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

Abdoul Razack Sare,M Haïssam Jijakli,Sebastien Massart,Mathilde Eck,Gilles Stouvenakers,Amber Lampens,Sofie Goormachtig

doi:10.3390/microorganisms8030342

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.

Highlights

Plant tissues provide several niches for microbial growth and a rough distinction can be made between the aboveground plant organs, referred to as phyllosphere and the belowground microbial niches: the rhizosphere; the rhizoplane; and the root endosphere
In order to assess the impact of these parameters, we evaluated the effect of successive washes and of different washing protocols on the composition and quantity of microbiota harvested from apple carposphere and lettuce roots
There is a need to pay attention to these parameters in further studies when harvesting microbial cells from plants. These two study cases indicate that the washing protocol significantly influences the quantity and the bacterial diversity of microorganisms harvested and that four successive washes can increase up to three-times the quantity of harvested microorganisms

Summary

Introduction

Plant tissues provide several niches for microbial growth and a rough distinction can be made between the aboveground plant organs, referred to as phyllosphere and the belowground microbial niches: the rhizosphere (the soil directly surrounding plant roots from which the physicochemical properties are influenced by the root); the rhizoplane (the root surface); and the root endosphere (the compartment formed by the apoplastic spaces between the root cells). When considering the root system from the outside to the inside, the microbial diversity decreases while the degree of specialization and the strength of attachment and interaction increases [1]. In order to survive the harsh environmental conditions and the oligotrophic environment, phyllosphere inhabitants, especially colonizers of the phylloplane, have developed specific features (e.g., pigments, motility, biofilm formation) and are populating specialized compartments such as substomatal chambers. The wax layer, the cuticle, and the spatial heterogeneity of the leaf play a pivotal role in shaping the phyllosphere microbial communities and their attachment to plant tissue [5]. They are not uniformly distributed on fruit surface, as it was shown with the fungal populations of the different parts of apple fruit [8]

Methods

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Discussion

Conclusion