Does the essential oil of Lippia sidoides Cham. (pepper-rosmarin) affect its endophytic microbial community?

Background: Lippia sidoides Cham., also known as pepper-rosmarin, produces an essential oil in its leaves that is currently used by the pharmaceutical, perfumery and cosmetic industries for its antimicrobial and aromatic properties. Because of the antimicrobial compounds (mainly thymol and carvacrol) found in the essential oil, we believe that the endophytic microorganisms found in L. sidoides are selected to live in different parts of the plant. Results: In this study, the endophytic microbial communities from the stems and leaves of four L. sidoides genotypes were determined using cultivation-dependent and cultivation-independent approaches. In total, 145 endophytic bacterial strains were isolated and further grouped using either ERIC-PCR or BOX-PCR, resulting in 76 groups composed of different genera predominantly belonging to the Gammaproteobacteria. The endophytic microbial diversity was also analyzed by PCR-DGGE using 16S rRNA-based universal and group-specific primers for total bacteria, Alphaproteobacteria, Betaproteobacteria and Actinobacteria and 18S rRNA-based primers for fungi. PCR-DGGE profile analysis and principal component analysis showed that the total bacteria, Alphaproteobacteria, Betaproteobacteria and fungi were influenced not only by the location within the plant (leaf vs. stem) but also by the presence of the main components of the L. sidoides essential oil (thymol and/or carvacrol) in the leaves. However, the same could not be observed within the Actinobacteria. Conclusion: The data presented here are the first step to begin shedding light on the impact of the essential oil in the endophytic microorganisms in pepper-rosmarin

Whole-Genome Sequence of Rummeliibacillus stabekisii Strain PP9 Isolated from Antarctic Soil

Submitted by sandra infurna ([email protected]) on 2016-07-03T20:58:34Z No. of bitstreams: 1 fabio_mota_etal_IOC_2016.pdf: 142051 bytes, checksum: 4e3c1cc47174651eb6ca5fd6f7587608 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-07-03T21:07:55Z (GMT) No. of bitstreams: 1 fabio_mota_etal_IOC_2016.pdf: 142051 bytes, checksum: 4e3c1cc47174651eb6ca5fd6f7587608 (MD5)Made available in DSpace on 2016-07-03T21:07:55Z (GMT). No. of bitstreams: 1 fabio_mota_etal_IOC_2016.pdf: 142051 bytes, checksum: 4e3c1cc47174651eb6ca5fd6f7587608 (MD5) Previous issue date: 2016Made available in DSpace on 2016-07-14T19:05:35Z (GMT). No. of bitstreams: 3 fabio_mota_etal_IOC_2016.pdf.txt: 6708 bytes, checksum: b14cbaa12e78f7b4b01c4cf614529c12 (MD5) fabio_mota_etal_IOC_2016.pdf: 142051 bytes, checksum: 4e3c1cc47174651eb6ca5fd6f7587608 (MD5) license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Computacional e Sistemas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil.The whole genome of Rummeliibacillus stabekisii PP9, isolated from a soil sample from Antarctica, consists of a circular chromosome of 3,412,092 bp and a circular plasmid of 8,647 bp, with 3,244 protein-coding genes, 12 copies of the 16S-23S-5S rRNA operon, 101 tRNA genes, and 6 noncoding RNAs (ncRNAs)

The Endophytic Bacterial Microbiota Associated with Sweet Sorghum (Sorghum bicolor) Is Modulated by the Application of Chemical N Fertilizer to the Field

Sweet sorghum (Sorghum bicolor) is a multipurpose crop used as a feedstock to produce bioethanol, sugar, energy, and animal feed. However, it requires high levels of N fertilizer application to achieve the optimal growth, which causes environmental degradation. Bacterial endophytes, which live inside plant tissues, play a key role in the health and productivity of their host. This particular community may be influenced by different agronomical practices. The aim of the work was to evaluate the effects of N fertilization on the structure, diversity, abundance, and composition of endophytic and diazotrophic bacterial community associated with field-grown sweet sorghum. PCR-DGGE, quantitative PCR, and high-throughput sequencing were performed based on the amplification of rrs and nifH genes. The level of N fertilization affected the structure and abundance but not the diversity of the endophytic bacterial communities associated with sweet sorghum plants. This effect was pronounced in the roots of both bacterial communities analyzed and may depend on the physiological state of the plants. Specific bacterial classes and genera increased or decreased when the fertilizer was applied. The data obtained here contribute to a better understanding on the effects of agronomical practices on the microbiota associated with this important crop, with the aim to improve its sustainability

Monitoring of the Parasite Load in the Digestive Tract of <i>Rhodnius prolixus</i> by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle

<div><p>Background</p><p>Here we report the monitoring of the digestive tract colonization of <i>Rhodnius prolixus</i> by <i>Trypanosoma cruzi</i> using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection.</p><p>Methodology/Principal Findings</p><p>qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the <i>T</i>. <i>cruzi</i> repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes.</p><p>Conclusions/Significance</p><p>These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.</p></div

Comparison of time-course development of epimastigotes and trypomastigotes expressing luciferase in the digestive tract of <i>R</i>. <i>prolixus</i> during the first 24 h p.i., in the presence or absence of <i>R</i>. <i>rhodnii</i>.

<p>(A) Representative values of the luminescence emission for epimastigotes and trypomastigotes at 6 h and 24 h. Epi 6 h vs. Epi 24 h, P<0.01; Trypo 6 h vs. Trypo 24 h, P<0.0001. ANOVA followed by Tukey's multiple comparisons test. (B) Quantification of the BLI signal emitted by adult insects infected with various amounts of epimastigotes (Epi) or trypomastigotes (Trypo). (C) Quantification of the BLI signal emitted by gut microbiota-free first instar nymphs fed with epimastigote (Epi) or trypomastigote (Trypo) forms (10⁷ cells/ml), in the presence or absence of <i>R</i>. <i>rhodnii</i>. In each stage ± bacteria, 6 h vs. 24 h, P<0.001. ANOVA followed by Tukey's multiple comparisons test.</p

BLI of infected insects after feeding.

<p><i>In vivo</i> (A, B) and e<i>x vivo</i> (C) BLI showing that at one to two weeks after refeeding, most of the parasites remains densely packed in the rectum, as evidenced by the punctuated luminescent signal located at the end of the digestive tract.</p

Real-time monitoring of <i>R</i>. <i>prolixus</i> gut colonization by <i>T</i>. <i>cruzi</i> in natural conditions.

<p>Follow-up of parasite development in the anterior midgut (AM), posterior midgut (PM) and hindgut (H) post-infection (p.i.) and post-feeding (p.f.) in epimastigotes (A) and metacyclics trypomastigotes (B). Adult insects were fed with 10⁷ cells/ml. The arrowhead and arrow indicates, respectively, the time of the initial infection a trypomastigotes and refeeding (21 days p.i.). At several time points, the insects were dissected, and total DNA was extracted individually from the different gut segments and used to assess the parasite number by qPCR. Each time point represents an experiment (n = 8).</p

BLI time-course development of <i>T</i>. <i>cruzi</i> expressing luciferase in the insect digestive tract during the first 24 h p.i.

<p>(A) The images show the drastic reduction of the luminescence signal in the AM. (B) Quantification of the BLI signal emitted by the whole intestine obtained at different times as indicated in A. (C) <i>Ex vivo</i> BLI confirming that the signal was located exclusively in PM 24 hours p.i.. (D) Quantification of the BLI signal emitted by AM (a) and PM (b) at 0 and 24 hours after infection. (E) Effect of the microbiota on parasite lysis and BLI reduction. The insects were infected with parasites alone (Tc) or with parasites plus 2.5 x 10⁷ or 2.5 x 10⁸<i>Rhodococcus rhodnii</i> per ml of blood. Two independent experiments (n = 16) were conducted, and the results were analyzed by one-way ANOVA.</p

Real-time monitoring of <i>T</i>. <i>cruzi</i> loads in the different gut segments after <i>R</i>. <i>prolixus</i> infection.

<p>(A) Follow-up of parasite development in the anterior midgut (AM), posterior midgut (PM) and hindgut (H) post-infection (p.i.) and post-feeding (p.f.). Adult insects were fed with 10⁷ cells/ml of blood. The arrowhead and arrow indicates, respectively, the time of the initial infection and refeeding with a blood meal without parasites (21 days p.i.). Each time point represents three independent experiments (n≥8). The percentage of metacyclic trypomastigote forms (mean ± SE; n = 10) determined in the hindgut contents at 7 and 14 days p.i. and 14 days p.f. are indicated. (B) Monitoring of the DNA clearance of heat-killed parasites. Infections were performed with live parasites or parasites killed by incubation at 65°C during 2 hours before injection. n≥4 for each time point. (C) Comparison between the gut colonization of <i>R</i>. <i>prolixus</i> by <i>T</i>. <i>cruzi</i> Dm28c (left panel) and CL Brener (right panel). Each time point represents three independent experiments (n≥8). At several time points, the insects were dissected, and total DNA was extracted individually from the different gut segments and used to assess the parasite number by qPCR.</p