Annual Plant Reviews (2019) 2, 1–34 http://onlinelibrary.wiley.com doi: 10.1002/9781119312994.apr0699 PHYTOBIOMES, THE REASON WHY MICROBIOLOGISTS AND BOTANISTS SHOULD WORK TOGETHER Michelle Snoeijenbos1, Martha Cárdenas1,3, Marcela Guevara-Suarez1,3, Adriana Bernal1, Pedro Jiménez2 and Silvia Restrepo1,3 1Biological Sciences Department, Universidad de los Andes, Bogotá, Colombia 2Facultad de ciencias básicas y aplicadas, Universidad Militar Nueva Granada, Bogotá, Colombia 3Vicerrectoría de Investigaciones, Universidad de los Andes, Bogotá, Colombia Abstract: Phytobiomes consist of plants, their environment, and their associated communities of macro and microorganisms. Within the phytobiome, the micro- biome consists of the microorganisms associated with the plants comprising the endophytes and epiphytes. Endophytes are microorganisms, mainly consisting of bacteria and fungi, which colonise internal plant tissues without causing any dis- ease symptom or tissue damage in different hosts. All these organisms associated to the plant contribute to its fitness. In this article, we revised a total of 103stud- ies containing the terms phytobiomes and microbiomes and endophytes, with the aims of reviewing: (i) the evolution of the term endophyte and the evolution of the endophyte condition; (ii) the current literature on studies considering the endo- phyte community within phytobiomes; and (iii) the literature on the bacteria living in fungal endophytes. In our analyses, we highlighted the biases that have been introduced in the phytobiomes’ studies. For example, the presence of endohyphal bacteria could have contributed to overestimating the number of bacterial endo- phytes in the scientific literature. This is the first article that includes studies that evaluate endophytes that are not considered alone but belonging to a complex interacting community, the phytobiome. Keywords: endohyphal bacteria, endophyte, microbiome, phytobiome Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 1 M Snoeijenbos et al. 1 Introduction: Plants Are Not Alone: A Continuum of Interactions That Define the Plant Phenotype Most endophytes, mainly bacteria and fungi, colonise internal plant tissues without causing disease symptoms or tissue damage to their hosts (Schulz and Boyle, 2005; Kogel et al., 2006). However, in one of the most influential reviews on endophytes, Schulz and Boyle showed that there is a continuum of phenotypes shown by endophytes within a plant (Schulz and Boyle, 2005). The microorganisms, for their whole – or nearly whole – life cycle can inconspicuously colonise tissues of healthy plants. There are several reports showing that this interaction can be mutualistic and, among other benefits, endophytes are able to protect plants against pathogens (Siegel et al., 1987; Stone et al., 2000). Furthermore, endophytes can also cause disease (Jumpponen, 2001; Sieber, 2002; Schulz and Boyle, 2005). There are multiple studies on endophytes but, to the best of our knowl- edge, there are no reviews published on the study of endophytes when the whole microbial community, the microbiome, or the whole environment and all micro- and macro-organisms living in, on, or around the plant, the phy- tobiome are considered. Prior to 2001, the term microbiome was also in use, mostly to infer a very small ecological niche incorporating plant and animal life. Nowadays, the microbiome is the collection of microorganisms that live on and within the tissues of plants. The microbiome is an important part of the phytobiome, which includes all organisms that could be in association with a given plant. The portion of the microbial community living inside the plant is known as the endophyte community. The phytobiome is now seen as an important partner of the plant, necessary to maintain homeostasis and general health of the plant itself. Thus, for the main sections of this article, we decided to focus on those studies that aimed to characterise the phytobiomes as a whole, but our analyses focused on the endophytic part of the microbiome. A total of 103 research articles on endophytes within plant microbiomes, published in English language peer-reviewed research journals, were located in the litera- ture during September 2018 (the 103 references appear in the section ‘Further Reading’). The articles were found in the online SCOPUS database using the keyword phrases ‘Phytobiome AND Plant microbiome AND Endophytic community’. Our aim in this article is threefold: (i) to very shortly revise the evolution of the term endophyte and the evolution of the endophyte condition, (ii) to review the current literature on studies considering the endophyte commu- nity within phytobiomes, and (iii) to review the literature on the bacteria living in fungal endophytes that are part of phytobiome communities. This last section will discuss important questions, pertinent to all studies con- ducted so far: (i) have we overestimated the plant bacterial endophytes when in fact they were endohyphal bacteria (EHB) of plant endophytes? (ii) Do Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 2 Phytobiomes these EHB influence the behaviour of the endophytes and the production of their secondary metabolites? 2 Endophyte Definition and Evolution of the Endophytic Lifestyle The term ‘endophyte’ first appeared in scientific publications in the works of the German botanist Anton de Bary during the nineteenth century (de Bary, 1866), and the first isolation of one of these organisms was performed in1904 from Lolium temulentum (Freeman, 1904). Since then, this word has become part of the key terminology in the fields of mycology, phytopathology, and botany but its meaning has changed, adapted and been debated extensively in the last 100 years. De Bary (1866) defined endophytes as ‘organisms occur- ring within plants’ during a time where it was generally believed that healthy and normal growing plants were sterile. Hence, for a long time, the term endophyte was directly related to pathogenicity. Then, along with studies proving the endophytic presence of beneficial or commensal microorganisms in plants (Schneider, 1894), and other studies showing that these organisms could inhabit different plant locations at different phases in their life cycles (Ibáñez et al., 2017), came a redefining of the term. The word began totake on a meaning of plant–microorganism relationship without any pathogenic- ity. More recently, many researchers have defined endophytes as organisms that inhabit plant organs and internal tissues at some point in their life cycle without causing any apparent harm to the plant (Petrini, 1991; Wilson, 1995). Before the development of molecular methods for identification of microor- ganisms, the term ‘endophyte’ referred only to organisms that could be iso- lated and cultivated. Currently, it is widely accepted that fungi and bacteria can be identified without cultivating them and this was duly included in the definition (Rossmann et al., 2017). Although algae, protozoa, amoebas, archaea, and other organisms can infect plant tissues (Trémouillaux-Guiller et al., 2002; Müller and Döring, 2009), the word is mostly used to refer to fungi and bacteria. Since de Bary’s time, so much has been discovered about endophytes that it has been impossible to maintain an exact and stable definition of the word. They are extremely flexible organisms with broad lifestyles. They can becom- mensal, mutualistic, beneficial, or pathogenic and can switch between these lifestyles depending on the phase of their life cycle, the host they are infecting or the environmental conditions in which they are growing (Hardoim et al., 2015), and thus they are considered to represent a continuum of lifestyles (Schulz and Boyle, 2005). As a result of this, the initially strict definition of the term has changed over time, depending on author and research topic, and it will probably continue to change with technological advances. Molecular and Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 3 M Snoeijenbos et al. genomic approaches to study microorganisms’ lifestyle have redefined the traditional concept of endophytic lifestyle. Alternatives to the traditional def- inition have been proposed, briefly, endophytic microorganisms that have a negative effect on host fitness are named pathogens and those that have a neu- tral or positive effect are considered classical endophytes (Partida-Martinez and Heil, 2011). Currently, researchers are questioning the definition of the word and recommend limiting it to a simple description of habitat, without considerations of pathogenicity or function in plant tissues (Hardoim et al. 2015). 3 Endophytic Lifestyles The most widely accepted hypothesis to explain the occurrence of the endo- phytic lifestyle states that it emerged from a pathogenic ancestor (Schardl and Clay, 1997). This would allow the pathogen to switch to a commensal or a mutualistic lifestyle (Newton et al., 2010; Schulz and Boyle, 2005; Xu et al., 2014). To support this, a previous study showed that a single base mutant in the genome of the plant pathogen Colletotrichum magna caused it to remain inside the plant tissues without causing any symptom (Freeman and Rodriguez, 1993). Nowadays, comparative genomics studies have added evi- dence to that obtained by traditional