Journal of Experimental Botany, Vol. 67, No. 4 pp. 995–1002, 2016 doi:10.1093/jxb/erv466 Advance Access publication 7 November 2015 REVIEW PAPER The plant microbiome explored: implications for experimental botany Gabriele Berg1,2, Daria Rybakova1, Martin Grube3 and Martina Köberl1,4 1 Graz University of Technology, Institute of Environmental Biotechnology, 8010 Graz, Austria 2 Austrian Centre of Industrial Biotechnology (ACIB GmbH), 8010 Graz, Austria 3 University of Graz, Institute of Plant Sciences, 8010 Graz, Austria 4 Pacific Northwest National Laboratory, Biological Sciences Division, Richland, WA 99352, USA Received 20 July 2015; Accepted 5 October 2015 Editor: Adam Price, University of Aberdeen Abstract The importance of microbial root inhabitants for plant growth and health was recognized as early as 100 years ago. Recent insights reveal a close symbiotic relationship between plants and their associated microorganisms, and high structural and functional diversity within plant microbiomes. Plants provide microbial communities with specific habi- tats, which can be broadly categorized as the rhizosphere, phyllosphere, and endosphere. Plant-associated microbes interact with their host in essential functional contexts. They can stimulate germination and growth, help plants fend off disease, promote stress resistance, and influence plant fitness. Therefore, plants have to be considered as metaorgan- isms within which the associated microbes usually outnumber the cells belonging to the plant host. The structure of the plant microbiome is determined by biotic and abiotic factors but follows ecological rules. Metaorganisms are co- evolved species assemblages. The metabolism and morphology of plants and their microbiota are intensively connected with each other, and the interplay of both maintains the functioning and fitness of the holobiont. Our study of the current literature shows that analysis of plant microbiome data has brought about a paradigm shift in our understanding of the diverse structure and functioning of the plant microbiome with respect to the following: (i) the high interplay of bacteria, archaea, fungi, and protists; (ii) the high specificity even at cultivar level; (iii) the vertical transmission of core microbi- omes; (iv) the extraordinary function of endophytes; and (v) several unexpected functions and metabolic interactions. The plant microbiome should be recognized as an additional factor in experimental botany and breeding strategies. Key words: Endosphere, holobiont, microbiome, phyllosphere, plant-microbe interaction, rhizosphere. Introduction The plant microbiome has been considered one of the key deter- structure and function of plant-associated microbial communi- minants of plant health and productivity for over 100 years, and ties of the model plant Arabidopsis were presented by Bulgarelli intensive research on this topic started with Lorenz Hiltner’s et al. (2012) and Lundberg et al. (2012), while another study work in 1901 (Hartmann et al., 2008). This long research period detailed a disease-suppressive rhizosphere microbiome in sugar was influenced by the continuous development of research beet (Mendes et al., 2011). The last century has been character- methods, but it was the application of molecular and omics ized by important, diverse, and unexpected discoveries relating techniques, as well as novel microscopic techniques combin- to plant-associated microorganisms that were made by apply- ing molecular and analytical tools, that led to the important ing several research methods, especially combinations thereof. milestones (Muyzer and Smalla, 1998; Caporaso et al., 2012; Several selected examples are as follows: (i) the potential of Jansson et al., 2012). For example, deeper insights into the root-associated microbes to suppress soil-borne pathogens, © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected] 996 | Berg et al. demonstrated by strain selection and field trials (Cook et al., known for many years that the rhizosphere enriches specific 1995; Weller et al., 2002); (ii) trans-kingdom communica- microbial species/genotypes in comparison to soil and inner tis- tion between plants and microbes, analysed by analytical and sues, but modern technologies provide much deeper insights and molecular methods (Hartmann and Schikora, 2012); (iii) plant expand our understanding of plant–microbe interactions (Bais species-specific rhizosphere microbial communities, obtained et al., 2006; Doornbos et al., 2012). A current model shows the by molecular fingerprints and molecular strain analysis (Berg occurrence of seed-borne microorganisms (Christin Zachow and Smalla, 2009; Hartmann et al., 2009); (iv) the rhizosphere and Gabriele Berg, personal communication) and the attrac- as a reservoir of facultative human pathogens, detected by iso- tion of microbes to nutrients such as carbohydrates and amino lation and characterization of strains (Berg et al., 2005), and acids (Moe, 2013) in combination with plant-specific secondary deep study of the lettuce metagenome (Berg et al., 2014a); (v) metabolites (Weston and Mathesius, 2013). Plant root exudates the high diversity and importance of the endophytic (myco) play important roles as both chemo-attractants and repellents biome visualized especially by fluorescencein situ hybridiza- (Badri and Vivanco, 2009). Additionally, plant defence signal- tion and microscopy (Omacini et al., 2001; Rodriguez et al., ling plays a role in this process (Doornbos et al., 2012). The 2009; Hardoim et al., 2015); and (vi) the detection of abundant importance of the rhizosphere microbiome can be underlined by endophytic Archaea in trees using molecular markers based on the number of species: in the metagenomes studied in our group, genomics of non-cultivable organisms (Müller et al., 2015). we found up to 1200 prokaryotic species (extracted 16S rRNA From protists to humans, all organisms are inhabited by genes annotated using the Greengenes reference database). microorganisms. According to the holobiont concept, metaor- Moreover, a higher number of species was found in medicinal ganisms are co-evolved species assemblages. Moreover, co-evo- and wild plants than on crops grown in intensive agriculture lution has resulted in intimate relationships forming between (Martina Köberl and Gabriele Berg, personal communication). microbes and their hosts that create specific and stable micro- For comparative analyses, all metagenomes were rarefied at a biomes. Therefore, all eukaryotic organisms can be considered sequencing depth of 1.7 × 107 sequences; the actual species diver- to be metaorganisms: an association of the macroscopic hosts sity is even much higher. The abundances measured sum up to and a diverse microbiome consisting of bacteria, archaea, 109–1011 bacterial cells colonizing each gram of the root, which fungi, and protists (even protists can have their own bacterial often not only outnumbers the cells of the host plants but also microbiota, and it has been argued that microbiota play an represent more microbes than people existing on Earth. While important role in the evolution of multicellularity; McFall- the well-studied rhizosphere represents the soil–plant interface, Ngai et al., 2013). Together, microbiota fulfil all important the phyllosphere forms the air–plant interface. This microhabitat functions for the holobiont themselves, and also for the eco- is also of special interest owing to its large and exposed surface system (Mendes and Raaijmakers, 2015; Vandenkoornhuyse area and its connection to the air microbiome, especially air- et al., 2015). Interestingly, in addition to the joint fulfilment of borne pathogens (Vorholt, 2012). In our metagenomes, we found tasks, many organisms have ‘outsourced’ some essential func- a lower microbial diversity in the phyllosphere than in the rhizo- tions, including those of their own development, to symbiotic sphere, but the overall diversity was quite large and comprised organisms living with them (Gilbert et al., 2012). up to 900 species (Armin Erlacher and Gabriele Berg, personal The realization that microbial communities colonize virtu- communication). In general, leaves have different strategies to ally every host and have central roles in health and disease trigger microbial colonization, for example (antimicrobial) wax throughout the entire life cycle of the hosts has been a revo- layers, (antimicrobial) secondary metabolites, trichomes, and lutionary advance in biological sciences, also directing plant hairs, and the microbial composition seems to be highly indi- research towards a more holistic view. In addition to the dis- vidual but also plant-dependent. However, an overview of a covery of an immense diversity associated with hosts, research broader range of plant phyla is still missing. Recently, the major- will move from describing the composition of microbial com- ity of the research has been focused on the endosphere of plants. munities to elucidating the principles that govern their assem- Although endophytes were defined by De Bary in 1866 as ‘any bly, dynamics, and functions (Waldor et al., 2015). Here, we organisms occurring within plant tissues’, their existence was report old and new insights into the plant microbiome with ignored until the end of the last century, and very often these a particular emphasis on the progress in the field, which has organisms were considered