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.

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.

RESUMO: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 inocula ção 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 ameri cana e Xanthomonas axonopodis foram os mais eficientes, com um controle entre 37 e 59%.

ABSTRACT: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 americanaand Xanthomonas axonopodis showed more efficiency, presenting control between 37 and 59%.

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.

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.

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.

Em geral, o acúmulo de exopolissacarídeos (EPS) produzidos pelos microrganismos ocorre na presença de excesso de substrato e condições limitantes de elementos essenciais ao crescimento, como nitrogênio, fósforo, enxofre, ou magnésio. A presença de EPS associada com células bacterianas é reconhecida em meio sólido para a formação de colônias mucoides e no meio líquido pelo aumento da viscosidade. Este trabalho propõe um método alternativo para realizar triagem de bactérias láticas produtoras de EPS utilizando meio sólido contendo discos de papel de filtro impregnado pela cultura. A seleção foi realizada em diferentes condições de cultivo, variando o tipo de açúcar, pH e temperatura. A produção de EPS foi visualizada pela presença da colônia mucoide sobre os discos, que foram confirmadas pelo precipitado, quando parte desta colônia foi misturada com álcool absoluto. As condições de cultivo em que houve maior produção de EPS foram: sacarose 10%, pH 7,5 a 28 ºC. Este método provou ser eficaz e econômico, uma vez que várias cepas podem ser testadas na mesma placa, sendo possível realizar uma prévia confirmação da produção do EPS.

The accumulation of exopolysaccharides (EPS) produced by microorganisms occurs in the presence of excess substrate and limiting conditions of elements that are essential to growth, such as nitrogen, phosphorus, sulfur, and magnesium. The presence of EPS produced by bacterial cells contributes to slime colonies formation in solid medium and increased viscosity in liquid medium. This paper proposes an alternative method for screening EPS-producing lactic acid bacteria using solid medium-containing discs of filter paper that are saturated with active cultures. The screening was carried out under different culture conditions varying the type of sugar, pH, and temperature. EPS production was visualized by the presence of mucoid colonies on the discs, which was confirmed by the formation of a precipitate when part of this colony was mixed with absolute alcohol. The established conditions for obtaining a high number of isolates producing EPS were 10% sucrose, pH 7.5 and 28 ºC. This method proved to be effective and economical because several strains could be tested on the same plate, with immediate confirmation.

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.

As micotoxinas são produzidas pelo metabolismo secundário de várias espécies de fungos e podem ser encontradas em quase 25% das commodities agrícolas. Esses compostos são tóxicos a humanos, animais e plantas e, portanto, esforços para evitar a contaminação de micotoxinas em alimentos e rações devem ser feitos. Além disso, até 25% das frutas e legumes em pós-colheita são perdidos em decorrência do ataque de fungos de armazenamento e/ou contaminações por micotoxinas. Vários métodos têm sido aplicados para mitigar os problemas de micotoxinas, mas a maioria deles se baseia no uso de fungicidas. Embora os produtos químicos sejam, muitas vezes, a primeira estratégia de defesa contra fungos micotoxigênicos, o uso indiscriminado de fungicidas vem despertando a percepção pública, em razão de seus efeitos nocivos sobre o meio ambiente e à saúde humana/animal. Assim, existe uma crescente pressão pública em busca de alternativas mais segura e não nocivas ao meio ambiente para controlar estes organismos. Nesse contexto, o controle biológico utilizando antagonistas microbianos, tais como bactérias, fungos e leveduras têm mostrado ser um substituto viável para reduzir a utilização de produtos químicos. Apesar dos resultados positivos, usando os agentes de controle biológico, poucos produtos apenas foram registrados e estão comercialmente disponíveis para controlar fungos produtores de micotoxinas. Esta revisão traz estratégias de controle biológico para evitar ou reduzir danos em commodities agrícolas causadas por fungos de armazenamento e a contaminação de alimentos e rações por micotoxinas.

Mycotoxins are produced by the secondary metabolism of many fungi and can be found in almost 25% of the world's agricultural commodities. These compounds are toxic to humans, animals, and plants and therefore, efforts should be made to avoid mycotoxin contamination in food and feed. Besides, up to 25% of all harvested fruits and vegetables are lost due to storage molds and/or mycotoxin contamination and many methods have been applied to mitigate these issues, but most of them rely on the use of fungicides. Although chemicals are often the first defensive line against mycotoxigenic fungi, the indiscriminate use of fungicides are awakening the public perception due to their noxious effects on the environment and human/animal health. Thus, there is an increasing public pressure for a safer and eco-friendly alternative to control these organisms. In this background, biological control using microbial antagonists such as bacteria, fungi and yeasts have been shown to be a feasible substitute to reduce the use of chemical compounds. Despite of the positive findings using the biocontrol agents only a few products have been registered and are commercially available to control mycotoxin-producing fungi. This review brings about the up-to-date biological control strategies to prevent or reduce harvested commodity damages caused by storage fungi and the contamination of food and feed by mycotoxins.

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.

Na busca de novos organismos e novos metabólitos secundários, um estudo foi conduzido visando avaliar a diversidade de bactérias endofíticas de etnovariedades de mandioca cultivadas por tribos indígenas da Amazônia brasileira e também para estudar metabólitos secundários produzidos por Bacillus pumilus. Sessenta e sete bactérias endofíticas de mandioca foram identificadas através do seqüenciamento do gene 16S rRNA e por meio da análise de ácidos graxos (FAME). Essas análises revelaram que 25% de todos os endofíticos pertenciam ao gênero Bacillus. O isolado Bacillus pumilus MAIIIM4a apresentou forte ação inibitória contra os fitopatógenos Rhizoctonia solani, Pythium aphanidermatum e Sclerotium rolfsii. Os metabólitos secundários deste isolado foram extraídos do sobrenadante usando-se hexano, diclorometano e acetato de etila. Esses extratos foram utilizados nas análises de bioautografia e LC-MS, as quais permitiram a identificação do composto pumilacidina, um antifúngico produzido por B. pumilus MAIIIM4a. A localização das bactérias endofíticas foi confirmada examinando-se o tecido celular da mandioca através de microscopia eletrônica.

In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the diversity of endophytic bacteria from ethnovarieties of cassava cultivated by Brazilian Amazon Indian tribes and also to study the secondary metabolites produced by a Bacillus pumilus strain. Sixty seven cassava endophytic bacteria were subjected to 16S rRNA sequencing and FAME analysis. The bacterial profile revealed that 25% of all endophytic isolates belonged to the genus Bacillus. The isolate B. pumilus MAIIIM4a showed a strong inhibitory activity against the fungi Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii. Secondary metabolites of this strain were extracted using hexane, dichloromethane and ethyl acetate. Extracts were subjected to bioautography and LC/MS analysis, which allowed the identification of pumilacidin, an antifungal compound produced by B. pumilus MAIIIM4a. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy.

O fungo Sclerotinia sclerotiorum, agente causal do mofo-branco, tem controle dificultado pela longevidade de seus escleródios no solo. Uma estratégia alternativa de controle é o uso do fungo antagonista Coniothyrium minitans, que parasita os escleródios de S. sclerotiorum e reduz a incidência da doença. O objetivo deste trabalho foi desenvolver uma formulação com C. minitans capaz de controlar o mofo-branco. Para tanto, picnídios deste fungo foram encapsulados com diferentes polímeros (alginato de sódio e pectina cítrica), caulim e substratos naturais (farinha de trigo e celulose). Das combinações obtidas, a formulação que continha 0,5% de alginato, 1,5% de celulose e 5% de caulim apresentou os melhores resultados quanto à viabilidade do fungo e controle da doença. Os grânulos da formulação armazenados a 4ºC apresentaram 100% de viabilidade do fungo. Nos grânulos armazenados a 28ºC, o fungo perdeu capacidade de crescer após os primeiros dois meses. O fungo formulado foi capaz de esporular sobre os grânulos incubados em meio de cultura BDA e no solo, como também foi capaz de reduzir a incidência da doença.

Sclerotinia sclerotiorum, causal agent of white mold, maintains its resistant structures viable for many years in the soil, and its control is difficult. An alternative method for controlling it is the use of Coniothyrium minitans, an antagonistic fungus efficient in attacking the sclerotia. The objective of this work was to develop a method to encapsulate picnidia of C. minitans to control the white mold, using different polymers, such as alginate and pectin supplemented with wheat flour or cellulose and kaolin. Picnidia of C. minitans formulated in alginate 0.5%, cellulose 1.5%, and kaolin 5% survived better at 4 than at 28ºC when stored during two months. After this period, only the formulation stored at 4ºC maintained its viability. When the formulation was stored at 28ºC, the fungus lost its ability to grow after two months. The fungus formulated grew abundantly on the pellets when they were inoculated on PDA. They also reduced completely the disease incidence in infested soil.

O objetivo deste trabalho foi realizar um levantamento da diversidade de microrganismos endofíticos, em plantas de mandioca (Manihot esculenta Crantz) coletadas de áreas comerciais, no Estado de São Paulo, e de etnovariedades dos estados do Amazonas e Bahia e, também, avaliar seu potencial para fixar N atmosférico e para produzir ácido indolacético. Nos três estados, foram identificadas 47 espécies de microrganismos pertencentes a 27 gêneros. Bacillus spp. foi o mais freqüente em todas as regiões. O maior número de gêneros foi encontrado em plantas provenientes do Estado do Amazonas, que apresenta a maior diversidade de microrganismos endofíticos. Amplificações por PCR do gene nifH foram avaliadas em espécies bacterianas pertencentes às gama-Proteobacteria. Isolados AIA positivos foram obtidos de material coletado em todos os estados, e foram representados por microrganismos pertencentes aos subgrupos gama-Proteobacteria, beta-Proteobacteria, Bacilli e Actinobacteria. A ocorrência de bactérias endofíticas em plantas de mandioca, com capacidade para fixar N atmosférico e produzir AIA in vitro, indica potencial para promover o crescimento da planta.

The aim of this work was to perform a survey of the diversity of endophytic microorganisms in cassava (Manihot esculenta Crantz) collected from commercial plantings, in the State of São Paulo, and in ethnovarieties collected in the States of Amazonas and Bahia, and also, to evaluate its potential of biological nitrogen fixation, and to produce indolacetic acid. In those States, 47 species of 27 genera were identified. Bacillus spp. was the most frequently found in all sampled regions. In Amazonas State, it was found the higher diversity of endophytic microorganisms. PCR amplifications of the nifH gene were evaluated in species of gamma-Proteobacteria subgroup. IAA production was found in microorganisms isolated in cassava grown in all sampled States and belonged to the following subgroups: gamma-Proteobacteria, beta-Proteobacteria, Bacilli and Actinobacteria. In cassava, the occurrence of endophytic bacteria, with N-fixing and in vitro IAA-producing abilities, indicates potential for plant growth promotion.

Os herbicidas representam 65% 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 35ºC, a diferença média de concentração do S-Metolachlor foi de 14.42%, e em 10 dias, 35.01%. Observou-se o aparecimento de um halo em volta das colônias, o que corrobora a hipótese de degradação microbiana do herbicida.

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 35ºC, the difference in the concentration of herbicide was 14.42% on average and after 10 days, 35.01%.

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% a incidência da doença, ao mesmo tempo que promoveu o crescimento de plantas.

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% of the disease and promoted the plant growth.