Pathogenic and non‐pathogenic Agrobacterium tumefaciens, A. rhizogenes and A. vitis strains form biofilms on abiotic as well as on root surfaces

Abstract

Pathogenic and non-pathogenic Agrobacterium tumefaciens, A. rhizogenes and A. vitis strains growing in minimal liquid medium adhered to different abiotic surfaces, forming biofilms at initial stages of development. Agrobacterium tumefaciens and A. vitis strains were able to attach to both polystyrene and polypropylene materials, whereas the A. rhizogenes strains only bound to polystyrene surfaces. Strains of the three species were also able to form biofilms on borosilicate coverslips. It is concluded that their ability to adhere to and form nascent biofilms on abiotic surfaces is dependent on the Agrobacterium species (biovar), surface material and growth conditions. Furthermore, tumorigenic A. tumefaciens and A. vitis strains, and the biological control agent A. rhizogenes strain K84, bound tightly to and formed complex biofilms on the surface of tomato root tips ex planta. More importantly, in planta assays confirmed that all three Agrobacterium spp. strains efficiently colonized tomato seedlings and also formed biofilms on roots. These complex structures, as revealed by scanning electron microscopy, were composed of numerous bacterial cells arranged in different ways: either dense and continuous carpets, large aggregates embedded in extra-cellular material or globular mushrooms traversed internally by channels. Confocal laser scanning microscopy, using GFP-marked derivative strains, corroborated the presence of live, three-dimensional and thick green fluorescent structures attached to plant material. This study illustrates that besides A. tumefaciens, strains of the species A. rhizogenes and A. vitis are also able to build biofilms on abiotic as well as on root surfaces.

Role for Rhizobium rhizogenes K84 Cell Envelope Polysaccharides in Surface Interactions

“…We have recently shown that the biocontrol agent R. rhizogenes (formerly A. radiobacter [56]) strain K84 is able to form biofilms not only on abiotic surfaces but also on plant root surfaces, where biofilms become complex and structured, with cells adhering to the surface and to each other (1). Based on these observations, we…”

“…In the first, large-scale screen, 10 l of overnight cultures in MG/L liquid medium was added to 200 l of ATM liquid medium in 96-well microtiter polypropylene plates, using one well per mutant. The amounts of planktonic (optical density at 570 nm [OD 570 ]) and surface-attached (A 570 ) cells were determined by a standard crystal violet (CV) assay after static incubation for 48 h at 26°C (1,39). Each potential mutant that was compromised in terms of biofilm formation in this first screen was then rescreened by individually adjusting the ATM culture to an OD 570 of 0.04, and growth was continued in microtiter plates.…”

“…Green fluorescent protein (GFP)-labeled derivative strains of strain K84 and its mutants were evaluated for biofilm formation on the surface of tomato root tips following a previously described methodology (1). Tightly bound bacteria were visualized on the surface of washed root tips by luminescent GFP detection using an LAS-3000 imaging system (Fujifilm).…”

“…We recently reported that the crown gall biocontrol agent R. rhizogenes K84 binds tightly to polypropylene and efficiently colonizes root surfaces as complex, multilayered biofilm structures (1). However, the genetic basis for these properties had remained undefined.…”

“…Aggregates, microcolonies, and highly structured biofilms are common multicellular developmental stages that are observed in both pathogenic and nonpathogenic root-colonizing bacteria (1,12,13,35,42,47). Agrobacterium tumefaciens strain C58 forms biofilms on roots that are enhanced by phosphorus, oxygen limitation, and cellulose production (14,32,33,47).…”