Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi

The molecular fingerprinting methods used to evaluate soil microbial diversity could also be used as effective biosensors for the purposes of monitoring ecological soil status. The biodiversity of microorganisms is a relevant index of soil activity and there is a necessity to develop tools to generate reliable results for an emerging approach in the field of environmental control using microbial diversity biosensors. This work reports a method under development for determining soil microbial diversity using high efficiency Multiplex PCR-Terminal Restriction Fragment Length Polymorphism (M-T-RFLP) for the simultaneous detection of bacteria, archaea and fungi. Three different primer sets were used in the reaction and the analytical conditions were optimized. Optimal analytical conditions were achieved using 0.5 µM of primer for bacteria and 1 µM for archaea and fungi, 4 ng of soil DNA template, and HaeIII restriction enzyme. Comparative tests using the proposed analytical approach and a single analysis of each microorganism group were carried out to indicate that both genetic profiles were similar. The Jaccard similarity coefficient between single and multiplexing approach ranged from 0.773 to 0.850 for bacteria and fungi, and 0.208 to 0.905 for archaea. In conclusion, the multiplexing and pooling approaches significantly reduced the costs and time required to perform the analyses, while maintaining a proper effectiveness.

[1]  R. Conrad,et al.  Acetoclastic and hydrogenotrophic methane production and methanogenic populations in an acidic West-Siberian peat bog. , 2004, Environmental microbiology.

[2]  P. Gillevet,et al.  Analyzing salt-marsh fungal diversity: comparing ARISA fingerprinting with clone sequencing and pyrosequencing , 2009 .

[3]  L. Kerkhof,et al.  Phylogeography of Sulfate-Reducing Bacteria among Disturbed Sediments, Disclosed by Analysis of the Dissimilatory Sulfite Reductase Genes (dsrAB) , 2005, Applied and Environmental Microbiology.

[4]  Norbert Sauberer,et al.  Surrogate taxa for biodiversity in agricultural landscapes of eastern Austria , 2004 .

[5]  K. Lindström,et al.  Novel group within the kingdom Crenarchaeota from boreal forest soil , 1997, Applied and environmental microbiology.

[6]  P. Balvanera,et al.  Quantifying the evidence for biodiversity effects on ecosystem functioning and services. , 2006, Ecology letters.

[7]  K. Jürgens,et al.  Interaction of Nutrient Limitation and Protozoan Grazing Determines the Phenotypic Structure of a Bacterial Community , 2003, Microbial Ecology.

[8]  T. Lueders,et al.  Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts , 2003, Applied and Environmental Microbiology.

[9]  W. Wade,et al.  Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA , 1998, Applied and Environmental Microbiology.

[10]  Hans H. Cheng,et al.  Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA , 1997, Applied and environmental microbiology.

[11]  O. Mathieu,et al.  High Microbial Diversity Promotes Soil Ecosystem Functioning , 2018, Applied and Environmental Microbiology.

[12]  D. Barker,et al.  The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA. , 1998, Nucleic acids research.

[13]  P. Schenk,et al.  Culture-independent molecular tools for soil and rhizosphere microbiology , 2013 .

[14]  Diversity of soil Archaea in boreal forest before, and after clear-cutting and prescribed burning , 1999 .

[15]  N. C. Gomes,et al.  Assessment of Variation in Bacterial Composition among Microhabitats in a Mangrove Environment Using DGGE Fingerprints and Barcoded Pyrosequencing , 2012, PloS one.

[16]  L. Karliński,et al.  Soil Microbial Biomass and Community Composition Relates to Poplar Genotypes and Environmental Conditions , 2020, Forests.

[17]  A. Gillison,et al.  Assessing biodiversity at landscape level in northern Thailand and Sumatra (Indonesia): the importance of environmental context , 2004 .

[18]  Zhenyu Zhu,et al.  The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases , 2008, Nucleic acids research.

[19]  P. Janssen,et al.  PCR-generated artefact from 16S rRNA gene-specific primers. , 2005, FEMS microbiology letters.

[20]  B. Dam,et al.  Isolation of high molecular weight and humic acid-free metagenomic DNA from lignocellulose-rich samples compatible for direct fosmid cloning , 2018, Applied Microbiology and Biotechnology.

[21]  J. Foster,et al.  MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes , 2007, Microbial Ecology.

[22]  J. Swings,et al.  Comparison of 16S ribosomal DNA sequences of all Xanthomonas species. , 1997, International Journal of Systematic Bacteriology.

[23]  S. Tsuneda,et al.  Long-term monitoring of the succession of a microbial community in activated sludge from a circulation flush toilet as a closed system. , 2006, FEMS microbiology ecology.

[24]  R. Conrad,et al.  Differential Effects of Nitrogenous Fertilizers on Methane-Consuming Microbes in Rice Field and Forest Soils , 2006, Applied and Environmental Microbiology.

[25]  J. Martiny,et al.  Testing the functional significance of microbial composition in natural communities. , 2007, FEMS microbiology ecology.

[26]  I. Anderson,et al.  Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium. , 2006, The New phytologist.

[27]  Mark V Brown,et al.  Community fingerprinting in a sequencing world. , 2014, FEMS microbiology ecology.

[28]  G. Berg,et al.  Pros and Cons of Ion-Torrent Next Generation Sequencing versus Terminal Restriction Fragment Length Polymorphism T-RFLP for Studying the Rumen Bacterial Community , 2014, PloS one.

[29]  M. V. D. van der Heijden,et al.  The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. , 2008, Ecology letters.

[30]  M. Moran,et al.  Analysis of Internal Transcribed Spacer (ITS) Regions of rRNA Genes in Fungal Communities in a Southeastern U.S. Salt Marsh , 2002, Microbial Ecology.

[31]  Jerzy Lipiec,et al.  Community Level Physiological Profiles (CLPP), Characterization and Microbial Activity of Soil Amended with Dairy Sewage Sludge , 2012, Sensors.

[32]  Jordan A. Fish,et al.  FunGene: the functional gene pipeline and repository , 2013, Front. Microbiol..

[33]  H. Ni,et al.  Land use change effects on diversity of soil bacterial, Acidobacterial and fungal communities in wetlands of the Sanjiang Plain, northeastern China , 2019, Scientific Reports.

[34]  T. Junier,et al.  TRiFLe, a Program for In Silico Terminal Restriction Fragment Length Polymorphism Analysis with User-Defined Sequence Sets , 2008, Applied and Environmental Microbiology.

[35]  B. Singh,et al.  Multiplex T-RFLP Allows for Increased Target Number and Specificity: Detection of Salmonella enterica and Six Species of Listeria in a Single Test , 2012, PloS one.

[36]  M. Viaud,et al.  Diversity of soil fungi studied by PCR–RFLP of ITS , 2000 .

[37]  D. Stahl,et al.  Recurring Seasonal Dynamics of Microbial Communities in Stream Habitats , 2006, Applied and Environmental Microbiology.

[38]  K. Nkongolo,et al.  Advances in monitoring soil microbial community dynamic and function , 2020, Journal of Applied Genetics.

[39]  Christoph Heller,et al.  A fully automated multicapillary electrophoresis device for DNA analysis , 1999 .

[40]  J. Cairney,et al.  Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. , 2004, Environmental microbiology.

[41]  A. Nikolić,et al.  Indirect diagnosis of haemophilia B by multiplex PCR/RFLP. , 2005, Clinical and laboratory haematology.

[42]  M. Friedrich,et al.  Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure , 2003, Applied and Environmental Microbiology.

[43]  T. Bruns,et al.  ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts , 1993, Molecular ecology.

[44]  P. Lemanceau,et al.  Going back to the roots: the microbial ecology of the rhizosphere , 2013, Nature Reviews Microbiology.

[45]  S. T. Buckland,et al.  Long-term datasets in biodiversity research and monitoring: assessing change in ecological communities through time. , 2010, Trends in ecology & evolution.

[46]  J. E. Christensen,et al.  Rapid molecular diagnosis of lactobacillus bacteremia by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene. , 2004, Clinical medicine & research.

[47]  R. Conrad,et al.  Diversity and ubiquity of thermophilic methanogenic archaea in temperate anoxic soils. , 2006, Environmental microbiology.

[48]  B. Singh,et al.  Use of Multiplex Terminal Restriction Fragment Length Polymorphism for Rapid and Simultaneous Analysis of Different Components of the Soil Microbial Community▿ , 2006, Applied and Environmental Microbiology.

[49]  Peter Ricke,et al.  Application of a Newly Developed ARB Software-Integrated Tool for In Silico Terminal Restriction Fragment Length Polymorphism Analysis Reveals the Dominance of a Novel pmoA Cluster in a Forest Soil , 2005, Applied and Environmental Microbiology.

[50]  H. Smidt,et al.  Successive DNA extractions improve characterization of soil microbial communities , 2017, PeerJ.