RESUMEN
Abstract The sugarcane in Brazil is passing through a management transition that is leading to the abolition of pre-harvest burning. Without burning, large amounts of sugarcane trash is generated, and there is a discussion regarding the utilization of this biomass in the industry versus keeping it in the field to improve soil quality. To study the effects of the trash removal on soil quality, we established an experimental sugarcane plantation with different levels of trash over the soil (0%, 50% and 100% of the original trash deposition) and analyzed the structure of the bacterial and fungal community as the bioindicators of impacts. The soil DNA was extracted, and the microbial community was screened by denaturing gradient gel electrophoresis in two different seasons. Our results suggest that there are no effects from the different levels of trash on the soil chemistry and soil bacterial community. However, the fungal community was significantly impacted, and after twelve months, the community presented different structures among the treatments.
Asunto(s)
Microbiología del Suelo , Bacterias/aislamiento & purificación , Saccharum/microbiología , Hongos/aislamiento & purificación , Estaciones del Año , Suelo/química , Bacterias/clasificación , Bacterias/genética , Brasil , Saccharum/crecimiento & desarrollo , Biodiversidad , Hongos/clasificación , Hongos/genéticaRESUMEN
The sugarcane in Brazil is passing through a management transition that is leading to the abolition of pre-harvest burning. Without burning, large amounts of sugarcane trash is generated, and there is a discussion regarding the utilization of this biomass in the industry versus keeping it in the field to improve soil quality. To study the effects of the trash removal on soil quality, we established an experimental sugarcane plantation with different levels of trash over the soil (0%, 50% and 100% of the original trash deposition) and analyzed the structure of the bacterial and fungal community as the bioindicators of impacts. The soil DNA was extracted, and the microbial community was screened by denaturing gradient gel electrophoresis in two different seasons. Our results suggest that there are no effects from the different levels of trash on the soil chemistry and soil bacterial community. However, the fungal community was significantly impacted, and after twelve months, the community presented different structures among the treatments.
Asunto(s)
Bacterias/aislamiento & purificación , Hongos/aislamiento & purificación , Saccharum/microbiología , Microbiología del Suelo , Bacterias/clasificación , Bacterias/genética , Biodiversidad , Brasil , Hongos/clasificación , Hongos/genética , Saccharum/crecimiento & desarrollo , Estaciones del Año , Suelo/químicaRESUMEN
Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples.
Asunto(s)
Carbón Orgánico , ADN Bacteriano/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Microbiología del Suelo , Suelo/química , Biota , ADN Bacteriano/genética , ADN de Hongos/genética , Electroforesis en Gel de Gradiente Desnaturalizante , Reacción en Cadena de la PolimerasaRESUMEN
Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit MiniprepTM (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples.
Asunto(s)
Análisis de Secuencia de ADN/métodos , Carbón Orgánico , Microbiología del Suelo , Reacción en Cadena de la PolimerasaRESUMEN
AIM: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. METHODS: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. RESULTS: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.
Asunto(s)
Bacterias/clasificación , Proteínas de Plantas/genética , ARN Ribosómico 16S/genética , Microbiología del Suelo , Bacterias/genética , Bacterias/aislamiento & purificación , Biomarcadores , ADN Bacteriano/análisis , ARN Polimerasas Dirigidas por ADN , Ecosistema , Electroforesis en Gel de Poliacrilamida , Variación Genética , FilogeniaRESUMEN
This manuscript is a review of the innovative methodologies that enable more precise evaluations of soil microbial diversity. Highlighting the molecular approach, which does not require the isolation of microorganisms and allows the inclusion of non-culturable genotypes in the analyses, the described methodologies revolutionised the environmental microbiology and opened gateways for an accurate understanding of the ecology and diversity of microorganisms. The application of techniques based on soil total DNA extraction, PCR amplification of genes or gene fragments, and sequence analysis revealed that the microbial universe is far more complex than ever imagined. Examples of applications of the molecular approach to study the diversity of soil diazotrophic bacteria are given.
Asunto(s)
Filogenia , Microbiología del Suelo , Variación Genética , Reacción en Cadena de la Polimerasa , ARN Ribosómico/análisis , Análisis de Secuencia de ADN , Análisis de Secuencia de ARNRESUMEN
With the aim of detecting Rhizobium species directly in the environment, specific PCR primers for Rh. tropici and Rh. leguminosarum were designed on the basis of sequence analysis of 16S-23S rDNA spacer regions of several Rh. tropici, Rh. leguminosarum and Agrobacterium rhizogenes strains. Primer specificity was checked by comparison with available rDNA spacer sequences in databases, and by PCR using DNA from target and reference strains. Sequence polymorphisms of rDNA spacer fragments among strains of the same species were detected by denaturing gradient gel electrophoresis (DGGE). The specific PCR primers designed in this study could be applied to evaluate the diversity of Rh. tropici and Rh. leguminosarum by analysing the polymorphisms of 16S-23S spacer rDNA amplified from either whole-cell or soil-extracted DNA.
Asunto(s)
ADN Ribosómico/genética , Rhizobium leguminosarum/clasificación , Rhizobium/clasificación , Cartilla de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Rhizobium/genética , Rhizobium leguminosarum/genética , Análisis de Secuencia de ADN , Microbiología del Suelo , Especificidad de la EspecieRESUMEN
Pyrolysis mass spectrometry (PyMS) and DNA fingerprinting (RAPD and RSalpha hybridization) were used to characterize soybean inoculant strains and root nodule isolates of bradyrhizobia from the Brazilian Cerrado soils. Most isolates were shown to be derived from the inoculant strains on the basis of genotype comparisons by DNA fingerprinting. Phenotypic analysis (using PyMS) of the strains and separately of the polysaccharides derived from them showed that the nodule isolates differed from the parental strains, suggesting adaptation to the Cerrado soil environment. The extent of the differences between the derivatives and inoculant strains was similar for comparisons made on the basis of whole-cell preparations or from the isolated polysaccharides, indicating that the adaptation was caused by changes in the composition of the polysaccharides produced.
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Adaptación Fisiológica , Bradyrhizobium/crecimiento & desarrollo , Bradyrhizobium/genética , Glycine max/microbiología , Polisacáridos Bacterianos/química , Microbiología del Suelo , Bradyrhizobium/química , Brasil , Espectrometría de Masas/métodos , Hibridación de Ácido Nucleico , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismo , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
The pattern of DNA fragments produced by the polymerase chain reaction using arbitrary primers is strongly influenced by the age of the colonies sampled. We show that the number, clarity and reproducibility of the bands produced is optimal when very young colonies are sampled.