Subunits of ribosomal RNA genes (rDNAs) characterized by PCR-based protocols have been the proxy for studies in microbial taxonomy, phylogenetics, evolution and ecology. However, relevant factors ...have shown to interfere in the experimental outputs in a variety of systems. In this work, a 'theoretical' to 'actual' delta approach was applied to data on culturable mock bacterial communities (MBCs) to study the levels of losses in operational taxonomic units (OTUs) detectability. Computational and lab-bench strategies based on 16S rDNA amplification by 799F and U1492R primers were employed, using a fingerprinting method with highly improved detectability of fragments as a case-study tool. MBCs were of two major types: in silico MBCs, assembled with database-retrieved sequences, and in vitro MBCs, with AluI digestions of PCR data generated from culturable endophytes isolated from cacao trees.
Interfering factors for the 16 s rDNA amplifications, such as the type of template, direct and nested PCR, proportion of chloroplast DNA from a tropical plant source (Virola officinalis), and biased-amplification by the primers resulted in altered bacterial 16S rDNA amplification, both on MBCs and V. officinalis leaf-extracted DNA. For the theoretical data, the maximum number of fragments for in silico and in vitro cuts were not significantly different from each other. Primers' preferences for certain sequences were detected, depending on the MBCs' composition prior to PCR. The results indicated overall losses from 2.3 up to 8.2 times in the number of OTUs detected from actual AluI digestions of MBCs when compared to in silico and in vitro theoretical data.
Due to all those effects, the final amplification profile of the bacterial community assembled was remarkably simplified when compared to the expected number of detectable fragments known to be present in the MBC. From these findings, the scope of hypotheses generation and conclusions from experiments based on PCR amplifications of bacterial communities was discussed.
Trichoderma species are found in soil and in association with plants. They can act directly or indirectly in the biological control of plant diseases and in the promotion of plant growth, being among ...the most used fungi in the formulation of bioproducts applied to agricultural systems. The main objective of this study was to characterize at a first-tier level a collection of 67 Trichoderma isolates from various tropical sources, based solely on sequencing of the internal transcribed spacer (ITS) region of the rRNA genes. Our goal was to provide a preliminary idea of the baseline diversity in this collection, to combine this information later with an array of other isolate-specific physiological data. This study provides a required knowledge at molecular level for assessment of this germplasm potential as a source of biotechnological products for beneficial effects in plants.
Sequencing of the ITS region showed that the 67 Trichoderma isolates belonged in 11 species: T. asperellum, T. atroviride, T. brevicompactum, T. harzianum, T. koningiopsis, T. longibrachiatum, T. pleuroticola, T. reesei, T. spirale, T. stromaticum and T. virens. A total of 40.3% of the isolates were very closely related to each other and similar to T. harzianum. The baseline genetic diversity found indicates that the collection has different genotypes, which can be exploited further as a source of bioproducts, aiming at providing beneficial effects to plants of interest to cope with biotic and abiotic stresses.
Bacillus thuringiensis (Bt) has been used successfully as a biopesticide for more than 60 years. More recently, genes encoding their toxins have been used to transform plants and other organisms. ...Despite the large amount of research on this bacterium, its true ecology is still a matter of debate, with two major viewpoints dominating: while some understand Bt as an insect pathogen, others see it as a saprophytic bacteria from soil. In this context, Bt's pathogenicity to other taxa and the possibility that insects may not be the primary targets of Bt are also ideas that further complicate this scenario. The existence of conflicting research results, the difficulty in developing broader ecological and genetics studies, and the great genetic plasticity of this species has cluttered a definitive concept. In this review, we gathered information on the aspects of Bt ecology that are often ignored, in the attempt to clarify the lifestyle, mechanisms of transmission and target host range of this bacterial species. As a result, we propose an integrated view to account for Bt ecology. Although Bt is indeed a pathogenic bacterium that possesses a broad arsenal for virulence and defense mechanisms, as well as a wide range of target hosts, this seems to be an adaptation to specific ecological changes acting on a versatile and cosmopolitan environmental bacterium. Bt pathogenicity and host-specificity was favored evolutionarily by increased populations of certain insect species (or other host animals), whose availability for colonization were mostly caused by anthropogenic activities. These have generated the conditions for ecological imbalances that favored dominance of specific populations of insects, arachnids, nematodes, etc., in certain areas, with narrower genetic backgrounds. These conditions provided the selective pressure for development of new hosts for pathogenic interactions, and so, host specificity of certain strains.
Intragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In ...this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner.
Total genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5-V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding
I-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the 'number of
I bands' and 'type of eletropherogram'.
Two types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the
I-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity.
The approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.
Bioinsecticides with lower concentrations of endospores/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus ...thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are unwanted (β-exotoxins), as they display widespread toxicity across taxa. A strain simultaneously providing distinct toxin activities in crystals and SN would be desirable for bioinsecticides development; however, strains secreting β-exotoxins should be discarded, independently of other useful entomotoxins. Entomotoxicity of crystals and SN from a Brazilian Bt tolworthi strain (Btt01) was tested against Spodoptera frugiperda to assess the potential for biocontrol-product development based on more than one type of toxin/activity. Tests showed that 10(7) endospores mL(-1) caused >80% of larvae mortality, suggesting Btt01 may be used in similar concentrations as those of other Bt-based biopesticides. When it was applied to cornfields, a significant 60% reduction of larvae infestation was observed. However, bioassays with Btt01 SN revealed a thermostable toxic activity. Physicochemical characterization strongly suggests the presence of unwanted β-exotoxins, with isolate-specific temporal variation in its secretion. Knowledge of the temporal pattern of secretion/activity in culture for all forms of toxins produced by a single strain is required to both detect useful activities and avoid the potential lack of identification of undesirable toxins. These findings are discussed in the contexts of commercial Bt product development, advantages of multiple-activity strains, and care and handling recommended for large-scale fermentation systems.
The goal of this study was to assess the presence and surfactant potential of naturally occurring microbes from a tropical soil with petrochemical contamination. Microorganisms in a soil sample from ...a Brazilian landfarm were isolated and grown on petroleum as the sole carbon source. Of 60 isolates screened for petroleum-based growth, 7 demonstrated surfactant activities by the drop-collapse methodology over various types of oils. From their growth profiles in liquid culture during 132 h, all had their first detection of surfactant activity after 96 h. Little is currently known about biosurfactant-producing microorganisms in tropical environments contaminated by hydrophobic compounds, and the search for them is essential for bioremediation and for oil recovery enhanced by microbes. Our results indicate that different petroleum-grown microorganisms showing surfactant activity can be recovered from landfarm soil in a tropical environment.
Amostras fecais de cães provenientes de distritos carentes do Município de Ilhéus / BA foram analisadas para a presença de Salmonella spp durante o período de junho de 2001 a abril de 2002. Dezoito ...(9,47%) dos 190 animais foram considerados positivos sendo que 66,6% destes casos ocorreram com animais até 1 ano de idade, 56% se alimentavam com comida caseira, 66% tinham acesso à fonte de água não tratada e 67% possuía comportamento domiciliar. A sintomatologia da doença não foi percebida em 83,3% dos animais. A S. Gafsa representou o sorotipo mais prevalente (38,9%), seguido pela S. Rubislaw (27,8), S. Carrau (16,7%) e S. enterica subsp. Houtenae (11,1%). Uma cepa não pôde ser identificada. O antibiograma revelou a gentamicina como sendo a droga mais potente, in vitro, para o tratamento das salmonelas encontradas na região de Ilhéus, seguida pela ampicilina. Nossos resultados confirmam que cães representam um importante reservatório de sorotipos exóticos de Salmonella e que ocorre padrões diferentes de sensibilidade a antibióticos entre eles.
Fecal samples from dogs residing in areas of poor living conditions in Ilhéus city / BA were examined for Salmonella spp from June 2001 to April 2002. Eighteen (9.47%) of 190 animals were found to be positive. 66.6% of those samples were taken from one year old puppies, 56% of the positive animals were fed by table scrap, 66% did not drink treated water and 67% had a domiciliary behavior. Symptoms of salmonellosis were not present in 83% of all positive animals. S. Gafsa represented the most prevalent serovar (38.9%), followed by S. Rubislaw (27.8%), S. Carrau (16.7%) and S. enterica subsp. Houtenae (11.1%). One sample could not be identified. Gentamicin was the most potent in vitro drug for the treatment of salmonelosis, followed by ampicilin. Our results confirmed that dogs represent an important reservoir of exotic Salmonella serovars and that there are different patterns of sensitivity to drugs among them.
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