Evaluation of Strategies to Separate Root-Associated Microbial Communities: A Crucial Choice in Rhizobiome Research

Abstract

Plants shape distinct, species-specific microbiomes in their rhizospheres. A main premise for evaluating microbial communities associated with root-soil compartments is their successful separation into the rhizosphere (soil-root interface), the rhizoplane (root surface), and the endosphere (inside roots). We evaluated different approaches (washing, sonication, and bleaching) regarding their efficiency to separate microbial cells associated with different root compartments of soil-grown rice using fluorescence microscopy and community fingerprinting of 16S rRNA genes. Vigorous washing detached 45% of the rhizoplane population compared to untreated roots. Additional sonication reduced rhizoplane-attached microorganisms by up to 78% but caused various degrees of root tissue destruction at all sonication intensities tested. Treatment with sodium hypochlorite almost completely (98%) removed rhizoplane-associated microbial cells. Community fingerprinting revealed that microbial communities obtained from untreated, washed, and sonicated roots were not statistically distinguishable. Hypochlorite-treated roots harbored communities significantly different from all other samples, likely representing true endospheric populations. Applying these procedures to other root samples (bean and clover) revealed that treatment efficiencies were strongly affected by root morphological parameters such as root hair density and rigidity of epidermis. Our findings suggest that a careful evaluation of separation strategies prior to molecular community analysis is indispensable, especially when endophytes are the subject of interest.

Recognizing Patterns: Spatial Analysis of Observed Microbial Colonization on Root Surfaces

“…SYBR Green I has been shown to give strong fluorescent signals that can be used to detect rhizoplane-colonizing microorganisms (Reinhold-Hurek et al, 2015;Richter-Heitmann et al, 2016). The signal-to-noise ratios obtained with SYBR Green I were found to be superior to other general nucleic acid stains such as DAPI.…”

“…For downstream microscopic analyses, only primary nodal roots were used to increase comparability among the replicates. Staining of rhizoplane-associated bacteria with SYBR-Green I (Lumiprobe, Germany) and preparation of the roots on objective slides for fluorescence microscopy was performed as described in Richter-Heitmann et al (2016). In addition, bacteria were visualized on replicate root samples via CARD-FISH as described in Schmidt and Eickhorst (2014) with slight modifications (see Supplementary Materials).…”

Effectiveness of Plant Beneficial Microbes: Overview of the Methodological Approaches for the Assessment of Root Colonization and Persistence

“…Indeed, to study microbial endophytes, it is necessary to surface sterilize the roots prior to grinding, chopping or blending them (McInroy and Kloepper, 1991). Several works describe a prior wash with 1% chloramine and cycles of washing/agitation treatments using ethanol and PBS (Ladha et al, 1997;Dennis et al, 2008;Richter-Heitmann et al, 2016). Cleaned roots to be analyzed by cultureindependent methods can be stored in a solution of PBS buffer and 70% ethanol (2:3 v/v) for a long time at -20°C (Dennis et al, 2008;Richter-Heitmann et al, 2016).…”

“…Several works describe a prior wash with 1% chloramine and cycles of washing/agitation treatments using ethanol and PBS (Ladha et al, 1997;Dennis et al, 2008;Richter-Heitmann et al, 2016). Cleaned roots to be analyzed by cultureindependent methods can be stored in a solution of PBS buffer and 70% ethanol (2:3 v/v) for a long time at -20°C (Dennis et al, 2008;Richter-Heitmann et al, 2016). However, fresh root samples used to evaluate the density of the cultivable microorganisms by plating on growth media should be analyzed within a short time (24-48 h).…”

The Impact of Soil-Applied Biochars From Different Vegetal Feedstocks on Durum Wheat Plant Performance and Rhizospheric Bacterial Microbiota in Low Metal-Contaminated Soil

“…To assess the possible changes caused by biochar incorporation and/or the durum genotype to the soil microbiome, we specifically focused on the rhizosphere, which is the interface between plant roots and soil, since the microorganisms colonizing the rhizosphere may contribute to plant growth and health (Richter-Heitmann et al, 2016); moreover, different studies have already established that the rhizosphere corresponds to the plant-soil compartment harboring a higher richness (Qian et al, 2019).…”