Cultivated bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae) from floodplains in Brazil

ABSTRACT Carnivorous plant species, such as Utricularia spp., capture and digest prey. This digestion can occur through the secretion of plant digestive enzymes and/or by bacterial digestive enzymes. To comprehend the physiological mechanisms of carnivorous plants, it is essential to understand the microbial diversity related to these plants. Therefore, in the present study, we isolated and classified bacteria from different organs of Utricularia breviscapa (stolons and utricles) and from different geographic locations (São Paulo and Mato Grosso). We were able to build the first bacterium collection for U. breviscapa and study the diversity of cultivable bacteria. The results show that U. breviscapa bacterial diversity varied according to the geographic isolation site (São Paulo and Mato Grosso) but not the analyzed organs (utricle and stolon). We reported that six genera were common to both sample sites (São Paulo and Mato Grosso). These genera have previously been reported to be beneficial to plants, as well as related to the bioremediation process, showing that these isolates present great biotechnological and agricultural potential. This is the first report of an Acidobacteria isolated from U. breviscapa. The role of these bacteria inside the plant must be further investigated in order to understand their population dynamics within the host.

Microbial interactions: ecology in a molecular perspective

ABSTRACT The microorganism-microorganism or microorganism-host interactions are the key strategy to colonize and establish in a variety of different environments. These interactions involve all ecological aspects, including physiochemical changes, metabolite exchange, metabolite conversion, signaling, chemotaxis and genetic exchange resulting in genotype selection. In addition, the establishment in the environment depends on the species diversity, since high functional redundancy in the microbial community increases the competitive ability of the community, decreasing the possibility of an invader to establish in this environment. Therefore, these associations are the result of a co-evolution process that leads to the adaptation and specialization, allowing the occupation of different niches, by reducing biotic and abiotic stress or exchanging growth factors and signaling. Microbial interactions occur by the transference of molecular and genetic information, and many mechanisms can be involved in this exchange, such as secondary metabolites, siderophores, quorum sensing system, biofilm formation, and cellular transduction signaling, among others. The ultimate unit of interaction is the gene expression of each organism in response to an environmental (biotic or abiotic) stimulus, which is responsible for the production of molecules involved in these interactions. Therefore, in the present review, we focused on some molecular mechanisms involved in the microbial interaction, not only in microbial-host interaction, which has been exploited by other reviews, but also in the molecular strategy used by different microorganisms in the environment that can modulate the establishment and structuration of the microbial community.

Microbial interactions: ecology in a molecular perspective

ABSTRACT The microorganism-microorganism or microorganism-host interactions are the key strategy to colonize and establish in a variety of different environments. These interactions involve all ecological aspects, including physiochemical changes, metabolite exchange, metabolite conversion, signaling, chemotaxis and genetic exchange resulting in genotype selection. In addition, the establishment in the environment depends on the species diversity, since high functional redundancy in the microbial community increases the competitive ability of the community, decreasing the possibility of an invader to establish in this environment. Therefore, these associations are the result of a co-evolution process that leads to the adaptation and specialization, allowing the occupation of different niches, by reducing biotic and abiotic stress or exchanging growth factors and signaling. Microbial interactions occur by the transference of molecular and genetic information, and many mechanisms can be involved in this exchange, such as secondary metabolites, siderophores, quorum sensing system, biofilm formation, and cellular transduction signaling, among others. The ultimate unit of interaction is the gene expression of each organism in response to an environmental (biotic or abiotic) stimulus, which is responsible for the production of molecules involved in these interactions. Therefore, in the present review, we focused on some molecular mechanisms involved in the microbial interaction, not only in microbial-host interaction, which has been exploited by other reviews, but also in the molecular strategy used by different microorganisms in the environment that can modulate the establishment and structuration of the microbial community.

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

Analysis of 16S rRNA and mxaF genes reveling insights into Methylobacterium niche-specific plant association

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51 percent and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.

The effect of different growth regimes on the endophytic bacterial communities of the fern, Dicksonia sellowiana hook (Dicksoniaceae)

Endophytic bacteria associated with the fern Dicksonia sellowiana were investigated. The bacterial communities from the surface-sterilized pinnae and rachis segments of the plants from the Brazilian Atlantic Rainforest that grew in native field conditions were compared with the bacterial communities from plants grown in greenhouses and plants that were initially grown in greenhouses and then transferred to the forest. From 540 pinnae and 540 rachis segments, 163 (30.2 percent) and 346 (64.2 percent) were colonized by bacteria, respectively. The main bacterial genera and species that were isolated included Bacillus spp. (B. cereus, B. megaterium, B. pumilus and B. subtilis), Paenibacillus sp., Amphibacillus sp., Gracilibacillus sp., Micrococcus sp. and Stenotrophomonas spp. (S. maltophilia and S. nitroreducens). B. pumilus was the most frequently isolated bacterial species. Amphibacillus and Gracilibacillus were reported as endophytes for the first time. Other commonly found bacterial genera were not observed in D. sellowiana, which may reflect preferences of specific bacterial communities inside this fern or detection limitations due to the isolation procedures. Plants that were grown in greenhouses and plants that were reintroduced into the forest displayed more bacterial genera and species diversity than native field plants, suggesting that reintroduction shifts the bacterial diversity. Endophytic bacteria that displayed antagonistic properties against different microorganisms were detected, but no obvious correlation was found between their frequencies with plant tissues or with plants from different growth regimes. This paper reports the first isolation of endophytic bacteria from a fern.

Genetic variability of Brazilian isolates of Alternaria alternata detected by AFLP and RAPD techniques / Variabilidade genética de isolados Brasileiros de Alternaria alternata por meio de marcadores moleculares de AFLP e RAPD

The Alternaria brown spot (ABS) is a disease caused in tangerine plants and its hybrids by the fungus Alternaria alternata f. sp. citri which has been found in Brazil since 2001. Due to the recent occurrence in Brazilian orchards, the epidemiology and genetic variability of this pathogen is still an issue to be addressed. Here it is presented a survey about the genetic variability of this fungus by the characterization of twenty four pathogenic isolates of A. alternata f. sp. citri from citrus plants and four endophytic isolates from mango (one Alternaria tenuissima and three Alternaria arborescens). The application of two molecular markers Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) had revealed the isolates clustering in distinct groups when fingerprintings were analyzed by Principal Components Analysis (PCA). Despite the better assessment of the genetic variability through the AFLP, significant modifications in clusters components were not observed, and only slight shifts in the positioning of isolates LRS 39/3 and 25M were observed in PCA plots. Furthermore, in both analyses, only the isolates from lemon plants revealed to be clustered, differently from the absence of clustering for other hosts or plant tissues. Summarizing, both RAPD and AFLP analyses were both efficient to detect the genetic variability within the population of the pathogenic fungus Alternaria spp., supplying information on the genetic variability of this species as a basis for further studies aiming the disease control.

A mancha marrom ou mancha de Alternaria é uma doença causada pelo fungo Alternaria alternata f. sp. citri, encontrada no Brasil desde 2001 em plantas de tangerina e seus híbridos. Por se tratar de uma doença recente no Brasil, a epidemiologia e variabilidade genética deste patógeno compõem importantes pontos a serem estudados. Este trabalho teve como objetivo avaliar a variabilidade genética deste patógeno por meio da caracterização de vinte e quatro isolados de A. alternata f. sp. citri de plantas de citros juntamente com quatro isolados endofíticos de manga, sendo um deles identificado como Alternaria tenuissima e outros três como Alternaria arborescens. A análise de componentes principais (PCA) do perfil de bandas obtidos pela aplicação de duas técnicas de marcadores moleculares, Amplificação Aleatória de Polimorfismos de DNA (RAPD) e Polimorfismo de Comprimento de Fragmentos Amplificados (AFLP), mostrou a formação de quatro grupos distintos. Apesar do mais amplo perfil de análise por meio da técnica de AFLP, não foi observado nenhuma modificação significativa dentro dos grandes grupos obtidos quando comparado ao RAPD, exceto no posicionamento dos isolados LRS 39/3 e 25M. Além disso, em ambas as análises, somente os isolados de plantas de limão agruparam entre si. Considerando outros hospedeiros ou tecidos de planta não foi possível encontrar grupos específicos. Concluindo, ambas as análises (RAPD e AFLP) são eficientes no estudo de variabilidade genética de Alternaria spp., fornecendo informações sobre a diversidade genética desta espécie, servindo como base para futuramente correlacionar este estudo com estudos adicionais objetivando o controle da doença.

Assessing the diversity of bacterial communities associated with plants / Avaliação da diversidade de comunidades bacterianas associadas às plantas

Plant-bacteria interactions result from reciprocal recognition between both species. These interactions are responsible for essential biological processes in plant development and health status. Here, we present a review of the methodologies applied to investigate shifts in bacterial communities associated with plants. A description of techniques is made from initial isolations to culture-independent approaches focusing on quantitative Polymerase Chain Reaction in real time (qPCR), Denaturing Gradient Gel Electrophoresis (DGGE), clone library construction and analysis, the application of multivariate analyses to microbial ecology data and the upcoming high throughput methodologies such as microarrays and pyrosequencing. This review supplies information about the development of traditional methods and a general overview about the new insights into bacterial communities associated with plants.

As interações planta-bactéria resultam de um reconhecimento recíproco de ambas espécies. Estas interações são responsáveis por processos biológicos essenciais para o desenvolvimento e a proteção das plantas. Este trabalho revisa as metodologias aplicadas na investigação de alterações nas comunidades bacterianas associadas às plantas. Uma descrição das técnicas é feita, desde o isolamento até a aplicação de técnicas independentes de cultivo, destacando as técnicas de qPCR, Gel de Eletroforese em Gradiente Desnaturante (DGGE), construção e análise de bibliotecas de clones, a aplicação de análise multivariada em dados de ecologia microbiana, e as novas metodologias de alto processamento de amostras como microarranjos e pirosequenciamento. Em resumo, esta revisão fornece informações sobre o desenvolvimento das técnicas tradicionais e uma visão geral sobre as novas tendências dos estudos de comunidades bacterianas associadas às plantas.

The biology and potential for genetic research of transposable elements in filamentous fungi

Recently many transposable elements have been identified and characterized in filamentous fungi, especially in species of agricultural, biotechnological and medical interest. Similar to the elements found in other eukaryotes, fungal transposons can be classified as class I elements (retrotransposons) that use RNA and reverse transcriptase and class II elements (DNA transposons) that use DNA. The changes (transposition and recombination) caused by transposons can supply wide-ranging genetic variation, especially for species that do not have a sexual phase. The application of transposable elements to gene isolation and population analysis is an important tool for molecular biology and studies of fungal evolution

Direct RAPD evaluation of bacteria without conventional DNA extraction

O presente trabalho mostra a amplificação de DNA das bactérias Pantoea agglomerans e Bacillus pumilus por meio da técnica de RAPD (Amplificação ao acaso de DNA polimórfico). Para esta análise, o DNA molde foi obtido sem a utilização de técnicas de extração convencional, ou seja, sem a purificação do DNA. Bactérias foram cultivadas por 20 horas em 5 mL de meio LB, centrifugado e ressuspendido em tampão TE. A suspensão resultante foi fervida por 5 min., diluída e 2,0 µL foram usados em reações de 15 µL. Os resultados mostraram que os padrões observados com o DNA obtido pela fervura das células não apresentou diferenças significativas daquele obtido com DNA extraído e purificado com fenol, sugerindo a possibilidade da utilização deste método para o estudo da variabilidade genética de populações microbianas.