Community (DNA) fingerprinting

For bacterial community TRFLP analysis, primers 8F and 1520R ([25]; S2 Table) were fluorescently labelled at the 5ʹ and 3’ ends with FAM and HEX, respectively [16]. Each 25 μl reaction mixture contained 400 nM of each primer, 1 × Bioline MyHSTaq reaction buffer, 1 U MyHSTaq DNA Polymerase (BioLine Pty Ltd.), and 2 μl of template DNA (1:100 dilution of 10 ng/μl DNA). Thermocycling conditions are given in S2 Table. Reactions were validated by agarose gel electrophoresis. PCR products were digested (separate reactions), with AluI and CfoI (Promega) to generate fluor-labelled terminal restriction fragments (TRFs) of varying size. Pre- and post-digestion PCR products were purified using AxyPrep Mag PCR paramagnetic bead solution and 96 well magnetic plates, according to the manufacturer’s protocol.

Restriction fragments were separated by capillary electrophoresis (ABI 3730 DNA Analyzer) at the Australian Genome Research Facility (Adelaide, Australia). The lengths (base pairs) of individual TRFs were calculated by comparison to the internal size standard GS500LIZ (Applied Biosystems). Electropherograms were imported into Peak Scanner (Applied Biosystems), visually inspected for sizing quality and peak areas (in base pairs) determined for TRFs 50–500 bp in length. The online tool T-Rex [26] was then used to distinguish true peaks from background fluorescence [27]. The custom R script ‘interactive binner’ [28] provided bacterial community fingerprints by binning peaks to operational taxonomic units (OTUs). Each peak was inferred to be an OTU and the height of each peak used as a measure of the relative abundance of each OTU.

A nested PCR approach was taken for the amplification of Pseudomonas-specific 16S rRNA gene fragments. Initially, Pseudomonas-specific PCR was conducted using primers F311Ps and R1459Ps ([29]; S2 Table). The presence of amplicons of the expected size was validated by agarose gel electrophoresis. Resultant PCR products were diluted 1/10 and used as template DNA for the second, general bacterial amplification using DGGE primers F968-GC and R1378 ([30]; S2 Table). Each 25 μl reaction mixture contained 200 nM of each primer, 1 × Bioline MyTaq reaction buffer, 1 U (Pseudomonas) or 0.625 U (Bacteria) MyHSTaq DNA Polymerase (BioLine Pty Ltd.), and 2 μl (20 ng) of template DNA. Thermocycling conditions (S2 Table) and DGGE methodology as described by Wakelin et al. [31]. The 50 PCR products were analyzed in a randomized order across three DGGE gels. Band location and intensity data were collected using TotalLab TL120 software (Nonlinear Dynamics, UK). Each band was inferred to be an OTU and band intensity data considered a measure of the abundance of each OTU.

Utilising a community approach, the primary focus of this study was to investigate relationships between indigenous bacterial and Pseudomonas communities, and edaphic, environmental, and farm-management factors. DNA fingerprinting techniques provide a useful exploratory approach to identify ecological patterns and, although affording lower taxonomic resolution than high throughput sequencing, have a similar capacity to correlate abiotic variables with separation in microbial community structure (β-diversity) [32, 33].