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      Standardization of Plant Microbiome Studies: Which Proportion of the Microbiota is Really Harvested?

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          Abstract

          Studies in plant-microbiome currently use diverse protocols, making their comparison difficult and biased. Research in human microbiome have faced similar challenges, but the scientific community proposed various recommendations which could also be applied to phytobiome studies. Here, we addressed the isolation of plant microbiota through apple carposphere and lettuce root microbiome. We demonstrated that the fraction of the culturable epiphytic microbiota harvested by a single wash might only represent one-third of the residing microbiota harvested after four successive washes. In addition, we observed important variability between the efficiency of washing protocols (up to 1.6-fold difference for apple and 1.9 for lettuce). QIIME2 analysis of 16S rRNA gene, showed a significant difference of the alpha and beta diversity between protocols in both cases. The abundance of 76 taxa was significantly different between protocols used for apple. In both cases, differences between protocols disappeared when sequences of the four washes were pooled. Hence, pooling the four successive washes increased the alpha diversity for apple in comparison to a single wash. These results underline the interest of repeated washing to leverage abundance of microbial cells harvested from plant epiphytic microbiota whatever the washing protocols, thus minimizing bias.

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          Roots shaping their microbiome: global hotspots for microbial activity.

          Land plants interact with microbes primarily at roots. Despite the importance of root microbial communities for health and nutrient uptake, the current understanding of the complex plant-microbe interactions in the rhizosphere is still in its infancy. Roots provide different microhabitats at the soil-root interface: rhizosphere soil, rhizoplane, and endorhizosphere. We discuss technical aspects of their differentiation that are relevant for the functional analysis of their different microbiomes, and we assess PCR (polymerase chain reaction)-based methods to analyze plant-associated bacterial communities. Development of novel primers will allow a less biased and more quantitative view of these global hotspots of microbial activity. Based on comparison of microbiome data for the different root-soil compartments and on knowledge of bacterial functions, a three-step enrichment model for shifts in community structure from bulk soil toward roots is presented. To unravel how plants shape their microbiome, a major research field is likely to be the coupling of reductionist and molecular ecological approaches, particularly for specific plant genotypes and mutants, to clarify causal relationships in complex root communities.
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            The Madness of Microbiome: Attempting To Find Consensus “Best Practice” for 16S Microbiome Studies

            ABSTRACT The development and continuous improvement of high-throughput sequencing platforms have stimulated interest in the study of complex microbial communities. Currently, the most popular sequencing approach to study microbial community composition and dynamics is targeted 16S rRNA gene metabarcoding. To prepare samples for sequencing, there are a variety of processing steps, each with the potential to introduce bias at the data analysis stage. In this short review, key information from the literature pertaining to each processing step is described, and consequently, general recommendations for future 16S rRNA gene metabarcoding experiments are made.
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              Biofilm formation in plant-microbe associations.

              Bacteria adhere to environmental surfaces in multicellular assemblies described as biofilms. Plant-associated bacteria interact with host tissue surfaces during pathogenesis and symbiosis, and in commensal relationships. Observations of bacteria associated with plants increasingly reveal biofilm-type structures that vary from small clusters of cells to extensive biofilms. The surface properties of the plant tissue, nutrient and water availability, and the proclivities of the colonizing bacteria strongly influence the resulting biofilm structure. Recent studies highlight the importance of these structures in initiating and maintaining contact with the host by examining the extent to which biofilm formation is an intrinsic component of plant-microbe interactions.
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                Author and article information

                Journal
                Microorganisms
                Microorganisms
                microorganisms
                Microorganisms
                MDPI
                2076-2607
                28 February 2020
                March 2020
                : 8
                : 3
                Affiliations
                [1 ]Laboratory of Integrated and Urban Phytopathology, Gembloux Agro-Bio Tech, University of Liège, 5030 Gembloux, Belgium; G.Stouvenakers@ 123456uliege.be (G.S.); Mathilde.Eck@ 123456uliege.be (M.E.); mh.jijakli@ 123456uliege.be (M.H.J.); sebastien.massart@ 123456uliege.be (S.M.)
                [2 ]VIB-UGent Center of Plant Systems Biology, 9052 Ghent, Belgium; Amber.Lampens@ 123456psb.vib-ugent.be (A.L.); sogoo@ 123456psb.vib-ugent.be (S.G.)
                [3 ]Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium
                Author notes
                [†]

                These authors contributed equally to this work.

                Article
                microorganisms-08-00342
                10.3390/microorganisms8030342
                7142977
                32121205
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

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