhibitory effects of elevated ATP can be eliminated by mild trypsin proteolysis, whereupon the reaction goes to completion at any ATP concentration. The turbidimetric responses of the axoneme-substrate suspensions are consistent with the extent and type of axoneme disintegration revealed by electron microscope examination of the various suspensions, suggesting that the turbidimetric assay may prove to be a reliable means for assessing the state of axoneme integrity.     

Molecular characterization of a β-1,4-endoglucanase from an endophytic Bacillus pumilus strain

Endophytes comprise mainly microorganisms that colonize inner plant tissues, often living with the host in a symbiotic manner. Several ecological roles have been assigned to endophytic fungi and bacteria, such as antibiosis to phytopathogenic agents and plant growth promotion. Nowadays, endophytes are viewed as a new source of genes, proteins and biochemical compounds that may be used to improve industrial processes. In this study, the gene EglA was cloned from a citrus endophytic Bacillus strain. The EglA encodes a -1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The predicted protein, EglA, has high homology to other bacterial cellulases and shows a modular structure containing a catalytic domain of the glycosyl hydrolase family 9 (GH9) and a cellulose-binding module type 3 (CBM3). The enzyme was expressed in Escherichia coli, purified to homogeneity, and characterized. EglA has an optimum pH range of 5–8, and remarkable heat stability, retaining more than 85% activity even after a 24-h incubation at pH 6–8.6. This characteristic is an important feature for further applications of this enzyme in biotechnological processes in which temperatures of 50–60°C are required over long incubation periods.     

Bacterial community characterization in the soils of native and restored rainforest fragments

The Brazilian Atlantic Forest (“Mata Atlântica”) has been largely studied due to its valuable and unique biodiversity. Unfortunately, this priceless ecosystem has been widely deforested and only 10 % of its original area is still untouched. Some projects have been successfully implemented to restore its fauna and flora but there is a lack of information on how the soil bacterial communities respond to this process. Thus, our aim was to evaluate the influence of soil attributes and seasonality on soil bacterial communities of rainforest fragments under restoration processes. Soil samples from a native site and two ongoing restoration fragments with different times of implementation (10 and 20 years) were collected and assayed by using culture-independent approaches. Our findings demonstrate that seasonality barely altered the bacterial distribution whereas soil chemical attributes and plant species were related to bacterial community structure during the restoration process. Moreover, the strict relationship observed for two bacterial groups, Solibacteriaceae and Verrucomicrobia, increasing from the more recently planted (10 years) to the native site, with the 20 year old restoration site in the middle, which may suggest their use as bioindicators of soil quality and recovery of forest fragments being restored.     

Brazilian Microbiome Project: Revealing the Unexplored Microbial Diversity—Challenges and Prospects

The Brazilian Microbiome Project (BMP) aims to assemble a Brazilian Metagenomic Consortium/Database. At present, many metagenomic projects underway in Brazil are widely known. Our goal in this initiative is to co-ordinate and standardize these together with new projects to come. It is estimated that Brazil hosts approximately 20 % of the entire world’s macroorganism biological diversity. It is 1 of the 17 countries that share nearly 70 % of the world’s catalogued animal and plant species, and is recognized as one of the most megadiverse countries. At the end of 2012, Brazil has joined GBIF (Global Biodiversity Information Facility), as associated member, to improve the access to the Brazilian biodiversity data in a free and open way. This was an important step toward increasing international collaboration and clearly shows the commitment of the Brazilian government in directing national policies toward sustainable development. Despite its importance, the Brazilian microbial diversity is still considered to be largely unknown, and it is clear that to maintain ecosystem dynamics and to sustainably manage land use, it is crucial to understand the biological and functional diversity of the system. This is the first attempt to collect and collate information about Brazilian microbial genetic and functional diversity in a systematic and holistic manner. The success of the BMP depends on a massive collaborative effort of both the Brazilian and international scientific communities, and therefore, we invite all colleagues to participate in this project.     

Abundance and Genetic Diversity of nifH Gene Sequences in Anthropogenically Affected Brazilian Mangrove Sediments

Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies.     

Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium–plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant cultivation.