Microorganisms colonizing plant roots co-exist in complex, spatially structured multispecies biofilm communities. However, little is known about microbial interactions and the underlying spatial organization within biofilm communities established on plant roots. Here, a well-established four-species biofilm model (Stenotrophomonas rhizophila, Paenibacillus amylolyticus, Microbacterium oxydans, and Xanthomonas retroflexus, termed as SPMX) was applied to Arabidopsis roots to study the impact of multispecies biofilm on plant growth and the community spatial dynamics on the roots. SPMX co-culture notably promoted root development and plant biomass. Co-cultured SPMX increased root colonization and formed multispecies biofilms, structurally different from those formed by monocultures. By combining 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization with confocal laser scanning microscopy, we found that the composition and spatial organization of the four-species biofilm significantly changed over time. Monoculture P. amylolyticus colonized plant roots poorly, but its population and root colonization were highly enhanced when residing in the four-species biofilm. Exclusion of P. amylolyticus from the community reduced overall biofilm production and root colonization of the three species, resulting in the loss of the plant growth-promoting effects. Combined with spatial analysis, this led to identification of P. amylolyticus as a keystone species. Our findings highlight that weak root colonizers may benefit from mutualistic interactions in complex communities and hereby become important keystone species impacting community spatial organization and function. This work expands the knowledge on spatial organization uncovering interspecific interactions in multispecies biofilm communities on plant roots, beneficial for harnessing microbial mutualism promoting plant growth.
Bacteria colonize plant roots and engage in reciprocal interactions with their hosts. However, the contribution of individual taxa or groups of bacteria to plant nutrition and fitness is not well characterized due to a lack of in\uffc2\uffa0situ evidence of bacterial activity.
To address this knowledge gap, we developed an analytical approach that combines the identification and localization of individual bacteria on root surfaces via gold\uffe2\uff80\uff90based in\uffc2\uffa0situ hybridization with correlative NanoSIMS imaging of incorporated stable isotopes, indicative of metabolic activity.
We incubated Kosakonia strain DS\uffe2\uff80\uff901\uffe2\uff80\uff90associated, gnotobiotically grown rice plants with 15N\uffe2\uff80\uff93N2 gas to detect in\uffc2\uffa0situ N2 fixation activity. Bacterial cells along the rhizoplane showed\uffc2\uffa0heterogeneous patterns of 15N enrichment, ranging from the natural isotope abundance levels up to 12.07 at% 15N (average and median of 3.36 and 2.85 at% 15N, respectively, n\uffe2\uff80\uff89=\uffe2\uff80\uff89697 cells).
The presented correlative optical and chemical imaging analysis is applicable to a broad range of studies investigating plant\uffe2\uff80\uff93microbe interactions. For example, it enables verification of the in\uffc2\uffa0situ metabolic activity of host\uffe2\uff80\uff90associated commercialized strains or plant growth\uffe2\uff80\uff90promoting bacteria, thereby disentangling their role in plant nutrition. Such data facilitate the design of plant\uffe2\uff80\uff93microbe combinations for improvement of crop management.
Bacteria colonize plant roots and engage in reciprocal interactions with their hosts. However, the contribution of individual taxa or groups of bacteria to plant nutrition and fitness is not well characterized due to a lack of in\uffc2\uffa0situ evidence of bacterial activity.
To address this knowledge gap, we developed an analytical approach that combines the identification and localization of individual bacteria on root surfaces via gold\uffe2\uff80\uff90based in\uffc2\uffa0situ hybridization with correlative NanoSIMS imaging of incorporated stable isotopes, indicative of metabolic activity.
We incubated Kosakonia strain DS\uffe2\uff80\uff901\uffe2\uff80\uff90associated, gnotobiotically grown rice plants with 15N\uffe2\uff80\uff93N2 gas to detect in\uffc2\uffa0situ N2 fixation activity. Bacterial cells along the rhizoplane showed\uffc2\uffa0heterogeneous patterns of 15N enrichment, ranging from the natural isotope abundance levels up to 12.07 at% 15N (average and median of 3.36 and 2.85 at% 15N, respectively, n\uffe2\uff80\uff89=\uffe2\uff80\uff89697 cells).
The presented correlative optical and chemical imaging analysis is applicable to a broad range of studies investigating plant\uffe2\uff80\uff93microbe interactions. For example, it enables verification of the in\uffc2\uffa0situ metabolic activity of host\uffe2\uff80\uff90associated commercialized strains or plant growth\uffe2\uff80\uff90promoting bacteria, thereby disentangling their role in plant nutrition. Such data facilitate the design of plant\uffe2\uff80\uff93microbe combinations for improvement of crop management.
Microorganisms colonizing plant roots co-exist in complex, spatially structured multispecies biofilm communities. However, little is known about microbial interactions and the underlying spatial organization within biofilm communities established on plant roots. Here, a well-established four-species biofilm model (Stenotrophomonas rhizophila, Paenibacillus amylolyticus, Microbacterium oxydans, and Xanthomonas retroflexus, termed as SPMX) was applied to Arabidopsis roots to study the impact of multispecies biofilm on plant growth and the community spatial dynamics on the roots. SPMX co-culture notably promoted root development and plant biomass. Co-cultured SPMX increased root colonization and formed multispecies biofilms, structurally different from those formed by monocultures. By combining 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization with confocal laser scanning microscopy, we found that the composition and spatial organization of the four-species biofilm significantly changed over time. Monoculture P. amylolyticus colonized plant roots poorly, but its population and root colonization were highly enhanced when residing in the four-species biofilm. Exclusion of P. amylolyticus from the community reduced overall biofilm production and root colonization of the three species, resulting in the loss of the plant growth-promoting effects. Combined with spatial analysis, this led to identification of P. amylolyticus as a keystone species. Our findings highlight that weak root colonizers may benefit from mutualistic interactions in complex communities and hereby become important keystone species impacting community spatial organization and function. This work expands the knowledge on spatial organization uncovering interspecific interactions in multispecies biofilm communities on plant roots, beneficial for harnessing microbial mutualism promoting plant growth.