produces interspecies biofilms and changes in colony morphology of A. brasilense Pseudomonas protegens CHA0 - Azospirillum brasilense Sp245 interaction

PAGNUSSAT LUCIANA; SALCEDO FLORENCIA; CREUS CECILIA; VALVERDE CLAUDIO

Workshop; 1 II LATIN AMERICAN PGPR WORKSHOP; 2014

Introduction Plant-growth-promoting rhizobacteria (PGPR) enhances growth of many terrestrial crop plants and are currently commercialized as simple or complex inoculants (i.e.Azospirillum brasilense and Pseudomonas fluorescens). PGPRs efficiency depends to a great extent on their capabilities to survive and interact with other bacteria in the rhizosphere. A prerequisite for these organisms to exert their effect is to achieve adequate inocula to insure root colonization. An important concern is that Pseudomona secretes several secondary metabolites, including antibiotics that can negatively affect- A. brasilense proliferation in mixed inoculants. However, previous results obtained in our laboratory indicate that co-cultivation of A. brasilense Sp245 with P. protegens not only doesn?t inhibit A. brasilense, but it promotes its growth and adherence in vitro. The aim of this work was to study interspecies biofilm structure and P. protegens effect on colony morphology of A. brasilense.   Materials and methods Bacterial strains: A. brasilense Sp245 Pmp2444 (Gmr, Plac-egfp) and P. protegens CHA0 (ex fluorescens) PME7134 (Tcr, Plac-dsRed). Polystyrene covered slides were placed on 105 or 106 bacteria.ml-1 A. brasilense-105P. protegens Nfb-NO3- and Fe static cultures for 2 days at 30ºC. Biofilm microscopic analysis was performed on a Nikon C1 confocal laser scanning microscope, using 40.0x/1.30/0.22 Oil spring-loaded lens. DsRed and GFP proteins were excited at 543 and 488 nm and detected at 550?650 nm and 650?750 nm respectively. The effect of P. protegens on colony morphology of A. brasilense was studied in Petri dishes. A. brasilense was spotted onto Nfb NO3-Fe agar plates, P. protegens lawn or P. protegens cell free media and incubated for five days at 30°C. Colony morphology was analyzed by optical magnifier, and UFCml-1 was calculated. Results Confocal images showed thicker biofilms on mixed cultures in comparison to single-species biofilms. A particular specie-specific distribution inside the biofilm was also observed. A. brasilense Sp245 strain was restricted on the bottom of the biofilm, while P. protegens was superficially localized, in contact with culture media. Azospirillum showed higher number of colonies (UFCml-1) when it was grown on Nfb-NO3--Fe with P. protegens lawn or P. protegens cell-free media. However those colonies were smaller compared to colonies developed on Nfb NO3-Fe agar plates. Conclusion P. protegens and A. brasilense are capable of forming mixed biofilms with specie-specific distribution. On the other hand Pseudomonas has an effect on the morphology and the number of colonies of Azopirillum. These results suggest that complex and dynamic inter-specific communication could modulate root colonization in mixed inoculants. Experiments are in progress to unravel the molecular mechanisms underlying these findings.