The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.Up to now, most of the microbial diversity studies conducted in complex ecosystems, such as soil, have been biased essentially by the unculturability of many microorganisms and the lack of sensitivity of traditional microbiological methods (4). In the past decade, applications of new molecular biology methods based primarily on amplification of soil-extracted nucleic acids have provided a pertinent alternative to classical culturebased microbiological methods, providing unique insight into the composition, richness, and structure of microbial communities (3, 6, 9, 11). However, the results of molecular analysis of microbial communities rely not only on the extraction of DNAs representative of the indigenous bacterial community composition but also on factors related to PCR, such as the choice of primers, the concentration of amplified DNA, errors in the PCR, or even the method chosen for analysis. Recently, numerous studies have investigated new methods to improve extraction, purification, amplification, and quantification of DNA from soils (8,13,14). Comparative studies have been performed to analyze the efficiency of methods for extraction and purification of soil DNA recovered, revealing that these methods suffer from low efficiency, mainly due to incomplete cell lysis and DNA sorption to soil particles (1, 5). However, the impact of the extraction method on the outcome of indigenous microbial community analysis has not been clearly established (5).The goal of this study was to evaluate the effect of DNA extraction methods on the bacterial diversity detected within DNA extracted from three soils exhibiting contrasting physicochemical characteristics and, in the context of an experimental study, from unamended soil and soils amended for 15 years with farmyard manure or sewage sludge. Two commercial DNA purification kits and a laboratory-devised method based on mechanical lysis were used to extract DNA directly from soils. Amplified ribosomal DNA restriction analysis (ARDRA) and ribosomal intergenic spacer analysis (RISA) were performed to estimate the effect of the DNA extraction procedure used on the bacterial diversity revealed.DNA extraction from soils.

Despite its importance in plant health and crop quality, the diversity of epiphytic bacteria on grape berries and other plant parts, like leaves and bark, remains poorly described, as does the role of telluric bacteria in plant colonization. In this study, we compare the bacterial community size and structure in vineyard soils, as well as on grapevine bark, leaves and berries. Analyses of culturable bacteria revealed differences in the size and structure of the populations in each ecosystem. The highest bacteria population counts and the greatest diversity of genera were found in soil samples, followed by bark, grapes and leaves. The identification of isolates revealed that some genera – Pseudomonas, Curtobacterium, and Bacillus – were present in all ecosystems, but in different amounts, while others were ecosystem-specific. About 50% of the genera were common to soil and bark, but absent from leaves and grapes. The opposite was also observed: grape and leaf samples presented 50% of genera in common that were absent from trunk and soil. The bacterial community structure analyzed by T-RFLP indicated similarities between the profiles of leaves and grapes, on the one hand, and bark and soil, on the other, reflecting the number of shared T-RFs. The results suggest an interaction between telluric bacterial communities and the epiphytic bacteria present on the different grapevine parts.

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Twenty‐five 2,4‐dichlorophenoxyacetic acid (2,4‐D) degrading bacteria from geographically diverse locations and presenting various degrees of similarity or no similarity to the tfdA and tfdB genes from Alcaligenes eutrophus JMP134 were analysed by PCR‐RFLP (restriction length fragment polymorphism). Primers for the 2,4‐D etherase gene were derived by sequence alignment of the tfdA genes from A. eutrophus JMP134 and Burkholderia sp. RASC. Primers for the 2,4‐dichlorophenolhydroxylase gene were based on the tfdB gene sequence from A. eutrophus JMP134 by taking codon degeneration and variations in amino acid residue sequences into consideration. PCR amplification using the tfdA primer set produced fragments of 0.3 kb from 17 strains which showed varying degrees of similarity to the tfdA gene probe from A. eutrophus JMP134. Significant variations in the gene sequences were confirmed by PCR‐RFLP analysis. DNA amplification using the tfdB primer set produced a 1.1 kb fragment from 19 strains. Amongst them, two did not show any similarity to the tfdB gene probe. The size and restriction pattern of the products obtained from A. eutrophus JMP134 were in accordance with the expected size calculated from the A. eutrophus tfdA and tfdB gene sequence and their theoretical PCR‐RFLP patterns. Some strains which did not amplify using the tfdA primer set did however amplify with the tfdB primer set. These results suggest the independent evolution of these two genes in the construction of the 2,4‐D metabolic pathway. Our tfdA and tfdB primer sets could be used for the detection of similar sequences in bacteria and soils. Moreover, PCR‐RFLP patterns could also be used to select subsets of strains for sequencing to study the phylogeny of the tfdA and tfdB genes.