Exopolysaccharide production, biofilm formation and adhesion as screening criteria for the selection of bacterial inocula functional to the promotion of plant drought tolerance

Plant growth-promoting (PGP) are gaining increasing attention for their possible use in bioinoculant formulations for sustainable agriculture. The production of extracellular polymeric substances (EPS) is a key driver of bacterial establishment in the rhizospere and the expression of PGP traits. However, biofilm formation and adhesion to solid surfaces are also important processes when screening for novel candidates, considering their importance for rhizosphere competence. In this work, EPS production, biofilm formation and adhesion capability were studied in 120 bacteria isolated from the fruiting body of Tuber borchii through an optimized step-wise protocol. The strains were qualitatively tested for EPS production on agar medium with a 40:1 C/N ratio, on which EPS producers form smooth, mucous colonies. The best performing EPS isolates were further characterized by EPS quantification using alcohol precipitation method and Congo red binding assay. Biofilm formation and matrix overproduction were assessed by Congo red agar (CRA) method. The ability to adhere to solid surfaces was quantitatively determined in 96-well microtiter plates by crystal violet (CV) binding assay. Most strains showed EPS accumulation on solid medium after 48 hours. The highest EPS production was 3.27-2.43 g of EPS l-1 of bacterial culture. Overall, several strains showed biofilm formation on CRA after 72h, although some showed early (16 hours) and others showed only late (72 hours) biofilm production. The ability to produce biofilm was always accompanied by good/very good EPS productivity. By contrast, CV assay adhesion capacity did not necessarily correlate with EPS/biofilm production, as strains resulting mild/poor EPS producers and late biofilm formers still showed significant adhesion in multiwell plates. Our results showed that although EPS production and biofilm formation are important biological aspects in relation to each other, adhesion capacity should always be included in the screening process and characterization of novel candidate characterized by drought-tolerance traits.