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Soil DNA (soil + dna)
Selected AbstractsDirect amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soilENVIRONMENTAL MICROBIOLOGY, Issue 6 2001Adolphe Zézé Sequences of nodD, a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii, R. leguminosarum biovar viciae and Sinorhizobium meliloti. The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii[11 restriction fragment length polymorphism (RFLP) types] and 15 clones identified as viciae (seven RFLP types). These identifications were confirmed by sequencing. There were no clones related to S. meliloti nodD. For comparison, 122 strains were isolated from nodules of white clover (Trifolium repens) growing at the field site, and 134 from nodules on trap plants of T. repens inoculated with the soil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii -like clones. We conclude that clover selects specific genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S. meliloti was rare. [source] Structure and activity of the nitrate-reducing community in the rhizosphere of Lolium perenne and Trifolium repens under long-term elevated atmospheric pCO2FEMS MICROBIOLOGY ECOLOGY, Issue 3 2004Kathrin Deiglmayr Abstract Rhizosphere soil was sampled in monocultures of Lolium perenne and Trifolium repens in June and October 2002, at two different nitrogen fertilisation levels (14 and 56 g N m,2 year,1) and under two pCO2 atmospheres (360 and 600 ppmv) at the Swiss FACE (Free Air Carbon dioxide Enrichment) site. Directly extracted soil DNA was analysed with restriction fragment length polymorphism (PCR-RFLP) by use of degenerated primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. The corresponding enzyme activity of the nitrate reductase was determined colorimetrically after 24 h of anaerobic incubation. The narG PCR-RFLP fingerprints showed that the structure of the nitrate-reducing community was primarily affected by season and pH of the sampling site, whereas CO2 enrichment, plant species or fertiliser treatment had no apparent effect. In contrast, the nitrate reductase activity responded to N fertilisation, CO2 enrichment and plant species in October, whereas in June drought stress most likely kept the enzyme activity at a low level in all treatments. Apparently, the respiratory nitrate-reducing community adapted to different treatments primarily by altered enzyme activity. [source] Analysis of the distribution of Phytophthora cinnamomi in soil at a disease site in Western Australia using nested PCRFOREST PATHOLOGY, Issue 2 2009N. Williams Summary The oomycete plant pathogen Phytophthora cinnamomi has infected a very large area of native vegetation in the south western corner of Australia. An important aspect of effective disease management depends on being able to accurately map areas of infestation. For this purpose, we have developed a nested polymerase chain reaction (PCR) protocol for the detection of P. cinnamomi in soil. The test uses two sets of primers developed from the rRNA ITS sequences of P. cinnamomi and can detect as little as 1 pg DNA. The degree of sensitivity was reduced with DNA extracted from soil although this depended on the type of soil. Soils with a high organic content, such as eucalypt forest soil and potting mix were more inhibitory than sandy soils. Inhibition by soil DNA could be reduced by the addition of bovine serum albumin and formamide to the reaction. Taq DNA polymerase was very sensitive to inhibitors compared with Tth+ or TaqF1*. In comparison with baiting (0,10% positive samples), nested PCR proved to be a very much more efficient (90,100% positive samples) method for the detection of P. cinnamomi in soil. [source] Evolution of a degradative bacterial consortium during the enrichment of naphtha solventJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2000L. Cavalca A microbial mixed culture able to degrade naphtha solvent, a model of hydrocarbon aromatic mixture, was isolated from a hydrocarbon-polluted soil. Composition of the population was monitored by phenotypic and molecular methods applied on soil DNA, on whole enrichment culture DNA, and on 85 isolated strains. Strains were characterized for their 16S rDNA restriction profiles and for their random amplified polymorphic DNA profiles. Catabolic capabilities were monitored by phenotypic traits and by PCR assays for the presence of the catabolic genes methyl mono-oxygenase ( xylA,M), catechol 2,3 dioxygenase (xylE) and toluene dioxygenase (todC1) of TOL and TOD pathways. Different haplotypes belonging to Pseudomonas putida, Ps. aureofaciens and Ps. aeruginosa were found to degrade aromatic compounds and naphtha solvent. The intrinsic catabolic activity of the microbial population of the polluted site was detected by PCR amplification of the xylE gene directly from soil DNA. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to detect ultra low population of Ralstonia solanacearum (Smith 1896) Yabuchi et al. (1996)LETTERS IN APPLIED MICROBIOLOGY, Issue 5 2009A. Grover Abstract Aims:, To develop a reliable and sensitive protocol for detection of Ralstonia solanacearum using MDA-PCR (Multiple displacement amplification,PCR amplification). Methods and Results:, MDA-PCR technique was performed on pure cell lysates as well as soil samples. Pure cell lysate as well as that of soil DNA was used as template in MDA reaction. MDA of template DNA was carried out in the presence of sample buffer, reaction buffer and enzyme mix (, 29 DNA polymerase and random hexamers). The MDA amplified DNA was used for PCR amplification using R. solanacearum -specific PCR primers. MDA-PCR could detect as low as 1 colony forming unit (CFU ml,1) of bacteria within 8 h including DNA isolation. Conclusion:, MDA followed by standard PCR facilitated the detection of pathogen from very low count samples. The method is of great importance in managing the brown rot disease of potato. Significance and Impact of study:, The ultrasensitive detection technique developed in the present study is sensitive and speedy enough to be included into integrated wilt disease control programmes. [source] Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGELETTERS IN APPLIED MICROBIOLOGY, Issue 4 2002R.S. Peixoto 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. [source] | ||||||||||||||||||||||