Interspecific variation of the bacterial community structure in the phyllosphere of the three major plant components of mangrove forests

Mangrove forests encompass a group of trees species that inhabit the intertidal zones, where soil is characterized by the high salinity and low availability of oxygen. The phyllosphere of these trees represent the habitat provided on the aboveground parts of plants, supporting in a global scale, a large and complex microbial community. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonizers, which is influenced by numerous environmental factors in addition to leaf physical and chemical properties. Here, a combination of culture-base methods with PCR-DGGE was applied to test whether local or plant specific factors shape the bacterial community of the phyllosphere from three plant species (Avicenia shaueriana, Laguncularia racemosa and Rhizophora mangle), found in two mangroves. The number of bacteria in the phyllosphere of these plants varied between 3.62 x 10(4) in A. schaeriana and 6.26 x 10³ in R. mangle. The results obtained by PCR-DGGE and isolation approaches were congruent and demonstrated that each plant species harbor specific bacterial communities in their leaves surfaces. Moreover, the ordination of environmental factors (mangrove and plant species), by redundancy analysis (RDA), also indicated that the selection exerted by plant species is higher than mangrove location on bacterial communities at phyllosphere

Endophytic fungi from the Amazonian plant Paullinia cupana and from Olea europaea isolated using cassava as an alternative starch media source

Endophytic fungi live inside plants, apparently do not cause any harm to their hosts and may play important roles in defense and growth promotion. Fungal growth is a routine practice at microbiological laboratories, and the Potato Dextrose Agar (PDA) is the most frequently used medium because it is a rich source of starch. However, the production of potatoes in some regions of the world can be costly. Aiming the development of a new medium source to tropical countries, in the present study, we used leaves from the guarana (a tropical plant from the Amazon region) and the olive (which grows in subtropical and temperate regions) to isolate endophytic fungi using PDA and Manihot Dextrose Agar (MDA). Cassava (Manihot esculenta) was evaluated as a substitute starch source. For guarana, the endophytic incidence (EI) was 90% and 98% on PDA and MDA media, respectively, and 65% and 70% for olive, respectively. The fungal isolates were sequenced using the ITS- rDNA region. The fungal identification demonstrated that the isolates varied according to the host plant and media source. In the guarana plant, 13 fungal genera were found using MDA and six were found using PDA. In the olive plant, six genera were obtained using PDA and 4 were obtained using MDA. The multivariate analysis results demonstrated the highest fungal diversity from guarana when using MDA medium. Interestingly, some genera were isolated from one specific host or in one specific media, suggesting the importance of these two factors in fungal isolation specificity. Thus, this study indicated that cassava is a feasible starch source that could serve as a potential alternative medium to potato medium.This work was supported by a grant from the Foundation for Research Assistance, São Paulo State and Amazon State, Brazil (grant n. 2009/53376-2) and by the National Council for Scientific and Technological Development (CNPq). We thank FAPESP for the M.C.Q. (grant no. 2010/50445-0), J.M. (grant no. 2011/18740-5) and S.T. (grant no. 2010/15192-4) and CNPq for E.F.S. and D.M.L. fellowships

The Microbiome of Brazilian Mangrove Sediments as Revealed by Metagenomics

Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H2S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments

Nitrogen transformations and Planctomycetes diversity in mangrove sediments

O nitrogênio é o quinto elemento mais abundante em nosso sistema solar, é essencial para a síntese de ácidos nucleicos e proteínas, os dois polímeros mais importantes da vida. Apesar da importância do nitrogênio e sua abundância na atmosfera, o N2 é praticamente inerte; assim, as formas de nitrogênio inorgânico fixados mais comuns são os íons nitrato e amônio que muitas vezes limitam a produtividade primária nos ecossistemas marinhos e terrestres. Uma biosfera ativa requer a incorporação do nitrogênio em moléculas biológicas por meio da fixação de nitrogênio, um processo no qual os domínios em procarióticos Bacteria e Archaea reduzem gás nitrogênio em amônia. Porém, a maioria dos organismos não pode fixar nitrogênio, mas sim obtê-lo na forma de nitrogênio orgânico a partir do ambiente, ou a partir da redução do nitrato. O amônio é retornado para o ambiente quando os organismos morrem e isto depende da presença do oxigênio nestes locais. Na presença do oxigênio, o amônio é oxidado em nitrato, numa via conhecida como nitrificação. Na ausência de oxigênio, o nitrato é utilizado por muitos microrganismos como um receptor de elétrons, como na redução dissimilatória de nitrato a amônia (DNRA), acoplada à oxidação anaeróbica de carbono orgânico; o nitrato pode ser convertido em gás dinitrogênio pela desnitrificação, ou ainda uma via alternativa pode transformar o nitrogênio fixado em N2, realizada por um grupo de bactérias conhecido como planctomycetes, onde a oxidação do amônio é acoplada a redução de nitrato, no processo chamado anammox. Neste trabalho de tese foi realizada a avaliação do ciclo do nitrogênio, buscando entendimento de três vias de transformação deste elemento. Neste sentido, estas vias foram avaliadas quanto as suas taxas de ocorrência, com uso incubações do sedimento de manguezais e nitrogênio marcado, como também por meio da identificação de genes funcionais envolvidos nestes três processos, por meio de pirosequenciamento. O grupo Planctomycetes ainda pode ser estudado utilizando a hibridização com sonda fluorescente in situ FISH. Os resultados indicam a presença marcante de genes identificados como parte da via desnitrificação, assim como as taxas de incubação observadas para esta via foram muito expressivas quando comparadas com as taxas obtidas para o processo anammox e DNRA. Os resultados obtidos com as análises metagenômicas e com a técnica FISH sustentam os resultados obtidos com as incubações. Pode-se observar que a filogenia sugere a tendência do agrupamento com organismos planctomycetes não descritos como responsáveis pelo processo anammox. Por meio da técnica FISH pode-se confirmar a detecção destas bactérias e visualizar sua menor presença nos manguezais amostrados, quando comparadas às bactérias totais encontradas nestas amostras.Nitrogen is the fifth most abundant element in our solar system, essential for the synthesis of nucleic acids and proteins, one of the most important polymers of life. Despite the importance of nitrogen and its overwhelming abundance in the atmosphere, N2 is practically inert, so the most commom forms of inorganic nitrogen fixed are nitrate and ammonium ions, which often limit primary productivity in marine and terrestrial ecosystems. An active biosphere requires the incorporation of nitrogen in biological molecules through nitrogen fixation, a process in which domains Archaea and Bacteria reduce nitrogen gas into ammonia. The most organisms can\'t fix nitrogen, but they can get it in inorganic nitrogen form from the environment or from the reduction of nitrate. The ammonia is returned to environment when organisms die and it depends on the presence of oxygen at these sites. In the presence of oxygen, ammonia is oxidized to nitrate, in a way known as nitrification, and in the absence of oxygen, the nitrate being used by many microbes as an electron acceptor, such as dissimilatory nitrate reduction to ammonia (DNRA), coupled to anaerobic oxidation of organic carbon. The nitrate can be converted to dinitrogen gas by denitrification, or an alternative route can turn fixed nitrogen in N2, performed by a bacteria group known as Planctomycetes, where oxidation is coupled to the ammonium nitrate reduction, in a process called anammox. In this work, with mangrove sediments from Bertioga city, State of São Paulo, the evaluation of these three pathways of nitrogen transformation was performed as their rates of occurrence, with incubations with labeled nitrogen, as well as through the identification of functional genes involved in these three cases, by pyrosequencing. The group Planctomycetes can also be studied using the hybridization probe FISH fluorescence in situ. The results show the strong presence of genes identified as part of the denitrification pathway, as well as the rates observed for incubation of this pathway were very significant when compared with the rates obtained for the process anammox and DNRA. The results obtained from the metagenomic analyze and the FISH technique support the results obtained with incubations. It can be observed that the trend suggests the phylogeny of the organisms group with Planctomycetes not described as responsible for anammox process. Through the FISH technique, it was possible to confirm the detection of these bacteria and view its reduced presence in mangrove sampled compared to total bacteria found in these samples

Molecular and physiological characterization of Burkholderia spp. associated with the sugar cane root

A cana-de-açúcar (Saccharum sp.) é uma planta que ocupa posição de destaque entre as culturas de importância econômica no cenário internacional, principalmente no Brasil, que é o maior produtor mundial. O estudo da diversidade microbiana associada a plantas, principalmente aquelas com interesse comercial, apresenta-se como importante alternativa para melhorar as características e a sustentabilidade destas culturas. Neste sentido, bactérias endofíticas e rizobactérias tem sido alvo de muitos estudos, pois apresentam efeitos benéficos para as plantas, como promoção de crescimento e inibição de patógenos. Um dos grupos de bactérias que colonizam a cana-de-açúcar é composto por espécies do gênero Burkholderia. Este gênero é composto de bactérias que podem ser encontradas em diferentes nichos ecológicos, como o solo, a água, ou em associação com plantas, fungos, e outros animais, além de humanos. Na interação bactéria-planta, as espécies de Burkholderia podem colonizar a rizosfera e o interior das raízes hospedeiro, onde podem estimular o crescimento do vegetal, contribuir para sua nutrição, como também protegê-lo da ação de fitopatógenos. Sendo assim, isolados de Burkholderia spp. do interior das raízes (endofíticos) e da rizosfera de cana-de-açúcar foram avaliados quanto à capacidade de fixar nitrogênio, produzir AIA, sideróforos, enzimas de interesse biotecnológico, solubilizar fosfato inorgânico e inibir patógenos desta cultura. Estes isolados também foram caracterizados geneticamente por análise de restrição e seqüenciamento dos genes 16S rDNA e gyrB, além da caracterização genotípica por BOX-PCR. Os resultados indicam que os isolados possuem potencial para promoção de crescimento vegetal, inibição de patógenos e produção de lipases. Filogeneticamente, a maioria dos isolados pertencem ao complexo Burkholderia cepacia com similaridade à B. cepacia e B. cenocepacia. Considerando a ocorrência dos isolados como endófitos ou rizosféricos, as metodologias fenotípicas e genotípicas não foram capazes de distinguir os membros componentes destas comunidades. Este trabalho evidencia a ampla associação deste grupo com cana-de-açúcar, e destaca as possíveis aplicações que tais bactérias podem ter no cultivo e sustentabilidade desta cultura.Sugarcane (Saccharum spp.) occupies a position of prominence among the economically important crops in the international scene, mainly in Brazil, which is the world\'s largest producer. The study of microbial diversity associated with plants, especially those with commercial interest, presents itself an important alternative to improve the performance and sustainability of these crops. In this sense, endophytic bacteria and rhizobacteria has been target of many studies, since they have beneficial effects for plants, such as plant growth promotion and inhibition of pathogens. One of the bacterial groups that colonize sugarcane is composed of species of the genus Burkholderia. This genus is composed of a bacterium that can be found in different ecological niches, such as soil, water, or in association with plants, fungi and other animals, as well as in humans. In the association bacterium-plant, the species of Burkholderia can colonize the rhizosphere and the inside of the host roots, which can stimulate the growth of the plant, contributing to its nutrition, but also protects it from the action of phytopathogens. Thus strains of Burkholderia spp. isolated from the inside of the roots (endophytic) and from the rhizosphere of sugarcane were evaluated for the ability to fix nitrogen, produce IAA, siderophores, enzymes of biotechnological interests, solubilize inorganic phosphate and inhibits pathogens of the same crop. These strains were also genetically characterized by the analysis of enzymatic restriction and sequencing of 16S rDNA and gyrB genes, in addition to the characterization by genotypic technique BOX-PCR. The results indicate that the strains have potential for plant growth promotion, inhibition of pathogens and production of lipases. Phylogenetically, the isolates were affiliated to Burkholderia cepacia complex, with mainly similarity to B. cepacia and B. cenocepacia. Considering the occurrence of isolated as endophytes or rhizosphere, the genotypic and phenotypic methods were not able to distinguish the members of these communities. This research work demonstrates the broad association that this group has with sugarcane, and highlights the possible applications that these bacteria may have in cultivation and sustainability of this crop

Part of a SEED-based functional analysis of mangrove metagenomes.

<p>Each item represents a functional role in the SEED and is labelled by the number of reads assigned in each dataset: (a) carbon fixation and methane metabolism; (b) nitrogen metabolism; and (c) sulphur metabolism. Boxes indicate the KEGG characteristic identified, and numbers in circles indicate the number of sequences from each metagenome affiliated with the KEGG function.</p

Principal component analysis of metagenomes based on taxonomic affiliation of reads determined using MG-RAST.

<p>The percentages of variance explained in each axis are indicated.</p

Comparison of the taxonomic assignment of reads using different annotation systems.

a<p>cut-off E-value 1e-10;</p>b<p>LCA parameters (maximum number of match per read = 5, min support = 5, min score = 5, top percent = 10).</p

Taxonomic affiliation of main microbial groups involved in methane, nitrogen, and sulphur transformations in mangroves.

<p>Reads assigned by MEGAN 4.0 software were based on BLASTX vs. NCBI-NR. Numbers refer to the complete table which is part Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038600#pone.0038600.s002" target="_blank">Figure S2</a>.</p