Materials and Methods
Isolation and culture of pseudomonads. A total of 170 leaf-colonizing Pseudomonas strains were isolated from nine sugar beet plants (Beta vulgaris var. Amethyst ) in three experimental plots at the University farm, Wytham, Oxford, UK in 1993. Some of these strains (108 isolates) were subjected to a previous multi-locus enzyme electrophoresis (MLEE) analysis wherein details of the sampling methods are provided (Haubold & Rainey, 1996). Briefly, each plant was divided into three leaf types (immature, mature and senescent), and for each leaf type two replicate leaves were placed in a sterile plastic bag containing 5 ml sterile water. After a 1 min “massage”, the bacterial suspensions were dilution-plated onto King’s Medium B (KB) supplemented with CFC (10 µg/ml cetrimide, 10 µg/ml fucidin, and 50 µg/ml cephalosporin) from Oxoid (Hampshire, UK). After incubation at 28 oC for 48 hrs, two colonies were randomly picked for each leaf sample and purity of the isolates was further checked by streaking onto KB agar plates. The obtained isolates were sub-cultured in Luria broth (LB), and subsequently stored frozen at -80 oC by mixing 1.0 ml culture with 0.8 ml glycerol saline solution (70% glycerol, 0.85% NaCl).
In 2004, five plants of the same B. vulgaris variety were grown at one site located in West Auckland, New Zealand; Pseudomonasstrains were isolated similarly using the KB + CFC selective plates. Isolates were coded similarly as strains from Oxford, indicating their origin of isolation: plot (1), plant (1 to 5), leaf type (1, senescent; 3, mature; 5, immature) and replicate leaf (a or b). A prefix X was assigned to distinguish the Auckland isolates from those from Oxford. For example, isolate X131b1 identifies isolate 1 from Auckland plot 1, from plant 3, from senescent (1) leaf “b”. Isolates from Oxford were additionally assigned a more simplified code from U100 to U269.
PCR amplification and DNA sequencing . Total DNA was extracted from bacterial cells using the CTAB-based method as previously described (Zhang et al., 2001). Briefly, bacteria were resuspended in 567 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to which 30 µl of 10% SDS and 3 µl of 20 mg/ml proteinase K were added. After lysis at 37oC for 1 hour, 100 µl of 5 M NaCl and 80 µl of CTAB/NaCl solution (10% hexadecyltrimethyl ammonium bromide, 0.7 m NaCl) were added and incubated at 65 oC for 10 min. DNAs were subsequently extracted with equal volume of phenol/chloroform and precipitated with 0.6 volume of isopropanol. PCR was performed in a 25 µl reaction containing 1x PCR buffer, 0.2 mM dNTPs, 1.6 mM MgCl2, 2% DMSO, 10 pmol for each primer, 30-100 ng template DNA and 0.2 units Taq polymerase (Invitrogen). After an initial denaturation at 94oC for 3 min, DNAs were amplified in 30 cycles of denaturation at 94 oC for 30 s, annealing at 58oC for 30 s elongation at 72 oC for I min, followed by a 3-min final extension at 72 oC. The PCR products were purified using the Exo-CIP Rapid PCR Cleanup Kit (New England Biolabs), before they were sent to Macrogen Inc (South Korea) for DNA sequencing.
Seven housekeeping genes were first tested in this work using available universal primers (Hwang et al., 2005): gapA , glyceraldehyde 3-phosphate
dehydrogenase; gltA , citrate synthase, acnB , aconitate hydratase; gyrB, gyrase B; coxC , cytochrome c oxidase;pgi , glucose- 6-phosphate isomerase; rpoD , RNA polymerase sigma factor D. Final MLSA was performed with three genes (gapA, gltA and acnB ) whose primer sequences are provided in Table 1. The three genes are almost equally distributed in the genome of P. fluorescens SBW25: gapA, gltA and acnB are annotated topflu4965 (5448514-5449515), pflu1815 (1979150-1980439)pflu3489 (3858403-3861012), respectively.
Assays for bacterial growth: The GN2 microtiter plates (Biolog Inc., Hayward, CA) were used following the manufacturer’s instructions. Inoculants were prepared by first growing stored bacteria in LB broth, and sub-cultured once in R2A broth (Reasoner & Geldreich, 1985). Cells were spun down and resuspended in the same volume of sterile deionized water; 100 µl of this bacterial suspension was then inoculated into 15 ml of the IF-0 inoculating fluid from Biolog, and subsequently starved at 28 oC for 2 hrs. Next, 150 µl of the starved cells was pipetted into each well of the GN2 MicroPlate and incubated at 28oC for 48 hrs. The initial and final cell density was estimated by measuring absorbance at the wavelength of 660 nM (A600 ) in a Synergy 2 plate reader equipped with the Gen5 software (BioTek Instruments). Growth utilization of histidine and urocanates was assessed with the minimal M9 salt medium as previously described (Zhang et al., 2012).
Data analysis: Geneious (Biomatters Ltd; Auckland, New Zealand) was used to manipulate the DNA sequences, including multiple sequence alignment, trimming and concatenation. For some analyses, unique sequence types (STs) were grouped into operational taxonomic units (OTUs) that included all STs that varied by the mean pairwise distance (0.06) of the total sample. Rarefaction analysis was performed using MOTHUR v.1.34.4 (Schloss et al., 2009). The numbers of STs and OTUs were determined by selecting the isolates at random from the population, and the procedure was repeated 1,000 times. The Simpson’s index (1-D ) of diversity was calculated to compare variability in isolates from Oxford and Auckland, and it takes into account both the number of taxa (i.e. STs or OTUs) and the relative abundance of each taxa (Simpson, 1949). Phylogenetic trees generated in Mothur on the MLSA data were visualized with FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Statistical testing of differences in genetic diversity was performed using the permutational multivariate analysis of variance (PERMANOVA) implemented in PRIMER V6 (Anderson, 2001). Pairwise distances were used as input and the testes were run with 9999 permutations. Visual comparison of isolates from different ecological sources (location, plot, plant and leaf type) were performed by multidimensional scaling (MDS) using PRIMER v6. Ancestral subpopulations of Pseudomonas were determined using STRUCTURE software (Pritchard et al., 2000).
Recombination was detected using the likelihood permutation test in LDhat v2.1 (McVean et al., 2002) as previously described (Kidd et al., 2012). Evidence of recombination breakpoints were further obtained from the aligned multilocus sequences using the SBP/GARD server (Kosakovsky Pond et al., 2006). GARD examined 2717 models in 00:16:25 wallclock time, at a rate of 2.76 models/second. The alignment contained 340 potential breakpoints, translating into the search space of 6550950 models with up to 3 breakpoints, of which 0.04% was explored by the genetic algorithm. Significance of the toplogical incongruence was inferred by KH test (Kishino & Hasegawa, 1989).
The BiologTM growth data (A660 ) were analyzed by first eliminating the 28 carbon sources which didn’t support any bacterial growth for all tested Pseudomonas isolates. To cluster the growth phenotypes, a distance matrix was generated from absorbance data of the remaining 67 carbon sources using PRIMER v6. The matrix was then subjected to PERMANOVA implemented in PRIMER V6 as described above for genotypic data. Correlation between phenotype and genotype was performed using Principal Component Analysis (PCA) and Canonical Variate Analysis (CVA) (Renaud et al., 2015).