Phylogenetic insights into the diversity of homocytous cyanobacteria from Amazonian rivers.

The Amazon Rainforest holds great tropical biodiversity, mainly because of its favourable climatic conditions. The high temperatures, luminosity and humidity coupled with the nutritional simplicity of cyanobacteria allow undiscovered diversity to flourish within this group of microorganisms. Some efforts to reveal this diversity have been attempted; however, most were focused on the microscopic observation of environmental samples without any genetic information. Very few studies focusing on morphological, ecological and molecular criteria have been conducted, and none have been devoted to homocytous cyanobacteria forms in Amazonia region. Therefore, the genetic relationships amongst strains retrieved from this ecosystem with regard to other environments from Brazil and the world have not been tested and, consequently, the Amazonian strains would naturally be assumed as novel to science. To examine these relationships, cultured homocytous cyanobacteria isolated from two Amazonian rivers (Amazonas and Solimões) were evaluated using a phylogenetic perspective, considering the 16S rRNA gene sequence. A total of eleven homocytous cyanobacterial strains were isolated. Morphologically, they were identified as Pseudanabaena, Leptolyngbya, Planktothrix and Phormidium, but genetically they were included in the typical clusters of Planktothrix, Pseudanabaena, Cephalothrix, Pantanalinema and Alkalinema. These three latter genera have been detected in other Brazilian ecosystems only (Pantanal, Atlantic Rainforest and Pampa), while those remaining have been extensively found in many parts of the world. The data provided here indicate that Amazonian rivers support a homocytous cyanobacterial diversity previously reported from other geographical and ecological environments.

Williamsia spongiae sp. nov., an actinomycete isolated from the marine sponge Amphimedon viridis.

A novel actinobacterium, designated isolate B138T, was isolated from the marine sponge, Amphimedon viridis, which was collected from Praia Guaecá (São Paulo, Brazil), and its taxonomic position was established using data from a polyphasic study. The organism showed a combination of chemotaxonomic and morphological characteristics consistent with its classification in the genus Williamsia and it formed a distinct phyletic line in the Williamsia 16S rRNA gene tree. It was most closely related to Williamsia serinedens DSM 45037T and Williamsia deligens DSM 44902T (99.0 % 16S rRNA gene sequence similarity) and Williamsia maris DSM 44693T (97.5 % 16S rRNA gene sequence similarity), but was distinguished readily from these strains by the low DNA-DNA relatedness values (62.3-64.4 %) and by the discriminatory phenotypic properties. Based on the data obtained, the isolate B138T (=CBMAI 1094T=DSM 46676T) should be classified as the type strain of a novel species of the genus Williamsia, for which the name Williamsia spongiae sp. nov. is proposed.

Effect of Monocerin, a Fungal Secondary Metabolite, on Endothelial Cells.

This study reports the isolation and identification of the endophytic fungus Exserohilum rostratum through molecular and morphological analysis using optical and transmission electron microscopy (TEM), as well as the procurement of its secondary metabolite monocerin, an isocoumarin derivative. Considering the previously observed biological activities of monocerin, this study was performed on human umbilical vein endothelial cells (HUVECs) that are widely used as an in vitro model for several different purposes. Important parameters, such as cell viability, senescence-associated ß-galactosidase, cellular proliferation by using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), apoptosis analysis with annexin, cellular morphology through scanning electron microscopy (SEM), and laser confocal analysis were evaluated after exposing the cells to monocerin. After 24 h of exposure to monocerin at 1.25 mM, there was more than 80% of cell viability and a low percentage of cells in the early and late apoptosis and necrosis. Monocerin increased cell proliferation and did not induce cell senescence. Morphological analysis showed cellular integrity. The study demonstrates aspects of the mechanism of action of monocerin on endothelial cell proliferation, suggesting the possibility of its pharmaceutical application, such as in regenerative medicine.

Wheat dwarfing influences selection of the rhizosphere microbiome.

The development of dwarf wheat cultivars combined with high levels of agrochemical inputs during the green revolution resulted in high yielding cropping systems. However, changes in wheat cultivars were made without considering impacts on plant and soil microbe interactions. We studied the effect of these changes on root traits and on the assembly of rhizosphere bacterial communities by comparing eight wheat cultivars ranging from tall to semi-dwarf plants grown under field conditions. Wheat breeding influenced root diameter and specific root length (SRL). Rhizosphere bacterial communities from tall cultivars were distinct from those associated with semi-dwarf cultivars, with higher differential abundance of Actinobacteria, Bacteroidetes and Proteobacteria in tall cultivars, compared with a higher differential abundance of Verrucomicrobia, Planctomycetes and Acidobacteria in semi-dwarf cultivars. Predicted microbial functions were also impacted and network analysis revealed a greater level of connectedness between microbial communities in the tall cultivars relative to semi-dwarf cultivars. Taken together, results suggest that the development of semi-dwarf plants might have affected the ability of plants to recruit and sustain a complex bacterial community network in the rhizosphere.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth.

Mangroves are ecosystems located in the transition zone between land and sea that serve as a potential source of biotechnological resources. Brazil's extensive coast contains one of the largest mangrove forests in the world (encompassing an area of 25,000km2 along all the coast). Endophytic bacteria were isolated from the following three plant species: Rhizophora mangle, Laguncularia racemosa and Avicennia nitida. A large number of these isolates, 115 in total, were evaluated for their ability to fix nitrogen and solubilize phosphorous. Bacteria that tested positive for both of these tests were examined further to determine their level of indole acetic acid production. Two strains with high indole acetic acid production were selected for use as inoculants for reforestation trees, and then the growth of the plants was evaluated under field conditions. The bacterium Pseudomonas fluorescens (strain MCR1.10) had a low phosphorus solubilization index, while this index was higher in the other strain used, Enterobacter sp. (strain MCR1.48). We used the reforestation tree Acacia polyphylla. The results indicate that inoculation with the MCR1.48 endophyte increases Acacia polyphylla shoot dry mass, demonstrating that this strain effectively promotes the plant's growth and fitness, which can be used in the seedling production of this tree. Therefore, we successfully screened the biotechnological potential of endophyte isolates from mangrove, with a focus on plant growth promotion, and selected a strain able to provide limited nutrients and hormones for in plant growth.

Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices.

Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.

Screening of Brazilian cacti rhizobacteria for plant growth promotion under drought.

Drought is one of the major problems worldwide. The search for new and efficient microorganisms, from unexplored environments, to be used in association with plants to alleviate the negative effects imposed by water stress, is an interesting alternative. Thus, cacti-associated bacteria from the Brazilian semi-arid region were isolated based on their ability to grow in medium with reduced water availability. Strains were tested for the production of exopolysaccharides (EPS), as well as in vitro plant growth promotion traits. A great proportion of the isolates belong to the genus Bacillus. From a total of forty-eight bacteria, 65% were able to grow in medium with reduced water availability (0.919Aw), exopolysaccharide production was observed for 65% of the strains. The production of indole acetic acid (IAA) exceeding 51µgmL(-1) was observed for 4% and the high solubilization of Ca-P was verified for 6% of the isolates. No strain was able to produce hydrogen cyanide (HCN), 71% produced ammonia and 79% showed a halo of carboxymethyl cellulose (CMC) degradation. Zea mays L. growth promotion under water stress (30% of field capacity) was achieved by two strains of Bacillus spp. This is the first report to describe cacti-associated bacteria from Brazilian semi-arid with plant growth-promoting abilities.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth

ABSTRACT Mangroves are ecosystems located in the transition zone between land and sea that serve as a potential source of biotechnological resources. Brazil's extensive coast contains one of the largest mangrove forests in the world (encompassing an area of 25,000 km2 along all the coast). Endophytic bacteria were isolated from the following three plant species: Rhizophora mangle, Laguncularia racemosa and Avicennia nitida. A large number of these isolates, 115 in total, were evaluated for their ability to fix nitrogen and solubilize phosphorous. Bacteria that tested positive for both of these tests were examined further to determine their level of indole acetic acid production. Two strains with high indole acetic acid production were selected for use as inoculants for reforestation trees, and then the growth of the plants was evaluated under field conditions. The bacterium Pseudomonas fluorescens (strain MCR1.10) had a low phosphorus solubilization index, while this index was higher in the other strain used, Enterobacter sp. (strain MCR1.48). We used the reforestation tree Acacia polyphylla. The results indicate that inoculation with the MCR1.48 endophyte increases Acacia polyphylla shoot dry mass, demonstrating that this strain effectively promotes the plant's growth and fitness, which can be used in the seedling production of this tree. Therefore, we successfully screened the biotechnological potential of endophyte isolates from mangrove, with a focus on plant growth promotion, and selected a strain able to provide limited nutrients and hormones for in plant growth.

Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices

ABSTRACT Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.

Avaliação de bactérias endofíticas para o controle biológico da mancha foliar de Exserohilum turcicum em milho / Assessing of endophytic bacteria for biological control of northern corn leaf blight (Exserohilum turcicum)

A eficácia de nove isolados de bactérias endofíticas foi avaliada no biocontrole da mancha foliar de Exserohilum turcicum, pela microbiolização das sementes e da parte aérea do milho híbrido AS-1548 (72 e 24 horas antes e no mesmo dia da inoculação do patógeno) em condições de casa de vegetação. Verificou-se que Bacillus subtilis 0G, Bacillus lentimorbus, Streptomyces sp. e Bacillus agaradhaerens se destacaram dos demais, quando aplicados na parte aérea, em todos os intervalos testados, com um controle na ordem de 42 a 61%. Quando as bactérias foram aplicadas nas sementes, Bacillus lentimorbus, Streptomyces sp., Ewingella americana e Xanthomonas axonopodis foram os mais eficientes, com um controle entre 37 e 59%.(AU)

The efficacy of nine endophytic bacterial strains was evaluated on biocontrol of northern corn leaf blight (Exserohilum turcicum), by microbiolization of seeds and aerial parts of maize, hybrid AS-1548 (72 and 24 hours before and at same day of the pathogen inoculation) in greenhouse conditions. It was found that Bacillus subtilis 0G, Bacillus lentimorbus, Streptomyces sp. and Bacillus agaradhaerens highlighted from the others, when applied on aerial parts of maize, in all intervals tested, presenting control in order of 42 to 61%. When the bacteria were applied on seeds, Bacillus lentimorbus, Streptomyces sp., Ewingella americana and Xanthomonas axonopodis showed more efficiency, presenting control between 37 and 59%.(AU)

Effects of different osmolarities on bacterial biofilm formation

Biofilm formation depends on several factors. The influence of different osmolarities on bacterial biofilm formation was studied. Two strains (Enterobacter sp. and Stenotrophomonas sp.) exhibited the most remarkable alterations. Biofilm formation is an important trait and its use has been associated to the protection of organisms against environmental stresses.

Screening of endoglucanase-producing bacteria in the saline rhizosphere of Rhizophora mangle

In screening the culturable endoglucanase-producing bacteria in the rhizosphere of Rhizophora mangle, we found a prevalence of genera Bacillus and Paenibacillus. These bacteria revealed different activities in endoglucolysis and biofilm formation when exposed to specific NaCl concentrations, indicating modulated growth under natural variations in mangrove salinity.

Biological control of phytopathogenic fungi by endophytic actinomycetes isolated from maize (Zea mays L.)

This work aimed a survey on the biodiversity of maize endophytic actinomycete, and an evaluation of their potential to control the phytopathogenic fungi. From several regions of São Paulo state, 40 strains were isolated from the healthy maize plants. The identification of these strains, based on morphological properties and fatty acid methyl ester (FAME) profile showed that most of them belonged to the Streptomyces genus. These isolates were first screened for the growth inhibition of phytopathogenic fungi and results showed that all the isolate were able to inhibit the development of at least one tested pathogen. Two selected isolates were then evaluated for the control of P. aphanidermatum in cucumber (Cucumis sativa L.) under greenhouse conditions. Isolate 16R3B was able to reduce up to 71% damping-off incidence whereas isolate 14F1D/2 reduced the disease incidence by 36%. Damping- off control in cucumber, mainly for the isolate 16R3B, suggested for its use in greenhouse cucumber producing fields and to be tested in field trials.

Changes in hyphal morphology due to chitosan treatment in some fungal species

In this work, changes in the hyphal morphology due to chitosan treatment in some fungal species were studied. Scanning electron microscope (SEM) observations revealed that chitosans with molar fraction of acetyl groups (F A 0.16 and 0.18) and degree of polymerization (DP 1,089 and 1,242) had a direct effect on the morphology of the chitosan-treated fungi, reflecting its potential for causing a delay in the growth of Alternaria alternata (500 µg × mL-1), Botrytis cinerea (1,000 µg × mL-1), Penicillium expansum (1,000 µg × mL-1) and Rhizopus stolonifer (500 µg × mL-1). Mycelial aggregation and structural changes such as excessive branching, swelling of the cell wall and hyphae size reduction were observed in the micrographs.

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.

Selection of microorganisms degrading S-Metolachlor herbicide

The aim of this work was to study herbicide degradation through selected microorganisms from humus and soil subjected to different plantation systems. The following bacterial species were identified: Klebsiella pneumoniae pneumoniae GC s.B strain 1, Pseudomonas alcaligenes, Enterobacter aerogenes GC s.A and Klebsiella pneumoniae pneumoniae GC s.B strain 2. Growth studies yet suggested the possibility of a very long lag phase. Although, culture with the herbicide presented biofilm formation and there were color changes in the herbicide that could have interfered with the espectrophotometry readings. After 5 days of incubation at 35°C, the difference in the concentration of herbicide was 14.42 percent on average and after 10 days, 35.01 percent.

Os herbicidas representam 65 por cento do consumo geral, sendo que o S-Metolachlor é um dos mais utilizados e está trazendo preocupações ambientais. Objetivamos detectar a degradação do S-Metolachlor por microorganismos de solos sob plantio. Foram identificadas as espécies bacterianas: Klebsiella pneumoniae pneumoniae GC s.B linhagem 1, Pseudomonas alcaligenes, Enterobacter aerogenes GC s.A e Klebsiella pneumoniae pneumoniae GC s.B linhagem 2. Resultados da curva de crescimento por espectrofotometria não permitiram definir diferentes fases, levando a pensar em uma fase Lag longa. Frascos de cultura demonstraram a formação de biofilme, provocando mudança na cor do herbicida, interferindo na leitura do crescimento. É possível a existência de fase Log, mas não detectável pelo método. Após 5 dias de incubação a 35°C, a diferença média de concentração do S-Metolachlor foi de 14.42 por cento, e em 10 dias, 35.01 por cento. Observou-se o aparecimento de um halo em volta das colônias, o que corrobora a hipótese de degradação microbiana do herbicida.

Antagonism of Serratia marcescens towards Phytophthora parasitica and its effects in promoting the growth of citrus

Phytophthora parasitica causes serious widespread, and difficult-to-control root rots in warmer regions. This oomycete is one of the most important pathogen of citrus. This paper reports the biological control of the pathogen by a strain of Serratia marcescens R-35, isolated from citrus rhizosphere. In greenhouse trials, the bacterium suppressed more than 50 percent of the disease and promoted the plant growth.

Phytophthora parasitica é um oomiceto que causa sérios problemas fitossanitários em diferentes espécies de plantas em regiões tropicais e o controle tem sido difícil. Este patógeno é um dos mais importante à citricultura. Este trabalho relata o controle biológico do patógeno por uma linhagem de Serratia marcescens R-35, isolada da rizosfera de citros. Em condições de casa-de-vegetação, a bactéria reduziu em mais de 50 por cento a incidência da doença, ao mesmo tempo que promoveu o crescimento de plantas.

Isolation and characterization of carbendazim degrading trichoderma harzianum rifai mutants

Mutants of Trichoderma harzianum Rifai, obtained after ultraviolet (UV) light exposure, showed high resistant to the fungicide benomyl. A mutant (2B6)was capable of degrading carbendazim, other fungicide of the benzimidazole fungicide. This mutant degraded 41.5% of the molecule within five days. This and others mutants (2B1 and 2B2) presented variation in size and frequency of uni-nucleated and/ or bi-nucleated spores compared to the wild type. Four primers generated RAPDs patterns that allowed the mutant to be differentiated from the wild-type. It is concluded that using UV mutagenization, it is feasible to obtain strains of T. harzianum with improved pesticide degradation ability.

Scanning electron microscopy of conidia of Trichoderma stromaticum, a biocontrol agent of witches' broom disease of cocoa

Estudos morfológicos de conídios de Trichoderma harzianun, um agente de biocontrole da vassoura-de-bruxa do cacaueiro, foram feitos sob microscopia eletrônica de varredura com emissão de campo. Características da superfície de conídios do fungo mostraram ser rugosas quando observadas em alta magnificação; fato esse impossível de ser visualizado por microscopia ótica. Também foram observados, com freqüência, massas de conídios completamente envolvidos por material mucilaginoso e detalhes de células ramificadas dicotomicamente que formam as pústulas.

Production of phenol-oxidases and peroxidases by fungi isolated from irrigated rice

The aim of this work was to study the potential of fungus strains considered as prospective degraders for the herbicides quinclorac and propanil, for ligninolytic enzyme production. Eight fungal strains were grown in King's B liquid culture medium supplemented with 0.05% Remazol Brilliant Blue R (RBBR) and in liquid culture medium containing wheat bran as substrate. The enzymatic system assessment were: lignin peroxidase, manganese peroxidase and laccases. Results indicated differential patterns of ligninolytic enzyme production; the highest enzymatic activities were related to the production of lignin peroxidase. Among the strains, two (P3SA1F and P11SA2F) showed RBBR discoloration, suggesting the possibility of their application in bioremediation studies.

A proposta deste trabalho foi estudar o potencial das linhagens fúngicas, consideradas potenciais degradadoras dos herbicidas quinclorac e propanil, para produção de enzimas ligninolíticas. Oito linhagens fúngicas foram cultivadas em meio de cultura líquido King's B suplementado com 0,05% de Remazol Brilliant Blue R (RBBR) e em meio de cultura líquido contendo farelo de trigo como substrato. Os sistemas enzimáticos avaliados foram: lignina peroxidase, manganês peroxidase e lacases. Os resultados demonstraram padrões diferenciados quanto à produção de enzimas ligninolíticas entre as linhagens, sendo que as maiores atividades enzimáticas estiveram relacionadas à produção de lignina peroxidase. Das oito linhagens, duas (P3SA1F e P11SA2F) apresentaram descoloração do RBBR, sugerindo a possibilidade de sua aplicabilicação em estudos de biorremediação