Early succession of a multispecies bacterial biofilm obtained from alfalfa rhizosphere

BOGINO, P.; ABOD, A.; NIEVAS, F.; NOCELLI, N.; GIORDANO, W.

La Falda (Córdoba)

Congreso; II Taller Latinoamericano Sobre Rizobacterias Promotoras del Desarrollo Vegetal; 2014

Universidad Nacional de Río Cuarto, Universidad Nacional de Quilmes y Universidad de Antioquía (Colombia)

Introduction Biofilms are microbial communities that adhere to biotic or abiotic surfaces and are enclosed in a protective matrix of extracellular compounds. It has been estimated that over 99% of all bacterial activity in natural ecosystems is associated with bacteria organized in biofilms. Although life in biofilm has several advantages for soil bacteria, the composition and functions of bacterial biofilms in soil microniches are poorly understood. The aim of this study was evaluate the early succession of multibacterial communities established as biofilm-like structures in the rhizosphere of Medicago sativa (alfalfa). Materials and methods Suspension of rhizospheric soils (RS) was obtained by shaking alfalfa roots in phosphate-buffered saline (PBS; pH 7.4) at 150 rpm for 1 h. RS suspension was placed in a device containing slides with water agar supplemented with alfalfa root exudates. This device was shaken at 40 rpm. At times 1 and 5 days (RS1 and RS5 respectively) the slides gently washed with sterile PBS. The material associated with the agar surface was harvested in 1 ml of sterile PBS in order to obtain the bacterial communities. Bacterial counting on Nutrient Agar (NA) medium and isolation of strains was carried out for each RS. The whole biofilm-forming ability (WBFA) for each community was carried out according staining methodology with crystal violet in microtiter plates. For each condition were selected strains with high BFA (HBFA) which were characterized by ARDRA and identified by 16S rRNA gen sequencing. Results Bacterial counting and WBFA for rhizospheric communities was higher in RS1 than in RS and RS5. ARDRA showed the presence of different ribotypes between the strains of HBFA in the RS in relation to the strains of HBFA obtained from succession (RS1 and RS5). For RS1 were obtained five ribotypes (R1-R5) whereas that after 5 days of established the microconsortium (RS5) were obtained four ribotypes (R2, R3, R4 y R6). The succession study showed the presence of (i) stable ribotypes (R2, R3 and R4), (ii) a predominant ribotype (R3) and (iii) replacement of ribotypes (R1 and R5 for R6). The phylogenetic analysis clearly differentiated between strains of RS with predominance of -proteobacteria and actinobacteria from RS1 and RS5 with prevalence of strains identified as -proteobacteria. Conclusion Our study demonstrates that the development of multibacterial communities established as biofilm-like structures in the rhizosphere of alfalfa is a dynamic process. The initial biofilm development highlights the presence of strains with high ability to adhere to the substrate (higher counting and WBFA in RS1). According to ARDRA, these bacterial members would not be able to stay and would be replaced by more competitive strains. The persistence of certain ribotypes would indicate the presence of species with high ability to establish and persist in the rhizospheric consortium. The appearance of new ribotypes would indicate the ability of strains to associating (replacement or co-existence) with a previously established structure of sessile bacteria in the biofilm of the alfalfa rhizosphere.