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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 culture-based 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. The physicochemical properties of the three soils used in this study are presented in Table 1. The field experiment was conducted in the Institut National de la Recherche Agronomique domain of Bordeaux, France (45°N, 1°W) on an acid, coarse, sandy soil (7). This experiment was limited to continuously growing corn (Zea maize, variety INRA 160), with four treatments, as follows: none (i) (i.e., N-P-K fertilizers only, with soil considered unamended), (ii) farmyard manure (10 tons of dry matter per ha each year), (iii) sewage sludge (10 tons of dry matter per ha each year), and (iv) sewage sludge (100 tons of dry matter per ha every 2 years), with blocks as for treatment in a randomized manner. Fresh soils samples were sieved (2-mm mesh) and stored at 4°C. Nucleic acids were extracted from three 250-mg aliquots of soils using two commercial kits, the UltraClean Soil DNA kit (MoBio Laboratories, Inc., Solana Beach, Calif.) and the Fast DNA spin sample kit (for soil; Bio 101, Lajolla, Calif.), according to the manufacturers' recommendations and using a procedure developed in our laboratory. Briefly, 1 ml of a solution containing 100 mM Tris (pH 8.0), 100 mM EDTA, 100 mM NaCl, 1% (wt/vol) polyvinylpyrrolidone, and 2% (wt/vol) sodium dodecyl sulfate was added to 250 mg of soil in a 2-ml mini-bead-beater tube containing 0.5 and 0.1 g of 106-µm- and 2-mm-diameter glass beads, respectively. Samples were then homogenized for 30 s at 1,600 rpm in a mini-bead-beater cell disruptor (Mikro-Dismembrator S; B. Braun Biotech International), after which the samples were centrifuged at 14,000 × g for 1 min at 4°C. The collected supernatants were incubated for 10 min on ice with 1/10 volume of 5 M sodium acetate and centrifuged at 14,000 × g for 5 min. After precipitation with 1 volume of ice-cold isopropanol, the nucleic acids were washed with 70% ethanol and purified using a Sepharose 4B spin column. The quality and the size of the soil DNAs were checked by electrophoresis on 1% agarose gels. DNA was quantified using a BioPhotometer (Eppendorf, Hamburg, Germany). Three replicates were used for DNA evaluation. A double-factor analysis of variance was performed to determine significant differences in the yield of soil DNA. The Newman-Keuls procedure (P < 0.05) was used to evaluate the impact of the different soils and extraction methods used on the DNA extraction yield.

ARDRA and RISA. To test the possible selectivity of the DNA extraction method, ARDRA and RISA were performed on DNA extracted directly from soils. Three independent replicates were included in each analysis, and the results were examined separately. Small subunit (SSU) ribosomal DNA (rDNA) genes were amplified from each soil replicate in thermocycler (PTC 200 gradient cycler; MJ Research, Waltham, Mass.). PCR amplification from 50 ng of extracted soil DNA was conducted with a total volume of 50 µl by using 1 µM concentrations of the universal primers 27_f (5'-AGA GTT TGA TCM TGG CTC AG-3') and 1492_r (5'-TAC GGH TAC CTT GTT ACG ACT T-3') (2) and 2.5 U of Taq DNA polymerase (Appligene) under the following conditions: 5 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C, plus an additional 15-min cycle at 72°C. The PCR products were then separated by electrophoresis on a 1% agarose gel. Bands were extracted and purified from the gel using the Qiaex II kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. Ten microliters of purified 16S rDNA from each sample was digested with 15 U of RsaI, HhaI, or MspI in 15-µl reaction mixtures.

PCR efficiency analysis. Three microliters of 16S rDNA PCR product was separated by electrophoresis on a 1% agarose gel run for 1 h at 100 V. Gels were stained with ethidium bromide and analyzed with the ImageQuant program. 16S-23S intergenic rDNA PCR products were purified with columns (Microcon PCR/Amicon Bioseparations; Millipore, Bedford, Mass.) according to the manufacturers' recommendations. A 2-µl volume of each purified PCR product was then diluted into 48 µl of sterile water, and the nucleic acid content was determined using a BioPhotometer (Eppendorf). For both 16S and 16S-23S intergenic rDNA PCR products, three repeats were carried out. Data were analyzed statistically as described above.

Effect of extraction methods on bacterial diversity analysis. The DNA yields obtained ranged between 0.2 and 2.5 µg g of soil¹ from sample to sample and differed with the extraction method (Table 2). The MS laboratory method yielded an amount of DNA that was significantly higher than those obtained with the kits from MoBio Laboratories and Bio 101 for Dijon and Epoisses soils. However, the quantity of DNA extracted from the sandy soil of Couhins was not significantly affected by the extraction method used. These results therefore suggest that the yield of DNA per gram of soil depends on the method used and on the properties of the soil considered. Patterns of restriction fragment length polymorphism of amplified 16S rDNA genes digested with RsaI restriction enzyme indicate that amplified 16S rDNA genes were very similar whatever extraction method used or soil tested (Fig. 1). Other enzymes (HhaI and MspI) were tested and also provided identical patterns for the three soils (data not shown). It is commonly admitted that ARDRA resolves microbial diversity up to the genus level. Our results therefore confirm that some genera are widely distributed and dominant in the three agricultural soils tested here. In addition, they suggest that at the genus level the particular DNA extraction method used does not influence bacterial diversity analysis. Evaluation of the impact of extraction methods on bacterial diversity was further conducted by means of RISA. Demonstration of the relevance and the sensitivity of the RISA approach for microbial diversity analysis has been previously reported (10). Similar RISA patterns were visualized for the three soil replicates, illustrating the reproducibility of the results for both DNA extraction and amplification (Fig. 2). However, it has to be noted that some differences between soils were observed: for DNAs extracted from the Dijon soil, additional bands were observable at 900, 300, and 220 bp (Fig. 2, lane 4 for Dijon soil). In addition, for each soil many differences were observed between the RISA patterns obtained for DNA extracted using different methods. For instance, a strong band between 242 and 320 bp and another one just above 501 bp were consistently observed in the RISA patterns generated for DNA extracted from the soil of Couhins using the MoBio Laboratories kit (Fig. 2, lanes 4 to 6). However, these two bands were not shown in the RISA patterns obtained with DNA extracted using the MS laboratory method (Fig. 2, lanes 1 to 3) and the Bio 101 kit (Fig. 2, lanes 7 to 9). Therefore, differences in the location of the dominant bands were evident for both Epoisses and Dijon soils when the RISA patterns obtained with the three different DNA extraction methods were compared. RISA patterns obtained with the soil of Couhins appeared to be more similar regardless of the extraction method used. This probably reflects the homogeneity of this soil due to its high percentage of sand (Table 1). Surprisingly, a strong-dominant band at 800 bp was observed for the three soils studied only in the RISA patterns obtained with DNA recovered with the Bio 101 kit, while it was not possible to distinguish this band with the two other extraction methods. Assuming that the brightness of this 800-bp band was expected to be identical for the same soil whatever the extraction method used, the discrepancies (variations in the positions and brightness of the bands) in the results obtained for the same soil using different DNA extraction methods strongly suggest a preferential DNA recovery and/or preferential DNA amplification, depending on the method used. It must be emphasized that such discrepancies were observed in comparing DNA extracted from soils using only methods based on mechanical lysis by bead beating, while other methods, based on sonication or lysozyme lysis, have been reported (5, 8). Diversity analyses based on cultivation methods are biased by the fact that cultivatable bacteria represent only a small proportion (<0.1%) of the soil microbial community (12). Similarly, our data show that DNA extraction from soil also suffers from biases that can distort the revealed community composition, richness, and structure and that the outcome of microbial community analysis is dependent on the DNA recovery method used. Because important variations in the brightness of the bands appeared in the RISA patterns for the different soil DNA extraction methods, it seems that the relative abundance of phylotypes in soil cannot be accurately estimated with these direct molecular approaches.

View larger version (80K): [in this window] [in a new window]   FIG. 1.   ARDRA. Shown is a SYBR green II-stained gel (6% acrylamide) of RsaI-digested PCR products amplified with 16S rDNA universal primers (27f and 1492r) from DNA extracted from Dijon, Couhins, and Epoisses soils following three different extraction methods: the MS laboratory method (lanes 1 to 3), the MoBio kit method (lanes 4 to 6), and the Bio 101 kit method (lanes 7 to 9). Lanes M, VIII Boehringer Mannheim molecular size markers (sizes indicated in base pairs at left and right).

View larger version (89K): [in this window] [in a new window]   FIG. 2.   RISA shown is a SYBR green II-stained gel (6% acryamide) of PCR products amplified with 16S rDNA intergenic spacer universal primers (38r and 72f) from DNA extracted from Dijon, Couhins, and Epoisses soils following three different extraction methods: the MS laboratory method (lanes 1 to 3), the MoBio kit method (lanes 4 to 6), and the Bio 101 kit method (lanes 7 to 9). Lanes M, VIII Boehringer Mannheim molecular size markers (sizes indicated in base pairs at left and right). Arrow, 800-bp band.

View larger version (80K): [in this window] [in a new window]   FIG. 3.   ARDRA. Shown is a SYBR green II-stained gel (6% acrylamide) of RsaI-digested PCR products amplified with 16S rDNA universal primers (27f and 1492r) from DNA extracted from unamended soil (U) and farmyard manure (FM)- and sewage sludge (SS10 and SS100)-treated plots of Couhins following three different extraction methods: the MS laboratory method (lanes 1 to 3), the MoBio kit method (lanes 4 to 6), and the Bio 101 kit method (lanes 7 to 9). Lanes M, VIII Boehringer Mannheim molecular size markers (sizes indicated in base pairs at left and right). SS10, 10 tons of dry matter/ha/years. SS100, 100 tons of dry matter/ha/2 years.

View larger version (69K): [in this window] [in a new window]   FIG. 4.   RISA. Shown is SYBR green II-stained gel (6% acryamide) of PCR products amplified with 16S ribosomal universal primers (38r and 72f) from DNA extracted from unamended soil (U) and farmyard manure (FM)- or sewage sludge (SS10 and SS100)-treated plots of Couhins soil following three different extraction methods: the MS laboratory method (MS) (lanes 1 to 3), the MoBio kit method (lanes 4 to 6), and the Bio 101 kit method (lanes 7 to 9). Lanes M, VIII Boehringer Mannheim molecular size markers (sizes indicated in base pairs at left and right). Arrow, 800-bp band.