Genotypic and phenotypic analyses reveal distinct population structures and ecotypes for sugar beet‐associated Pseudomonas in Oxford and Auckland

Abstract

Fluorescent pseudomonads represent one of the largest groups of bacteria inhabiting the surfaces of plants, but their genetic composition in planta is poorly understood. Here, we examined the population structure and diversity of fluorescent pseudomonads isolated from sugar beet grown at two geographic locations (Oxford, United Kingdom and Auckland, New Zealand). To seek evidence for niche adaptation, bacteria were sampled from three types of leaves (immature, mature, and senescent) and then characterized using a combination of genotypic and phenotypic analysis. We first performed multilocus sequence analysis (MLSA) of three housekeeping genes (gapA, gltA, and acnB) in a total of 152 isolates (96 from Oxford, 56 from Auckland). The concatenated sequences were grouped into 81 sequence types and 22 distinct operational taxonomic units (OTUs). Significant levels of recombination were detected, particularly for the Oxford isolates (rate of recombination to mutation (r/m) = 5.23 for the whole population). Subsequent ancestral analysis performed in STRUCTURE found evidence of six ancestral populations, and their distributions significantly differed between Oxford and Auckland. Next, their ability to grow on 95 carbon sources was assessed using the Biolog™ GN2 microtiter plates. A distance matrix was generated from the raw growth data (A660) and subjected to multidimensional scaling (MDS) analysis. There was a significant correlation between substrate utilization profiles and MLSA genotypes. Both phenotypic and genotypic analyses indicated presence of a geographic structure for strains from Oxford and Auckland. Significant differences were also detected for MLSA genotypes between strains isolated from immature versus mature/senescent leaves. The fluorescent pseudomonads thus showed an ecotypic population structure, suggestive of adaptation to both geographic conditions and local plant niches.

Polar accumulation of pyoverdin and exit from stationary phase

“…Measurement of the amount of pyoverdin in cells that localize the iron chelator showed that it is the same as that Polarization test in natural isolates, left to right and top to bottom: U106 (liquid KB medium for 24 h); U177 (SMM agarose pad for 24 h); U149 (liquid SMM with DP 100 µg/ml for 24 h); U180 (liquid SMM with DP 100 µg/ml for 24 h); U181 (SMM agarose pad for 16 h); T24 V1a (SMM agarose pad for 24 h); T24 V9b (SMM agarose pad for 24 h); T24 H1b (SMM agarose pad for 24 h); T24 H9b (liquid SMM for 24 h). Natural isolates were collected in Oxford, UK [33] (first and second row) and in Paris, France [32] (Bottom row)(locations are roughly marked with a pink circle on the map). Color represents the identified Pseudomonas species.…”

“…Paris, France [32]: T24 V1a, T24 V9b, T24 H1b, T24H9b; all classified as P. putida . Oxford, UK [33]: P. marginalis U106, P. putida U177, P . aureofaciens U149, U180, and U181.…”