Análise da comunidade de fungos em solos da Amazônia por Eletroforese em Gel com Gradiente Desnaturante (DGGE)

Abstract

The aim of the present work was the adaptation of a DGGE methodology for the analysis of the fungal community structure in soils under natural vegetation in the Brazilian Amazon and the evaluation of the impact of different land-uses on the soil fungal communities. The area studied is located in the region known as "Alto Solimões", close to the municipality of Benjamin Constant, Amazon State. Fifty composite soil samples were collected from undisturbed tropical forest and from sites nearby, submitted to five different land uses by local indigenous people. Metagenomic DNA was extracted directly from soil with a commercial kit and amplified by nested PCR using two sets of primers, NS1+EF3 and NS1+FRIGC, which are considered specific for the amplification of a 1650 bp fragment of fungal 18S rDNA gene. Satisfactory band separation in DGGE was accomplished with a gradient of denaturants ranging from 25 to 38%, under a constant voltage of 200 V and temperature of 55 °C, in an electrophoretic run of 15 hours. Under these conditions it was possible to detect variations in the fungal community structure with a polymorphic DGGE band pattern, characteristic of each land use evaluated. The DGGE band patterns among samples of the same land use were more similar than for samples from different origins. However, except for pasture, no statistically significant differences among land uses were detected, indicating that agricultural practices commonly adopted in the region have low impact on the fungal community structure. Information about identity and function of fungal species could now be obtained by construction of a genomic library based on extracted metagenomic DNA and excision and sequencing of amplicons.