Differentiation of Closely Related Oak-Associated Gram-Negative Bacteria by Label-Free Surface Enhanced Raman Spectroscopy (SERS)

Abstract

Due to the harmful effects of chemical fertilizers and pesticides, the need for an eco-friendly solution to improve soil fertility has become a necessity, thus microbial biofertilizer research is on the rise. Plant endophytic bacteria inhabiting internal tissues represent a novel niche for research into new biofertilizer strains. However, the number of species and strains that need to be differentiated and identified to facilitate faster screening in future plant-bacteria interaction studies, is enormous. Surface enhanced Raman spectroscopy (SERS) may provide a platform for bacterial discrimination and identification, which, compared with the traditional methods, is relatively rapid, uncomplicated and ensures high specificity. In this study, we attempted to differentiate 18 bacterial isolates from two oaks via morphological, physiological, biochemical tests and SERS spectra analysis. Previous 16S rRNA gene fragment sequencing showed that three isolates belong to Paenibacillus, 3—to Pantoea and 12—to Pseudomonas genera. Additional tests were not able to further sort these bacteria into strain-specific groups. However, the obtained label-free SERS bacterial spectra along with the high-accuracy principal component (PCA) and discriminant function analyses (DFA) demonstrated the possibility to differentiate these bacteria into variant strains. Furthermore, we collected information about the biochemical characteristics of selected isolates. The results of this study suggest a promising application of SERS in combination with PCA/DFA as a rapid, non-expensive and sensitive method for the detection and identification of plant-associated bacteria.