Identification and differentiation of Pseudomonas species in field samples using an rpoD amplicon sequencing methodology

Species of the genus Pseudomonas are used for several biotechnological purposes, including plant biocontrol and bioremediation. To exploit the Pseudomonas genus in environmental, agricultural or industrial settings, the organisms must be profiled at species level as their bioactivity potential differs markedly between species. Standard 16S rRNA gene amplicon profiling does not allow for accurate species differentiation. Thus, the purpose of this study was to develop an amplicon-based high-resolution method targeting a 760 nt region of the rpoD gene enabling taxonomic differentiation of Pseudomonas species in soil samples. The method was benchmarked on a sixteen membered Pseudomonas species mock community. All 16 species were correctly and semi-quantitatively identified using rpoD gene amplicons, whereas 16S rRNA V3V4 amplicon sequencing only correctly identified one species. We analysed the Pseudomonas profile in thirteen soil samples in northern Zealand, Denmark, where samples were collected from grassland (3 samples) and agriculture soil (10 samples). Pseudomonas species represented up to 0.7% of the microbial community, of which each sampling site contained a unique Pseudomonas composition. Thirty culturable Pseudomonas strains were isolated from each grassland site and ten from each agriculture site and identified by Sanger sequencing of the rpoD gene. In all cases, the rpoD-amplicon approach identified more species than found by cultivation, including hard-to-culture non-fluorescent pseudomonads, as well as more than found by 16S rRNA V3V4 amplicon sequencing. Thus, rpoD profiling can be used for species profiling of Pseudomonas, and large scale prospecting of bioactive Pseudomonas may be guided by initial screening using this method. ImportanceA high throughput sequence-based method for profiling of Pseudomonas species in soil microbiomes was developed and identified more species than 16S rRNA gene sequencing or cultivation. Pseudomonas species are used as biocontrol organisms and plant-growth promoting agents, and the method will allow tracing of specific species of Pseudomonas as well as enable screening of environmental samples for further isolation and exploitation.