"Phytobiomes, the Reason Why Microbiologists and Botanists

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 microbiome 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 disease 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 endophyte 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 endophytes 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

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 knowledge, 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 phytobiome 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 literature 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 community 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 conducted 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 occurring 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 pathogenicity. 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 microorganisms, the term ‘endophyte’ referred only to organisms that could be isolated 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 definition 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 endophytic 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 evidence to that obtained by traditional approaches of isolation, and endophytes belonging to genera that had been reported as plant pathogens have been described. A comparative genomic analysis among members of the genus Magnaporthe, pathogens of rice, and a phylogenetically related endophyte, Harpophora oryzae, revealed that the endophyte emerged from a pathogenic ancestor (Xu et al., 2014). Extensive genomic analyses indicated that the gain or loss of orphan genes, DNA duplications, gene family expansions, and the frequent translocation of transposon-like elements have been the drivers in the evolution of the endophyte from a pathogenic ancestor (Xu et al., 2014). Even today with all the genomic data available, no genomic signatures of the endophytic lifestyle have been identified. Recently, Knapp et al. (2018) sequenced two dark septate endophytes (DSE) Ascomycota from semiarid areas. When compared to other Ascomycota with different lifestyles, the DSE genomes did not show common properties that could be attributed to an endophytic lifestyle and, instead, a low level of convergence was observed in their genomic content (Knapp et al., 2018). Nonetheless, genome comparative analyses have shown that endophyte isolates are distinct from its congeneric or conspecific partners with different lifestyles, pathogens or saprotrophs. This is the case of Staphylococcus epidermidis, a bacterium that has a historic association with humans. However, S. epidermidis has also been related with rodents and with plants, especially as rice endophytic seeds or the so-called RESE (rice endophytic Staphylococcus

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 4 Phytobiomes epidermidis). Comparative genome analysis of S. epidermidis from different origins revealed that the RESE isolates had a set of unique genes related to recombination, replication, and repair (Chaudhry and Patil, 2016). These processes are involved in the response and tolerance to reactive oxygen species (ROS), an important plant defence mechanism. Hence, this set of genes was proposed as involved in the adaptations of RESE to the endophytic lifestyle.

4 The Endophytic Community Within the Phytobiome

In the following four sections, we will review the literature that contains the knowledge gathered on endophytes in the last years. We first introduced the general idea of each section by reviewing the most cited publications on endophytes (referenced in each section) and then, we focused on the retrieved 103 studies that we found in SCOPUS database using the above-mentioned keywords.

4.1 The Taxonomic Diversity of Endophytes Plant microbiomes harbour a variety of microbial organisms including archaea, protists, fungi, and bacteria; but in this article, we will focus solely on bacteria and fungi. Even though it has been shown that protists and archaea have important interactions and metabolite production within plants, the lack of knowledge about them can be attributed to the difficulty in cultivating them, the reduced amount of taxonomic and functional information available from databases and, in the case of archaea, the fact that not many pathogens of this taxon are known (Taffner et al., 2018; de Araujo et al. 2018). Most articles that study plant microbiomes focus on sequencing fungi and bacteria with the objective of discovering the interactions among them and with the plant and the natural products they synthesise. Within the 103 articles, we found 44% focused on sequencing and identifying only bacteria (46 articles), whereas 33% were limited to fungi (34 articles). The rest of the articles (23) looked for and classified both organisms in the plant. Fungi living within the plant endosphere are in constant interaction with other microorganisms. Fungi and bacteria can live in competition for resources and space, be antagonistic with each other, or they can have no effect on each other. Studies have also found that members of these two different kingdoms have the ability to work together to circumvent plant defences (Pozo and Azcón-Aguilar, 2007) or synergistically promote plant growth (Johansson et al., 2004). When researchers began to study endophytes, the research would normally target one specific taxon of organisms, but with an increment in knowledge and methods came the study of complete microbiomes and taxon interactions, mainly focusing on

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 5 M Snoeijenbos et al. mycorrhizal interactions. Within our reviewed literature, only two of the 23 articles that studied both bacteria and fungi were published before 2015, which shows that interest in full microbiomes and interactions between bacterial and fungal endophytes is a recent issue. The diversity of fungal endophytes that can be found within a single plant is tremendous, considering that they can enter through various independent infections and hyphal growth within the plant can be extensive (Rodriguez et al., 2009). Fungal communities in plants are even harder to study due to the complicated taxonomy of these organisms that has historically been revised, reviewed, and restructured many times (Hibbett et al., 2007). A few decades ago issues in fungal taxonomy were the result of the different fungal life cycles and morphologies as well as the different nomenclature for sexual and asexual stages of the same fungi (Guarro et al., 1999). Nowadays, changes have been proposed based on ambiguities that arise among classifications of cultured fungi, phylogenetic relationships, or direct sequencing methods mainly using the internal transcribed spacer (ITS) of ribosomal RNA. As a result, there are many different fungal trees of life as well as databases with dissimilar classifications (Hibbett et al., 2016). Zygomycota was an accepted fungal phylum for many years until the monophyletic nature of the group was challenged. In a comprehensive restructuring of fungal classifications in 2007, the organisms that belonged to Zygomycota were distributed into subphyla Mucoromycotina, Ento- mophthoromycotina, Kickxellomycotina, and Zoopagomycotina (Hibbett et al., 2007). Two phyla are currently recognised, namely Mucoromycota (Subphyla: Mucoromycotina, Glomeromycotina, and Mortierellomycotina) and Zoopagomycota (Subphyla: Zoopagomycotina, Kickcellomycotina, and Entomophthoromycotina) (Spatafora et al., 2016). Nonetheless, we found

Fungi

Blastocladiomycota

Ex-phylum ‘Glomeromycota’

Chytridiomycota

Phylum Ex-phylum ‘Zygomycota’

Basidiomycota

Ascomycota

0 102030405060 No. of studies

Figure 1 Fungal phyla found in 57 studies on plant microbiomes.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 6 Phytobiomes in our revision eight recently published studies that still use Zygomycota as a taxon at the phylum level. This demonstrates how difficult it is for researchers to adapt to taxonomic restructuring. Fungi belonging to the phylum Ascomycota were found in 53 articles that studied fungi (57 studies), in all plants studied and in every location sampled (Figure 1). This is the biggest and most widespread phylum of fungi with an extensive range of lifestyles and metabolic pathways. In second place, we found members of the phylum Basidiomycota, being detected in 32 studies (Figure 1) and they also corresponded to the second phylum in species numbers. Basidiomycota do not have the variability in life cycles and metabolites present in Ascomycota and they are less frequently found as plant pathogens or mutualists. At the genus level, Alternaria was found in most of the articles, which is in line with general endophyte research because this is a group of fungi that includes many plant pathogens, saprobes, and mutualists (Table 1). It is a genus of fungi responsible for a significant percentage of agricultural yield losses due to disease. For example, Alternaria brassicae is a pathogen that causes blight and has been found in every continent of the world, while plant resistance to this pathogen is not common and it is hard to control as a pest (Meena et al., 2016). Alternaria is also often found as a beneficial organism for plants; for example, it has a known role in locoweed plants as swainsonine producer, a compound toxic to grazing mammals and thus a way to avoid herbivory (Harrison et al., 2018). These varied relationships show that this genus of approximately 280 species (Woudenberg et al., 2013) has many different mechanisms to colonise and survive in plants as communities. The second most commonly detected fungal genus was Phoma. This group is not as large as Alternaria, but it also includes several plant pathogens, as well as plant growth promoting fungi. These are coelomycetous fungi often found in soil prone to root colonisation. In fact, in some cases, their growth-promoting activity is due to the competitive blocking of root infection sites impeding the access of pathogens (Shivanna et al., 1996). Aspergillus, Cladosporium, Fusarium,andPenicillium were each mentioned in around 17% of the articles that mentioned Fungi, all corresponding to commonly found and widely studied fungi (Table 1). When exploring higher taxonomic levels such as phyla, the bacterial diversity between plants or environments is similar, but when considering family or genus it becomes clear that different bacteria are adapted to colonise and survive in different plants, thanks to specific metabolic and structural adaptations. It is also important to consider that endophytic bacteria at these taxonomic levels have specialised strategies for using the carbon sources of specific hosts and circumventing their defences. Owing to characteristics such as mutation frequency, gene transfer, and genome size, the taxonomic classification of bacteria (and microorganisms in general) is complicated and con- stantly changing. Recently, a complete restructuring of the bacterial tree of life

Table 1 List of fungal genera found in our revision of 103 studies and identiﬁed in at least two studies.

Fungal genus No. of studies

Alternaria 21 Phoma 11 Fusarium 10 Cladosporium 10 Penicillium 9 Aspergillus 9 Colletotrichum 8 Phaeosphaeria 6 Diaporthe 6 Aureobasidium 6 Xylaria 5 Lewia 5 Epicoccum 5 Cryptococcus 5 Chaetomium 5 Preussia 4 Pleospora 4 Phomopsis 4 Microdochium 4 Leptosphaeria 4 Trichosporon 3 Trichoderma 3 Podospora 3 Paraconiothyrium 3 Nigrospora 3 Mycosphaerella 3 Mortierella 3 Guignardia 3 Gibberella 3 Curvularia 3 Cochliobolus 3 Cladophialophora 3 Chalara 3 Acremonium 3 Zygosaccharomyces 2 Venturia 2 Ulocladium 2 Talaromyces 2 Sporormiella 2 Setosphaeria 2 Saccharomyces 2 Rhodotorula 2 Pyrenophora 2 Phyllosticta 2 Phialophora 2

Table 1 (Continued)

Fungal genus No. of studies

Pestalotiopsis 2 Neurospora 2 Microdiplodia 2 Melampsora 2 Lophiostoma 2 Ilyonectria 2 Hormonema 2 Emericella 2 Embellisia 2 Drechslera 2 Cytospora 2 Coccomyces 2 Cadophora 2 Bipolaris 2

was proposed by Parks et al. (2018) in which phylogeny was inferred based on the concatenation of 120 ubiquitous single copy marker genes (Parks et al., 2018). This newly proposed tree differs from the current one, which is based on small subunit (SSU) of ribosomal RNA genes and has been proven to have inconsistencies and errors (Hug et al., 2016). In this article, however, we will base our results on previous taxonomies, since most articles reviewed used 16S rRNA to identify the bacterial endophytic communities. The most common phylum of bacteria found in the 69 articles revised that identified bacteria was Proteobacteria. A total of 56 of the articles foundmem- bers of this phylum in their studied plant, and in most papers, they corresponded to the most abundant phylum (Figure 2). Proteobacteria is one of the largest and most diverse groups of bacteria and one of the most studied phyla. It includes many beneficial nitrogen-fixing bacteria as well as pathogenic bacteria (Santoyo et al., 2016). Other phyla that were frequently found in the studies that mention bacteria were Actinobacteria (62%), Firmicutes (45%), Bacteroidetes (33%), and Acidobacteria (32%). These are the phyla with the highest number of identified species relevant to human health, agriculture, and industrial processes. Their abundance coupled with the fact that their sequences are highly reported and easily found in microbial databases could explain their prevalence in the results of many articles focusing on microbiomes (Hassani et al., 2018) (Figure 2). Of the 69 articles that identified bacteria, 44 of them managed to identify them down to the genus level (Table 2). Pseudomonas was the most commonly detected genus, appearing in approximately one-third of the 69 studies. The genus Pseudomonas is a large group of diverse bacteria in which it is easy to find plant growth promoting bacteria as well as pathogenic bacteria

Bacteria Saccharibacteria Spirochaete Tenericutes Nitrospira Gemmatimonadetes Fusobacteria Planctomycetes Deinococcus-Thermus Chloroflexi Phylum Cyanobacteria Verrucomicrobia Acidobacteria Bacteroidetes Firmicutes Actinobacteria Proteobacteria 0 102030405060 No. of studies

Figure 2 Bacterial phyla found in 69 studies on plant microbiomes. with similar ecological characteristics. Possibly, plants need to develop special mechanisms to recognise different Pseudomonas to either defend against or develop a symbiotic relationship with. The diversity of species within Pseudomonas also gives them the capacity to colonise leaf surfaces, rhizosphere, and intercellular spaces in leaves and roots (Preston, 2004). The second most commonly found genus was Bacillus, present in 20% of the reviewed literature that cited bacteria (69 studies). This is a genus of growth-promoting and pathogen-attacking bacteria that have the ability to produce plant hormones such as auxins (Ahmad et al., 2008). Therefore, Bacillus is deeply studied as a potential biological control for agricultural applications. Both Bacillus and Pseudomonas have species that are model organisms in scientific researchBacillus ( subtilis and Pseudomonas aeruginosa). These bacteria have proven easy to cultivate and therefore both bacterial genera have been extensively researched and sequenced. The genus Rhizobium was found in 15% of the 69 papers in which bacteria are mentioned (Table 2) and its abundance can be explained by the fact that it includes bacteria that form nitrogen-fixing symbiosis with legumes and other plants, creating nodules in the roots (Ahmad et al., 2008). Most articles reported a definite enrichment of Rhizobium in the root samples (Gourion et al., 2015). Other genera found in at least 11% of the 69 articles, in which bacteria are studied, were Burkholderia, Methylobacterium, Sphingomonas, Staphy- lococcus, and Xanthomonas. They belong to either Proteobacteria or Firmicutes

Table 2 List of bacterial genera found in our revision of 103 studies and identiﬁed in at least two studies.

Bacterial genus No. of studies

Pseudomonas 21 Bacillus 14 Rhizobium 10 Methylobacterium 9 Sphingomonas 9 Staphylococcus 8 Burkholderia 8 Xanthomonas 7 Erwinia 7 Streptomyces 6 Pantoea 6 Flavobacterium 6 Corynebacterium 6 Ralstonia 5 Enterobacter 5 Rhodococcus 4 Escherichia 4 Devosia 4 Paracoccus 4 Caulobacter 4 Niastella 4 Klebsiella 4 Dyella 4 Pedobacter 4 Serratia 4 Sphingobacterium 4 Deinococcus 3 Nocardioides 3 Agrobacterium 3 Acinetobacter 3 Sphingobium 3 Stenotrophomonas 3 Gluconacetobacter 3 Microbacterium 3 Micrococcus 3 Clavibacter 3 Streptococcus 3 Massilia 2 Acidovorax 2 Phenylobacterium 2 Lactococcus 2 Pectobacterium 2 Glomeribacter 2 Spirochaeta 2

Table 2 (Continued)

Bacterial genus No. of studies

Terrimonas 2 Pseudonocardia 2 Rubellimicrobium 2 Brevundimonas 2 Pandoraea 2 Salmonella 2 Mesorhizobium 2 Curtobacterium 2 Lactobacillus 2 Acetobacter 2 Bradyrhizobium 2 Pseudoxanthomonas 2 Achromobacter 2 Agrococcus 2 Herbaspirillum 2 Leptothrix 2

and are abundant bacteria in different environments, but most specifically, in plants. Table 2 and Figure 2 show the abundance of bacteria at both taxonomic levels (phylum and genus) in a more detailed way. When thinking about an explanation for these results, it is important to keep some things in mind. For example, Methylobacterium, Sphingomonas,andXanthomonas have been shown to be common contaminants of DNA extraction kits and could, therefore, unfairly enrich the sequencing results (Salter et al., 2014). Another factor to be aware of is that sequences identified as Cyanobacteria could be derived from plant chloroplast DNA since these organelles evolved from this group of bacteria. Because many plant microbiome studies extract DNA from entire plant tissues, it is often difficult to distinguish sequences from cyanobacteria and those from chloroplasts (Bengtsson-Palme et al., 2015).

4.2 The Thin Line Between Pathogens and Nonpathogens As discussed above, it is thought that the endophytic lifestyle evolved from a pathogen ancestor and that this would allow the pathogen to switch to a commensal or a mutualistic lifestyle (Newton et al., 2010). However, there are multiple reports of endophytes switching from their mutualistic, or commensal, lifestyle to a pathogenic behaviour. What factors induce an endophyte to switch to a destructive mode? Several studies have addressed this issue. Most of the time, parasitism is observed when an imbalance occurs in the plant resulting in what we have known for decades as a compatible interaction that leads to disease. In other

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 12 Phytobiomes words, if the endophyte is not perceived, it is because the defence responses are balanced (Kogel et al., 2006). The switch from mutualism to parasitism can depend on a single mutation of a gene that originates this imbalance. This is the case in a fungus associated to grass, Epichloë festucae, in which a single change in a fungal gene involved in the ROS metabolism induced the parasitic behaviour (Tanaka et al., 2006). In plant pathology, it is known that the oxidative burst plays a very important role in disease resistance, and the production of reactive species of oxygen is a characteristic early feature of the hypersensitive response that ensues perception of the pathogen signals (Lamb and Dixon, 1997). In a more general line, endophytes can become pathogens in stressful (artificial) conditions for the plant (Junker et al., 2012). In a simple experiment, some endophytes isolated from Arabidopsis thaliana induced symptoms when re-inoculated under artificial conditions (Junker et al. 2012). It has also been shown that a stressful situation for the microorganism also results in parasitism. Chemical, physical, or biotic conditions in a mycorrhizal association, in particular, the nutrient status of the soil, can cause that net costs exceed net benefits of this association. In these circumstances, the fungus behaves as a parasite (Johnson et al., 1997). These results reinforce the idea of the fine balance between the virulence of an endophyte and the defence of the host, and how parasitism arises when the host fitness is reduced (Redman et al., 2001). For all these reasons, Kloepper et al. (1992) included in their definition of endophytes all the microorganisms found inside roots, specifically the cor- tical colonists or vascular colonists, where we can find pathogens and latent pathogens (James and Olivares, 1998). In our literature revision of phytobiomes, retrieving only the microorganisms that were identified to the species level, we found several pathogenic species of bacteria and fungi. While only 26 of the articles analysed managed to identify fungi or bacteria to the species level, a total of 355 species were identified, of which 115 have been registered as phytopathogenic: 83 fungal species and 12 bacterial species (the same species can be registered several times in different studies). These results support the idea that latent pathogens live in the endosphere of plants. In the case of fungi, very well-known pathogenic species were identified: Fusarium oxysporum, Fusarium solani (currently Neocosmospora solani), and Magnaporthe oryzae. Several species of the genus Colletotrichum were also found: C. acutatum, C. gloeosporioides, C. lineola, C. linicola, C. phormi,andC. tropicale; as well as several species of the genus Alternaria: A. alternata, A. arborescens, A. brassicae, A. oregonensis, A. tenuissima, and A. triticina. In the case of bacteria, the pathogenic species were as follows: Achromobacter marplantensis, Bacillus pumilus (reported several times), Curtobacterium flaccumfaciens, Ensifer adherens, Mesorhizobium huakii, Pantoea agglomerans, Pantoea ananatis, Pseu- domonas graminis, Pseudomonas mucosa, Pseudomonas syringae, Rhodococcus fascians, and Xanthomonas translucens.

4.3 Geographic Distribution of Studies on Phytobiomes As is the case for animals and plants, endophytes are expected to show a higher diversity in the tropics than in temperate or boreal zones (Zimmerman and Vitousek, 2012). It is worth mentioning that the global distribution of endophytes has been traditionally determined by culture-dependent approaches, probably introducing biases to the taxonomic descriptions and diversity estimations. With this methodological approach, for the kingdom Fungi, an increase in the frequency of asymptomatic plant tissue colonisation and in endophytic species richness has been shown at lower latitudes (Arnold et al., 2000; Arnold and Lutzoni, 2007; Roy and Banerjee 2018). However, a higher diversity of fungal endophytes in the tropics has been confirmed, only recently, using next-generation sequencing (NGS) (Zimmerman and Vitousek, 2012). When considering a single host, endophytic communities have shown to differ among geographic localities (Herrera et al. 2010; Christian et al., 2016). The structure of the endophytic community of a particular host is shaped by abiotic environmental factors, which define a locality and that also affect the host development (Hoffman and Arnold, 2008). These factors include UV radiation, rainfall, temperature, altitude, macronutrient distribution (Müller et al., 2016), and climate (U’ren et al. 2012). A culture-independent approach to characterise the fungal endophytic community of a tree (Metrosideros poly- morpha) in an altitude range in Hawaii, revealed that the elevation and the rainfall have determined the endophytic community composition in the trees (Zimmerman and Vitousek, 2012). In addition, the variation of endophytes from the same host among sites could also be explained by their transmission mode. Fungal and bacterial endophytes are mainly transmitted in a horizontal way, from plant to plant of the same generation, allowing the local microbiota to affect the structure of the endophytic community (Christian et al., 2016). There are clear biases in the countries where the study was published and sampling sites around the world when it comes to studies on microbiomes, some countries are more represented than others. These trends cannot nec- essarily be attributed to the biogeographical distribution of the bacteria and fungi studied. As opposed to macroorganisms, it is difficult to determine the geographic scales at which microorganisms, such as bacteria and fungi oper- ate, especially when they are susceptible to a variety of interactions with other endophytic organisms. Although there have been studies proving that microbial communities are affected by geographic location (Wallace et al., 2018; Zimmerman and Vitousek, 2012; Tahtamouni et al. 2016), their high dispersal capacity and ability to survive, in a variety of environments and hosts, implies that local biotic interactions are very important (Harrison et al., 2018). This suggests that, besides the often-biased distribution of sampling, there are other driving forces determining endophytes’ geographical distribution.

The literature reviewed for this article reveals that the plant sampling sites for 103 articles on microbiomes are not evenly distributed around the world. As observed in Figure 3, the United States dominates with roughly 33% of the sites studied, for what may seem like obvious reasons. A total of 25% of sampling sites are in Europe, followed by China (8%) and Canada (6%). Sampling sites are very often the same as the publication country, and these most common sampling sites correspond to where more publications are pro- duced, according to the UNESCO Science Report on total scientific publications worldwide (UNESCO, 2017). The resources available in first world countries support a literature productivity and potential of studying plant microbiomes. Cultural practices, such as the high use of medicinal plants in Chinese medicine, have also driven interest and funding on endophyte research (Chen et al., 2018). It is also clear, however, that there is a large information gap in Africa, where only two of the reviewed articles studied the microbial community of plants, maize and banana in South Africa and Uganda, respectively (Mashiane et al., 2017; Köberl et al., 2016). Worldwide, publications that come out of Africa account for less than 1% of all scientific output, and reports have shown that, within these publications, there is an overrepre- sentation of medical science as a topic (Uthman et al., 2015). This indicates a huge amount of undiscovered microbial plant communities, considering the variety of endemic (and medicinal) plants that are found in Africa. A review that studied 139 papers on endophytic fungi in Africa showed that in the last two decades research has increased in this field mostly geared towards bioprospecting, but remains a continent with tremendous opportunities for discovery (Sibanda et al., 2018). When we consider the fact that 20% of the world’s plant species are found in Brazil, it should be a country with a higher proportion of publications on endophytic research. In the literature revised, Brazilian plants were sampled in only 5 of the 111 sampling sites used in all the 103 publications (Figure 3). In general, South America also seems to be a region with a lot of potential new species or natural products. There is still a lot of research left to do in the field, and it might be worthwhile to take a look at these underrepresented regions of the world for new insights into microbiome communities, their interactions and their natural products and metabolites.

4.4 The Plants and Their Speciﬁc Tissues Hosting Endophytes Several studies have examined the factors affecting the composition of endophytic communities including fertilisation schemes, the cultural practices, and the environment. However, relatively fewer studies have assessed whether the endophytes prefer a tissue or organ of the plant as their niche and the location where endophytes penetrate their hosts. There is evidence that endophytic bacteria, penetrate plants at lateral root junctions, most

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 15 M Snoeijenbos et al. 37 Distribution of a total of 111 sampling sites around the world in 103 studies on endophyte microbiomes. 1 Figure 3

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 16 Phytobiomes likely at natural cracks but although the openings are natural, the process of entry may be active given that cell-wall degrading enzymes (CWDEs) are expressed (James and Olivares 1998; Turner et al., 2013). Some passive ways of entry include natural openings in roots and aerial parts, or by vegetative propagation (Turner et al., 2013); endophytes are inside cuttings or other vegetative material used for propagation. Endophytic bacteria that enter through natural openings in the roots then move to the apoplast, where they then move on to the aerial parts (Bodenhausen et al., 2013; Afzal, et al., 2014; Beattie and Lindow, 1999). We and others have shown the presence of shared taxa across plant tiers; the upper tier refers to the young leaves, the middle tier (midtier) is composed of fully mature leaves, and the necromass tier is composed of senescent leaves (Ruiz-Pérez et al., 2016), suggesting that the endophytic communities are interconnected and that bacteria can travel from the rhizosphere towards the leaves and vice versa, as reported (Bodenhausen et al., 2013; Afzal, et al., 2014; Beattie and Lindow, 1999). However, the movement of bacteria from roots to shoots is sometimes selective, probably limited to bacteria that can express CWDEs or a Type-3 secretion system (Compant et al., 2010; James et al. 2002; Monteiro et al. 2012). Several studies have shown that the numbers of bacteria and their diversity are higher in roots than in shoots (Monteiro et al. 2012; Sarria-Guzmán et al., 2016). This observation could be due to other factors, such as the different conditions that the microorganisms encounter in the different organs (i.e. roots versus shoots) but could also be explained by the selective movement of bacteria through the plant. From all these results, the movement of bacterial endophytes within the plants is still a subject to be explored in future studies. The leaf endophytic communities can thus be formed by the endophytes moving through the vascular stream from other organs of the plant but also by the epiphytic microorganisms present on leaf surfaces that arrived there via water splashes or air dispersal and subsequently entered host tissues. Two lines of evidence support that leaf endophytes can enter the leaf directly. First, as we will show in the following paragraphs root and leaf endophytes can be significantly different in some plants (although we have shown the contrary, Ruiz-Pérez et al., 2016). Second, the epiphytic and endophytic communities of leaves can be very similar. In a previous study performed in our laboratory, we showed that epiphytic and endophytic community structures of the Páramo plant genus Espeletia were very similar in taxonomy, they shared most operational taxonomic units (OTUs), and were no different in richness or diversity indices (Ruiz-Pérez et al., 2016). Certainly, one important issue when studying endophytes is their colonisation strategies, particularly their entry point, but a second one is their pos- sible preference for a host organ or tissue. Plant tissues provide different biological niches for the endophytes due to their composition and function. Hence, differences in number and composition among the microorganism

Fruits Shoots 4% Flowers 4% 3%

Branches 5% Leaves 35% Seeds 9%

Stem 12%

Roots 28%

Figure 4 Type of plant tissues studied in 103 studies.

communities living inside plants are expected. Roots, for example, are the photosynthetic sinks and therefore, one of the preferred sites for establishment of the endophytes. Leaves, on the contrary, are more exposed to stressful conditions such as UV radiation, water, and nutrient limitations, which could explain the lower numbers of endophytes and their taxonomic differences. One study showed that bacterial endophytic communities differed in number, being more abundant in roots for all phyla except the Firmicutes (Robinson et al., 2016). Interestingly, the composition was also tissue specific, with Proteobacteria being more prevalent in roots and Firmicutes and Acti- nobacteria, the Gram-positive phyla, more prevalent in the leaves (Robinson et al., 2016). In our literature revision of phytobiomes, the most common tissues sampled to study the microorganism communities were leaves (35%), roots (28%), and stems (12%) (Figure 4). The taxonomy of plants used showed a bias to cer- tain divisions and classes (Table 3). Only five plant divisions were found, and the most represented divisions were Magnoliophyta (the flowering plants), followed by Pinophyta (the conifers).

Table 3 Distribution of 199 plant species studied in the 103 studies according to class and division.

No. of studies

Plant Division Magnoliophyta 160 Coniferophyta (Pinophyta) 13 Bryophyta 4 Lycopodiophyta 2 Pteridophyta 1 Plant Class Magnoliopsida 114 Liliopsida 43 Coniferae 17 Pinopsida 13 Sphagnopsida 4

5 Endohyphal Bacteria or Bacteria Within Fungal Hyphae: and Overestimation of Endophytic Bacteria?

Various interactions between fungi and bacteria have been documented, some of these interactions are related to the mycelosphere, the myceloplane, and within living structures such as hyphae (Araldi-Brondolo et al., 2017). Several studies have shown the presence of bacteria in fungal hyphae, mostly in filamentous endophytes (Hoffman and Arnold, 2010; Hoffman et al. 2013). Although bacteria were first reported in the 1970s to be present in arbuscular mycorrhizal fungi (Protsenko, 1975), the significance of these bacteria, and their role within the host have only just begun to be evaluated. EHB (endobacteria, endofungal bacteria, or bacterial endosymbionts) have been reported in different fungal taxonomic groups (including some mycorrhizae), such as phyla Ascomycota (mainly Pezizomycetes, Doth- ideomycetes, Eurotiomycetes, and Sordariomycetes), Basidiomycota, the former Glomeromycota, and Mucoromycotina (Arendt et al., 2016; Hoffman and Arnold, 2010). In addition, EHB have been found in a free-living state in soil and as endophytes of seeds and tissues such as roots and leaves, showing that the relationship between EHB and their fungal host is not an obligate one. EHB have been classified into three classes: class 1 EHB (referred to Mollicutes-related endobacteria), class 2 EHB (mostly members of Burkholde- riaceae family), and class 3 EHB (includes members of phylum Proteobacteria and Firmicutes). This classification was proposed according to host information, bacterial taxonomy, genomic data, and associated traits relevant to bacteria/fungi interactions and ecology (Araldi-Brondolo et al., 2017). Interestingly, several studies indicate that EHB could be tightly related to fungal processes such as sexual and asexual reproduction, induction

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 19 M Snoeijenbos et al. of secondary metabolite production, phytohormone production (e.g. indole-3-acetic acid), fermentation of carbohydrates, enzyme production, thermotolerance, virulence, and the influence of interactions between fungal endophytes and the host plants (Table 4) (Hoffman et al., 2013; Mondo et al., 2017; Shaffer et al. 2017, 2018). Some genera of the EHB have also been reported as endophytes, e.g. Burkholderia, Curtobacterium, Erwinia, Luteibacter, Pantoea, Paraburkholderia (Elbeltagy et al., 2000; Compant et al., 2005; Feng et al., 2006; da Silva et al., 2018). Although some studies have allowed an evaluation of the role of these bacteria as true endophytes using culture-dependent techniques, the following questions arise: Considering the existence of EHB in fungal endophytes, could the number of endophytic bacteria be overestimated in diversity studies that use the currently most common non-culture-dependent approach? What proportion of the endophytic bacterial diversity estimated by culture-independent approaches corresponds to EHB? Recently, Arnold et al. (2018) presented a patent that includes methods to affect phenotypic activity of endophytic fungi colonised by EHB. This patent reveals the importance of including studies with a culture-dependent approach for estimating bacterial endophytes in order to avoid overestimation. To our knowledge, to date, there are no studies that evaluate the presence of EHB in the plant microbiome. Therefore, we suggest including strategies to estimate and quantify EHB using NGS and culture-dependent techniques.

6 Conclusions and Perspectives

Endophytes are extremely flexible organisms with broad lifestyles that com- prise commensal, mutualistic, beneficial, or pathogenic states and can switch among these lifestyles depending on the phase of their life cycle, the host or the organ they are infecting or the environmental conditions in which they are growing. Many studies have been conducted to characterise endophytes but rarely have they been studied in the context of the microbiome or the phytobiome. Here, we only reviewed published documents that considered endophytes as part of the whole plant community. The main findings of our revision are the following: • More studies focus on endophytic bacteria than fungi. The ease of isolation combined with the advances in identification and the amount of data gathered in taxonomic databases make bacteria the preferred microorganism to be characterised and studied. • Within the kingdom Fungi, the most common phylum found within plants is Ascomycota and the most common genus is Alternaria, followed by Phoma.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 20 Phytobiomes Arnold et al. (2018) Hertweck (2005) Bianciotto et al. (2004) Arnold et al. (2018) Hoffman et al. (2013), Shaffer et al. (2017) Ruiz-Herrera et al. (2015) Arnold et al. (2018) Arnold et al. (2018) establishment production acid (IAA), fungal growth, enzymes production and asexual reproduction) available atmospheric nitrogen within fungal cells production production Growth promoterToxin production (rhizoxin) Sharma et al. (2008) Partida-Martínez and sp. Fungal growth, enzymes Luteibacter sp., sp. sp. Fungal growth, enzymes sp. Seed germination Shaffer et al. (2018) sp. Fermentation of carbohydrates Shaffer et al. (2017) Erwinia sp. Seed germination Shaffer et al. (2018) sp. Inﬂuence in sexuality (sexual sp. Fungal growth, enzymes Glomeribacter gigasporarum Hyphal growth and mycorrhizal sp. Production of indole-3-acetic sp. Seed germination Shaffer et al. (2018) sp., sp. The bacterium ﬁxes and makes Enterobacter Pantoea Chitinophaga Paraburkholderia Luteibacter Sphingomonas Burkholderia ) (formerly sp. sp. sp. sp. Endohyphal bacteria and their effect on fungi. sp. Gliocladiopsis Microdiplodia Neocosmospora keratoplastica Fusarium keratoplasticum Piriformospora indicaPestalotiopsis Rhizopus Rhizobium microspores radiobacter R. microsporesUstilago Burkholderia maydisXylaria cubensis Burkholderia rhizoxinica Bacillus Ralstonia Cladosporium Fusarium concolorGigaspora margarita Candidatus Streptococcus Table 4 FungusAlternaria Bacterium Fungal processes References

• The most common phylum of bacteria found in the 69 articles revised was Proteobacteria. Pseudomonas was the most commonly detected genus, appearing in at least one-third of the studies. The second most commonly found genus was Bacillus present in 20% of the reviewed literature. • In our literature revision of phytobiomes, we found several pathogenic species of bacteria and fungi among endophytes. In the 103 studies, 355 species were identified, of which 115 have been registered as phytopathogenic: 83 fungal species and 12 bacterial species (the same species can be registered several times in different studies). • The literature reviewed for this article reveals that the plant sampling sites for 103 articles on microbiomes are not evenly distributed around the world. The United States dominates with roughly 33% of the sites studied. A total of 25% of sampling sites are in Europe, followed by China (8%) and Canada (6%). The resources available in first world countries results in the highest amount of studies on plant microbiomes. • There is still a lot of research left to do in the field, and it might be worthwhile to take a look at the underrepresented regions of the world for new insights into microbiome communities, their interactions, and their natural products and metabolites. • The most common tissue used to study the microorganism communities were leaves (35%), roots (28%), and stems (12%). • The most represented plant divisions studied were Magnoliophyta (the flowering plants), followed by Pinophyta (the conifers). In the last section of this article, we focused on the EHB, bacteria living with the mycelia of fungi. The research on these bacteria is recent but our aim was to highlight the importance of these microorganisms as they can modulate the behaviour of the fungi, switch their lifestyles and have an impact on the plant fitness and therefore its yield.

Acknowledgements

We would like to thank Timothy Tranbarger who invited us to write this article.

Bacterial Pathogenicity Fungal Pathogenicity—Establishing Infection Plant–Microbe Interactions and Secondary Metabolites with Antibacterial, Antifungal and Antiviral Properties Microbial Communities in the Phyllosphere Filamentous Fungi on Plant Surfaces

References

Afzal, M., Khan, Q.M., and Sessitsch, A. (2014). Endophytic bacteria: prospects and applications for the phytoremediation of organic pollutants. Chemosphere 117: 232–242. Ahmad, I., Pichtel, J., and Hayat, H. ed. (2008). Plant-Bacteria Interactions Strategies and Techniques to Promote Plant Growth. Weinheim: Wiley. Araldi-Brondolo, S.J., Spraker, J., Shaffer, J.P. et al. (2017). Bacterial endosymbionts: master modulators of fungal phenotypes. Microbiology Spectrum 5 (5): FUNK-0056-2016. Arendt, K.R., Hockett, K.L., Araldi-Brondolo, S.J. et al. (2016). Isolation of endohyphal bacteria from foliar Ascomycota and in vitro establishment of their symbiotic associations. Applied and Environmental Microbiology 82: 2943–2949. Arnold, A.E., Maynard, Z., Gilbert, G.S. et al. (2000). Are tropical fungal endophytes hyperdiverse? Ecology Letters 3 (4): 267–274. Arnold, A.E. and Lutzoni, F. (2007). Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots? Ecology 88 (3): 541–549. Arnold, A.E., Arendt, K., and Baltrus, D.A. (2018). Method for affecting phenotypic activity of endophytic fungi. U.S. Patent Application No. 15/952,104. Bianciotto, V., Genre, A., Jargeat, P. et al. (2004). Vertical transmission of endobacteria in the arbuscular mycorrhizal fungus Gigaspora margarita through generation of vegetative spores. Appl. Environ. Microbiol. 70 (6): 3600–3608. Beattie, G.A. and Lindow, S.E. (1999). Bacterial colonization of leaves: a spectrum of strategies. Phytopathology 89 (5): 353–359. Bengtsson-Palme, J., Hartmann, M., Eriksson, K.M. et al. (2015). METAXA2: improved identification and taxonomic classification of small and large subunit rRNAin metagenomic data. Molecular Ecology Resources 15 (6): 1403–1414. Bodenhausen, N., Horton, M.W., and Bergelson, J. (2013). Bacterial communities associated with the leaves and the roots of Arabidopsis thaliana. PLoS One 8 (2): e56329. Chaudhry, V. and Patil, P.B. (2016). Genomic investigation reveals evolution and lifestyle adaptation of endophytic Staphylococcus epidermidis. Scientific Reports 6: 19263. doi: 10.1038/srep19263. Chen, H., Wu, H., Yan, B. et al. (2018). Core microbiome of medicinal plant Salvia miltiorrhiza seed: a rich reservoir of beneficial microbes for secondary metabolism? International Journal of Molecular Sciences 19 (3): 672. Christian, N., Sullivan, C., Visser, N.D. et al. (2016). Plant host and geographic location drive endophyte community composition in the face of perturbation. Microbial Ecology 72 (3): 621–632. Compant, S., Reiter, B., Sessitsch, A. et al. (2005). Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Applied and Environmental Microbiology 71 (4): 1685–1693. Compant, S., Clément, C., and Sessitsch, A. (2010). Plant growth-promoting bacteria in the rhizo-and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biology and Biochemistry 42 (5): 669–678. da Silva, P.R.A., Simões-Araújo, J.L., Vidal, M.S. et al. (2018). Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium. Brazilian Journal of Microbiology 49 (2): 210–211.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts. © 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd. 23 M Snoeijenbos et al. de Araujo, A.S.F., Mendes, L.W., Lemos, L.N. et al. (2018). Protist species richness and soil microbiome complexity increase towards climax vegetation in the Brazilian Cerrado. Communications Biology 1 (1): 135. de Bary, H.A. (1866). Morphologie und physiologie der pilze, flechten und myxomyceten. Munich: W. Engelmann. Elbeltagy, A., Nishioka, K., Suzuki, H. et al. (2000). Isolation and characterization of endophytic bacteria from wild and traditionally cultivated rice varieties. Soil Science and Plant Nutrition 46 (3): 617–629. Feng, Y., Shen, D., and Song, W. (2006). Rice endophyte Pantoea agglomerans YS19 pro- motes host plant growth and affects allocations of host photosynthates. Journal of Applied Microbiology 100 (5): 938–945. Freeman, E. (1904). The seed-fungus of Lolium temulentum, L., the Darnel. Proceeding of the Royal Society of London 71 (467–476): 27–30. Freeman, S. and Rodriguez, R.J. (1993). Genetic conversion of a fungal plant pathogen to a nonpathogenic, endophytic mutualist. Science 260 (5104): 75–78. Gourion, B., Berrabah, F., Ratet, P. et al. (2015). Rhizobium–legume symbioses: the crucial role of plant immunity. Trends in Plant Science 20 (3): 186–194. Guarro, J., Gené, J., and Stchigel, A.M. (1999). Developments in fungal taxonomy. Clin- ical Microbiology Reviews 12 (3): 454–500. Hardoim, P.R., Van Overbeek, L.S., Berg, G. et al. (2015). The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiology and Molecular Biology Reviews 79 (3): 293–320. Harrison, J.G., Parchman, T.L., Cook, D. et al. (2018). A heritable symbiont and host-associated factors shape fungal endophyte communities across spatial scales. Journal of Ecology 106 (6): 2274–2286. doi: 10.1111/1365-2745.12967. Hassani, M.A., Durán, P., and Hacquard, S. (2018). Microbial interactions within the plant holobiont. Microbiome 6: 58. Herrera, J., Khidir, H.H., Eudy, D.M. et al. (2010). Shifting fungal endophyte communities colonize Bouteloua gracilis: effect of host tissue and geographical distribution. Mycologia 102 (5): 1012–1026. doi: 10.1007/s10965-016-1156-5. Hibbett, D.S., Binder, M., Bischoff, J.F. et al. (2007). A higher-level phylogenetic classification of the Fungi. Mycological Research 111 (5): 509–547. Hibbett, D., Abarenkov, K., Kõljalg, U. et al. (2016). Sequence-based classification and identification of Fungi. Mycologia 108 (6): 1049–1068. Hoffman, M.T. and Arnold, A.E. (2008). Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees. Mycological Research 112 (3): 331–344. Hoffman, M.T. and Arnold, A.E. (2010). Diverse bacteria inhabit living hyphae of phylogenetically diverse fungal endophytes. Applied and Environmental Microbiology 76 (12): 4063–4075. Hoffman, M.T., Gunatilaka, M.K., Wijeratne, K. et al. (2013). Endohyphal bacterium enhances production of indole-3-acetic acid by a foliar fungal endophyte. PLoS One 8 (9): e73132. Hug, L.A., Baker, B.J., Anantharaman, K. et al. (2016). A new view of the tree of life. Nature Microbiology 1 (5): 16048. Ibáñez, F., Tonelli, M.L., Muñoz, V. et al. (2017). Bacterial endophytes of plants: diversity,invasion mechanisms and effects on the host. In: Endophytes: Biology and Biotech- nology (ed. D.K. Maheshwari), 25–40. Switzerland: Springer.

James, E.K., Gyaneshwar, P., Mathan, N. et al. (2002). Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67. Molecular Plant-Microbe Interactions 15 (9): 894–906. James, E.K. and Olivares, F.L. (1998). Infection and colonization of sugar cane and other graminaceous plants by endophytic diazotrophs. Critical Reviews in Plant Sci- ences 17 (1): 77–119. Johansson, J.F., Paul, L.R., and Finlay, R.D. (2004). Microbial interactions in the myc- orrhizosphere and their significance for sustainable agriculture. FEMS Microbiology Ecology 48 (1): 1–13. Johnson, N.C., Graham, J., and Smith, F.A. (1997). Functioning of mycorrhizal associations along the mutualism–parasitism continuum. New Phytologist 135 (4): 575–585. Jumpponen, A. (2001). Dark septate endophytes – are they mycorrhizal? Mycorrhiza 11 (4): 207–211. Junker, C., Draeger, S., and Schulz, B. (2012). A fine line – endophytes or pathogens in Arabidopsis thaliana. Fungal Ecology 5 (6): 657–662. Kloepper, J.W., Schippers, B., and Bakker, P. (1992). Proposed elimination of the term endorhizosphere. Phytopathology 82 (7): 726–727. Knapp, D.G., Németh, J.B., Barry, K. et al. (2018). Comparative genomics provides insights into the lifestyle and reveals functional heterogeneity of dark septate endophytic fungi. Scientific Reports 8 (1): 6321. Köberl, M., Dita, M., Nimusiima, J. et al. (2016). The banana microbiome: stability and potential health indicators. X International Symposium on Banana: ISHS-ProMusa Symposium on Agroecological Approaches to Promote Innovative Banana, vol. 1196, 1–8. Kogel, K.-H., Franken, P., and Hückelhoven, R. (2006). Endophyte or parasite – what decides? Current Opinion in Plant Biology 9 (4): 358–363. Lamb, C. and Dixon, R.A. (1997). The oxidative burst in plant disease resistance. Annual Review of Plant Biology 48 (1): 251–275. Mashiane, R.A., Ezeokoli, O.T., Adeleke, R.A. et al. (2017). Metagenomic analyses of bacterial endophytes associated with the phyllosphere of a Bt maize cultivar and its isogenic parental line from South Africa. World Journal of Microbiology and Biotech- nology 33 (4): 80. Meena, P.D., Awasthi, R.P., Chattopadhyay, C. et al. (2016). Alternaria blight: a chronic disease in rapeseed-mustard. Journal of Oilseed Brassica 1 (1): 1–11. Mondo, S.J., Lastovetsky, O.A., Gaspar, M.L. et al. (2017). Bacterial endosymbionts influence host sexuality and reveal reproductive genes of early divergent fungi. Nature Communications 8 (1): 1843. Monteiro, R.A., Balsanelli, E., Wassem, R. et al. (2012). Herbaspirillum-plant interactions: microscopical, histological and molecular aspects. Plant and Soil 356 (1–2): 175–196. Müller, D.B., Vogel, C., Bai, Y., and Vorholt, J.A. (2016). The plant microbiota: systems-level insights and perspectives. Annual Review of Genetics 50: 211–234. Müller, P. and Döring, M. (2009). Isothermal DNA amplification facilitates the identification of a broad spectrum of bacteria, fungi and protozoa in Eleutherococcus sp. plant tissue cultures. Plant Cell, Tissue and Organ Culture (PCTOC) 98 (1): 35–45. Newton, A.C., Fitt, B.D., Atkins, S.D. et al. (2010). Pathogenesis, parasitism and mutualism in the trophic space of microbe – plant interactions. Trends in Microbiology 18 (8): 365–373.

Parks, D.H., Chuvochina, M., Waite, D.W. et al. (2018). A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nature Biotechnology 36: 996–1004. Partida-Martinez, L.P. and Hertweck, C. (2005). Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 437 (7060): 884. Partida-Martinez, L.P.P. and Heil, M. (2011). The microbe-free plant: fact or artifact? Frontiers in Plant Science 2: 100. Petrini, O. (1991). Fungal endophytes of tree leaves. In: Microbial Ecology of Leaves (ed. J.H. Andrews and S.S. Hirano), 179–197. Switzerland: SpringerLink. Pozo, M.J. and Azcón-Aguilar, C. (2007). Unraveling mycorrhiza-induced resistance. Current Opinion in Plant Biology 10 (4): 393–398. Preston, G.M. (2004). Plant perceptions of plant growth-promoting Pseudomonas. Philo- sophical Transactions of the Royal Society, B: Biological Sciences 359 (1446): 907. Protsenko, M.A. (1975). Microorganism in the hyphae of mycorrhiza-forming fungus. Mikrobiologiia 44 (6): 1121–1124. Redman, R.S., Dunigan, D.D., and Rodriguez, R.J. (2001). Fungal symbiosis from mutualism to parasitism: who controls the outcome, host or invader? New Phytologist 151 (3): 705–716. Robinson, R.J., Fraaije, B.A., Clark, I.M. et al. (2016). Endophytic bacterial community composition in wheat (Triticum aestivum) is determined by plant tissue type, developmental stage and soil nutrient availability. Plant and Soil 405 (1–2): 381–396. Rodriguez, R.J., White Jr, J.F., Arnold, A.E., and Redman, A.R.A. (2009). Fungal endophytes: diversity and functional roles. New Phytologist 182 (2): 314–330. Rossmann, M., Sarango-Flores, S.W., Chiaramonte, J.B. et al. (2017). Plant microbiome: composition and functions in plant compartments. In: The Brazilian Microbiome (ed. V. Pylro and L. Roesch), 7–20. Switzerland: Springer. Roy, S. and Banerjee, D. (2018). Diversity of endophytes. In: Tropical Forests, in Endo- phytes of Forest Trees (ed. A.M. Pirttilä and C. Frank), 43–62. Switzerland: Springer. Ruiz-Pérez, C.A., Restrepo, S., and Zambrano, M.M. (2016). Microbial and functional diversity within the phyllosphere of Espeletia sp.inanAndeanhigh mountain ecosystem. Applied and Environmental Microbiology 82: 1807–1817. doi: 10.1128/AEM.02781-15. Ruiz-Herrera, J., León-Ramírez, C., Vera-Nuñez, A. et al. (2015). A novel intracellular nitrogen-fixing symbiosis made by Ustilago maydis and Bacillus spp. New Phytolo- gist, 207 (3): 769–777. Salter, S.J., Cox, M.J., Turek, E.M. et al. (2014). Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biology 12 (1): 87. Santoyo, G. et al. (2016). Plant growth-promoting bacterial endophytes. Microbiological Research 183: 92–99. Sarria-Guzmán, Y., Chávez-Romero, Y., Gómez-Acata, S. et al. (2016). Bacterial communities associated with different Anthurium andraeanum L. plant tissues. Microbes and Environments 31 (3): 321–328. doi: 10.1264/jsme2.ME16099. Schardl, C.L. and Clay, K. (1997). Evolution of mutualistic endophytes from plant pathogens. In: Plant Relationships Part B. The Mycota (A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research), vol. 5B (ed. G.C. Carroll and P. Tudzynski). Berlin, Heidelberg: Springer. Schneider, A. (1894). Mutualistic symbiosis of algae and bacteria with Cycas revoluta. Botanical Gazette 19 (1): 25–32.

Schulz, B. and Boyle, C. (2005). The endophytic continuum. Mycological Research 109 (6): 661–686. Shaffer, J.P., U’Ren, J.M., Gallery, R.E. et al. (2017). An endohyphal bacterium (Chitinophaga, Bacteroidetes) alters carbon source use by Fusarium keratoplasticum (F. solani species complex, Nectriaceae). Frontiers in Microbiology 8: 350. Shaffer, J.P., Zalamea, P.C., Sarmiento, C. et al. (2018). Context-dependent and vari- able effects of endohyphal bacteria on interactions between fungi and seeds. Fungal Ecology 36: 117–127. Sharma, M., Schmid, M., Rothballer, M. et al. (2008). Detection and identification of bacteria intimately associated with fungi of the order Sebacinales. Cell. Microbiol. 11: 2235–2246. Shivanna, M.B., Meera, M.S., and Hyakumachi, M. (1996). Role of root colonization ability of plant growth promoting fungi in the suppression of take-all and common root rot of wheat. Crop Protection 15 (6): 497–504. Sibanda, E.P., Mabandla, M., and Mduluza, T. (2018). A review of endophytic fungi bioprospecting in Africa-1994 to 2014. Current Biotechnology 7 (2): 80–88. Sieber, T. (2002). Fungal Root Endophytes. In: Plant Roots: The Hidden Half (ed. Y. Waisel, Amram Eshel, Tom Beeckman, and Uzi Kafkafi), 887–917. New York: Marcel Drekker. Siegel, M.R., Latch, G.C.M., and Johnson, M.C. (1987). Fungal endophytes of grasses. Annual Review of Phytopathology 25 (1): 293–315. Spatafora, J.W., Chang, Y., Benny, G.L. et al. (2016). A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia 108 (5): 1028–1046. Stone, J.K., Bacon, C.W., and White, J.F. Jr. (2000). An overview of endophytic microbes: endophytism defined. In: Microbial Endophytes (ed. C. W. Bacon and J. White), 17–44. Taffner, J., Erlacher, A., Bragina, A. et al. (2018). What is the role of Archaea in plants? New insights from the vegetation of Alpine bogs. mSphere 3 (3): e00122–e00118. Tahtamouni, M.E., Lucero, M., Sigala, J. et al. (2016). Diversity of endophytes across the soil-plant continuum for Atriplex spp. in arid environments. Journal of Arid Land 8 (2): 241–253. Tanaka, A., Christensen, M.J., Takemoto, D. et al. (2006). Reactive oxygen species play a role in regulating a fungus–perennial ryegrass mutualistic interaction. The Plant Cell 18 (4): 1052–1066. Trémouillaux-Guiller, J., Rohr, T., Rohr, R. et al. (2002). Discovery of an endophytic alga in Ginkgo biloba. American Journal of Botany 89 (5): 727–733. Turner, T.R., James, E.K., and Poole, P.S. (2013). The plant microbiome. Genome Biology 14 (6): 209. U’Ren, J.M., Lutzoni, F., Miadlikowska, J. et al. (2012). Host and geographic structure of endophytic and endolichenic fungi at a continental scale. American Journal of Botany 99 (5): 898–914. UNESCO (2017). UNESCO Science Report. Internal Oversight Service. https://en .unesco.org/about-us/ios (accessed 10 November 2018). Uthman, O.A., Wiysonge, C.S., Ota, M.O. et al. (2015). Increasing the value of health research in the WHO African Region beyond 2015—reflecting on the past, cele- brating the present and building the future: a bibliometric analysis. British Medical Journal Open 5 (3). doi: 10.1136/bmjopen-2014-006340.

Wallace, J., Laforest-Lapointe, I., and Kembel, S.W. (2018). Variation in the leaf and root microbiome of sugar maple (Acer saccharum) at an elevational range limit. PeerJ 6: e5293. Wilson, D. (1995). Endophyte: the evolution of a term, and clarification of its use and definition. Oikos 73 (2): 274–276. Woudenberg, J.H.C., Groenewald, J.Z., Binder, M. et al. (2013). Alternaria redefined. Studies in Mycology 75: 171–212. Xu, X.-H. et al. (2014). The rice endophyte Harpophora oryzae genome reveals evolution from a pathogen to a mutualistic endophyte. Scientific Reports 4: 5783. Zimmerman, N.B. and Vitousek, P.M. (2012). Fungal endophyte communities reflect environmental structuring across a Hawaiian landscape. Proceedings of the National Academy of Sciences of the United States of America 109 (32): 13022–13027.

Further Reading (The 103 studies reviewed in this manuscript to obtain the data presented)

Adam, E., Bernhart, M., Müller, H. et al. (2018). The Cucurbita pepo seed microbiome: genotype-specific composition and implications for breeding. Plant and Soil 422 (1–2): 35–49. Agler, M.T., Ruhe, J., Kroll, S. et al. (2016). Microbial hub taxa link host and abiotic factors to plant microbiome variation. PLoS Biology 14 (1): e1002352. Akinsanya, M.A., Goh, J.K., Lim, S.P. et al. (2015). Metagenomics study of endophytic bacteria in Aloe vera using next-generation technology. Genomics Data 6: 159–163. Alves, M., Pereira, A., Vicente, C. et al. (2018). The role of bacteria in Pine Wilt Disease: insights from microbiome analysis. FEMS Microbiology Ecology 94 (7). Ardanov, P., Sessitsch, A., Häggman, H. et al. (2012). Methylobacterium-induced endophyte community changes correspond with protection of plants against pathogen attack. PLoS One 7 (10): e46802. Baldan, E., Nigris, S., Populin, F. et al. (2014). Identification of culturable bacterial endophyte community isolated from tissues of Vitis vinifera “Glera”. Plant Biosys- tems 148 (3): 508–516. Beckers, B., De Beeck, M., Weyens, N. et al. (2016). Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome. Proceedings of the National Academy of Sciences of the United States of America 113 (8): 2312–2317. Beckers, B., De Beeck, M., Thijs, S. et al. (2016). Performance of 16s rDNA primer pairs in the study of rhizosphere and endosphere bacterial microbiomes in metabarcod- ing studies. Frontiers in Microbiology 7: 650. Beckers, B., De Beeck, M.O., Weyens, N. et al. (2017). Structural variability and niche differentiation in the rhizosphere and endosphere bacterial microbiome of field-grown poplar trees. Microbiome 5 (1): 25. Bergottini, V., Hervé, V., Sosa, D.A. et al. (2017). Exploring the diversity of the root-associated microbiome of Ilex paraguariensis St. Hil. (Yerba Mate). Applied Soil Ecology 109: 23–31. Bragina, A., Oberauner-Wappis, L., Zachow, C. et al. (2013). Insights into functional bacterial diversity and its effects on Alpine bog ecosystem functioning. Scientific Reports 3: 1955.

Bragina, A. et al. (2014). The Sphagnum microbiome supports bog ecosystem functioning under extreme conditions. Molecular Ecology 23 (18): 4498–4510. Bullington, L.S. and Larkin, B.G. (2015). Using direct amplification and next- generation sequencing technology to explore foliar endophyte communities in experimentally inoculated western white pines. Fungal Ecology 17: 170–178. Cao, H.X., Schmutzer, T., Scholz, U. et al. (2015). Metatranscriptome analysis reveals host-microbiome interactions in traps of carnivorous Genlisea species. Frontiers in Microbiology 6: 526. Carper, D.L., Carrell, A.A., Kueppers, L.M. et al. (2018). Bacterial endophyte communities in Pinus flexilis are structured by host age, tissue type, and environmental factors. Plant and Soil 428 (1–2): 335–352. Carrell, A.A. and Frank, A.C. (2014). Pinus flexilis and Picea engelmannii share a simple and consistent needle endophyte microbiota with a potential role in nitrogen fixation. Frontiers in Microbiology 5: 333. Carrell, A.A. and Frank, A.C. (2015). Bacterial endophyte communities in the foliage of coast redwood and giant sequoia. Frontiers in Microbiology 6: 1008. Carrell, A.A., Carper, D.L., and Frank, A.C. (2016). Subalpine conifers in different geographical locations host highly similar foliar bacterial endophyte communities. FEMS Microbiology Ecology 92 (8): fiw124. Cevallos, S., Herrera, P., Sánchez-Rodríguez, A. et al. (2018). Untangling factors that drive community composition of root associated fungal endophytes of Neotropical epiphytic orchids. Fungal Ecology 34: 67–75. Coleman-Derr, D., Desgarennes, D., Fonseca-Garcia, C. et al. (2016). Plant compart- ment and biogeography affect microbiome composition in cultivated and native Agave species. New Phytologist 209 (2): 798–811. Colin, Y., Nicolitch, O., Van Nostrand, J.D. et al. (2017). Taxonomic and functional shifts in the beech rhizosphere microbiome across a natural soil toposequence. Sci- entific Reports 7 (1). Correa-Galeote, D., Bedmar, E.J., and Arone, G.J. (2018). Maize endophytic bacterial diversity as affected by soil cultivation history. Frontiers in Microbiology 9: 484. David, A.S., Quiram, G.L., Sirota, J.I. et al. (2016). Quantifying the associations between fungal endophytes and biocontrol-induced herbivory of invasive purple loosestrife (Lythrum salicaria L.). Mycologia 108 (4): 625–637. David, A.S., Seabloom, E.W., and May, G. (2016). Plant host species and geographic distance affect the structure of aboveground fungal symbiont communities, and environmental filtering affects belowground communities in a coastal dune ecosystem. Microbial Ecology 71 (4): 912–926. Dawkins, K. and Esiobu, N. (2018). The invasive Brazilian Pepper Tree (Schinus tere- binthifolius) is colonized by a root microbiome enriched with alphaproteobacteria and unclassified spartobacteria. Frontiers in Microbiology 9: 876. De Souza, R.S.C., Okura, V.K., Armanhi, J.S.L. et al. (2016). Unlocking the bacterial and fungal communities assemblages of sugarcane microbiome. Scientific Reports 6: 28774. Ek-Ramos, M.J., Zhou, W., Valencia, C.U. et al. (2013). Spatial and temporal variation in fungal endophyte communities isolated from cultivated cotton (Gossypium hirsutum). PLoS One 8 (6): e66049. Eusemann, P., Schnittler, M., Nilsson, R.H. et al. (2016). Habitat conditions and phenological tree traits overrule the influence of tree genotype in the needle

mycobiome-Picea glauca system at an arctic treeline ecotone. The New Phytologist 211 (4): 1221–1231. Fitzpatrick, C.R., Copeland, J., Wang, P.W. et al. (2018). Assembly and ecological function of the root microbiome across angiosperm plant species. Proceedings of the National Academy of Sciences of the United States of America 115 (6): 1157–1165. Fonseca, C., Coleman-Derr, D., Garrido, E. et al. (2016). The Cacti microbiome: inter- play between habitat-filtering and host-specificity. Frontiers in Microbiology 7: 150. Fonseca, E.d.S., Peixoto, R.S., Rosado, A.S. et al. (2018). The microbiome of eucalyp- tus roots under different management conditions and its potential for biological nitrogen fixation. Microbial Ecology 75 (1): 192. Gallart, M., Adair, K.L., Love, J. et al. (2018). Host genotype and nitrogen form shape the root microbiome of Pinus radiata. Microbial Ecology 75 (2): 419–433. Garcias-Bonet, N., Arrieta, J.M., de Santana, C.N. et al. (2012). Endophytic bacterial community of a Mediterranean marine angiosperm (Posidonia oceanica). Frontiers in Microbiology 3: 342. Gdanetz, K. and Trail, F. (2017). The wheat microbiome under four management strategies, and potential for endophytes in disease protection. Phytobiomes 1 (3): 158–168. Geisen, S., Kostenko, O., Cnossen, M.C. et al. (2017). Seed and root endophytic fungi in a range expanding and a related plant species. Frontiers in Microbiology 8: 1645. Graham, L.E., Graham, J.M., Knack, J.J. et al. (2017). A Sub-Antarctic peat moss metagenome indicates microbiome resilience to stress and biogeochemical functions of early Paleozoic terrestrial ecosystems. International Journal of Plant Sciences 178 (8): 618–628. Guerreiro, M.A., Brachmann, A., Begerow, D. et al. (2018). Transient leaf endophytes are the most active fungi in 1-year-old beech leaf litter. Fungal Diversity 89 (1): 237–251. Gundale, M.J., Almeida, J.P., Wallander, H. et al. (2016). Differences in endophyte communities of introduced trees depend on the phylogenetic relatedness of the receiving forest. Journal of Ecology 104 (5): 1219–1232. Hampel, L.D., Cheeptham, N., Flood, N.J. et al. (2017). Plants, fungi, and freeloaders: examining temporal changes in the “taxonomic richness” of endophytic fungi in the dwarf mistletoe Arceuthobium americanum over its growing season. Botany 95 (3): 323–335. Haruna, E., Zin, N.M., Kerfahi, D. et al. (2018). Extensive overlap of tropical rainforest bacterial endophytes between soil, plant parts, and plant species. Microbial Ecology 75 (1): 88–103. Huang, Y., Kuang, Z., Wang, W. et al. (2016). Exploring potential bacterial and fungal biocontrol agents transmitted from seeds to sprouts of wheat. Biological Control 98: 27–33. Huang, Y., Kuang, Z., Deng, Z. et al. (2017). Endophytic bacterial and fungal communities transmitted from cotyledons and germs in peanut (Arachis hypogaea L.) sprouts. Environmental Science and Pollution Research 24 (19): 16458–16464. Huang, Y.L., Devan, M.N., U’Ren, J.M. et al. (2016). Pervasive effects of wildfire on foliar endophyte communities in Montane Forest trees. Microbial Ecology 71 (2): 452–468. Ibrahim, M., Sieber, T.N., and Schlegel, M. (2017). Communities of fungal endophytes in leaves of Fraxinus ornus are highly diverse. Fungal Ecology 29: 10–19.

Jin, H., Yang, X., Lu, D. et al. (2015). Phylogenic diversity and tissue specificity of fungal endophytes associated with the pharmaceutical plant, Stellera chamaejasme L. revealed by a cultivation-independent approach. Antonie van Leeuwenhoek Interna- tional Journal of General and Molecular Microbiology 108 (4): 835–850. Johnston, P.R., Johansen, R.B., Williams, A.F. et al. (2012). Patterns of fungal diversity in New Zealand Nothofagus forests. Fungal Biology 116 (3): 401–412. Kandalepas, D., Blum, M.J., and Van Bael, S.A. (2015). Shifts in symbiotic endophyte communities of a foundational salt marsh grass following oil exposure from the deepwater horizon oil spill. PLoS One 10 (4): e0122378. Khalaf, E.M. and Raizada, M.N. (2016). Taxonomic and functional diversity of cultured seed associated microbes of the cucurbit family. BMC Microbiology 16 (1): 131. Köberl, M., Dita, M., Nimusiima, J. et al. (2018). The banana microbiome: stability and potential health indicators. Acta Horticulturae (1196): 1–8. LaBonte, N.R., Jacobs, J., Ebrahimi, A. et al. (2018). Data mining for discovery of endophytic and epiphytic fungal diversity in short-read genomic data from deciduous trees. Fungal Ecology 35:1–9. Laforest-Lapointe, I., Paquette, A., Messier, C. et al. (2017). Leaf bacterial diversity mediates plant diversity and ecosystem function relationships. Nature 546 (7656): 145–147. Laforest-Lapointe, I., Messier, C., and Kembel, S.W. (2017). Tree leaf bacterial community structure and diversity differ along a gradient of urban intensity. MSystems 2 (6): e00087–e00017. Lamit, L.J., Lau, M.K., Sthultz, C.M. et al. (2014). Tree genotype and genetically based growth traits structure twig endophyte communities. American Journal of Botany 101 (3): 467–478. Lê Van, A., Quaiser, A., Duhamel, M. et al. (2017). Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera. PeerJ 5: 3454. Long, H.H., Sonntag, D.G., Schmidt, D.D. et al. (2010). The structure of the culturable root bacterial endophyte community of Nicotiana attenuata is organized by soil composition and host plant ethylene production and perception. New Phytologist 185 (2): 554–567. López, S., Pastorino, G., Franco, M. et al. (2018). Microbial endophytes that live within the seeds of two tomato hybrids cultivated in Argentina. Agronomy 8 (8): 136. Louca, S., Jacques, S.M., Pires, A.P. et al. (2017). Functional structure of the bromeliad tank microbiome is strongly shaped by local geochemical conditions. Environmental Microbiology 19 (8): 3132–3151. Lu, D., Jin, H., Yang, X. et al. (2016). Characterization of rhizosphere and endophytic fungal communities from roots of Stipa purpurea in alpine steppe around Qinghai Lake. Canadian Journal of Microbiology 62 (8): 643–656. Lumibao, C.Y., Formel, S., Elango, V. et al. (2018). Persisting responses of salt marsh fungal communities to the Deepwater Horizon oil spill. Science of the Total Environ- ment 642: 904–913. Lundberg, D.S., Lebeis, S.L., Paredes, S.H. et al. (2012). Defining the core Arabidopsis thaliana root microbiome. Nature 488 (7409): 86–90. Manter, D.K., Delgado, J.A., Holm, D.G. et al. (2010). Pyrosequencing reveals a highly diverse and cultivar-specific bacterial endophyte community in potato roots. Micro- bial Ecology 60 (1): 157–166.

Martinson, E.O., Herre, E.A., Machado, C.A. et al. (2012). Culture-free survey reveals diverse and distinctive fungal communities associated with developing figs (Ficus spp.) in Panama. Microbial Ecology 64 (4): 1073–1084. Mezzasalma, V., Sandionigi, A., Bruni, I. et al. (2017). Grape microbiome as a reliable and persistent signature of field origin and environmental conditions in Cannonau wine production. PLoS One 12 (9): e0184615. Mishra, A., Gond, S.K., Kumar, A. et al. (2012). Season and tissue type affect fungal endophyte communities of the Indian medicinal plant Tinospora cordifolia more strongly than geographic location. Microbial Ecology 64 (2): 388–398. Moler, E.R.V. and Aho, K. (2018). Whitebark pine foliar fungal endophyte communities in the southern Cascade Range, USA: host mycobiomes and white pine blister rust. Fungal Ecology 33: 104–114. Montanari-Coelho, K.K., Costa, A.T., Polonio, J.C. et al. (2018). Endophytic bacterial microbiome associated with leaves of genetically modified (AtAREB1) and conven- tional (BR 16) soybean plants. World Journal of Microbiology and Biotechnology 34 (4): 56. Moyes, A.B., Kueppers, L.M., Pett-Ridge, J. et al. (2016). Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer. New Phytologist 210 (2): 657–668. Ortega, R.A., Mahnert, A., Berg, C. et al. (2016). The plant is crucial: specific composition and function of the phyllosphere microbiome of indoor ornamentals. FEMS Microbiology Ecology 92 (12): fiw173. Ottesen, A.R., Gorham, S., Pettengill, J.B. et al. (2015). The impact of systemic and copper pesticide applications on the phyllosphere microflora of tomatoes. Journal of the Science of Food and Agriculture 95 (5): 1116–1125. Pawłowska, J., Wilk, M., Sliwi´ nska-Wyrzychowska,´ A. et al. (2014). The diversity of endophytic fungi in the above-ground tissue of two Lycopodium species in Poland. Symbiosis 63 (2): 87–97. Pinto, C., Pinho, D., Sousa, S. et al. (2014). Unravelling the diversity of grapevine microbiome. PLoS One 9 (1): e85622. Rahman, M.M., Flory, E., Koyro, H.W. et al. (2018). Consistent associations with beneficial bacteria in the seed endosphere of barleyHordeum ( vulgare L.). Systematic and Applied Microbiology 41 (4): 386–398. Rajala, T., Velmala, S.M., Vesala, R. et al. (2014). The community of needle endophytes reflects the current physiological state of Norway spruce. Fungal Biology 118 (3): 309–315. Robles, C.A., Lopez, S.E., McCargo, P.D. et al. (2015). Relationships between fungal endophytes and wood-rot fungi in wood of Platanus acerifolia in urban environments. Canadian Journal of Forest Research 45 (7): 929–936. Rybakova, D., Mancinelli, R., Wikström, M. et al. (2017). The structure of the Brassica napus seed microbiome is cultivar-dependent and affects the interactions of sym- bionts and pathogens. Microbiome 5 (1): 104. Saminathan, T., García, M., Ghimire, B. et al. (2018). Metagenomic and metatranscrip- tomic analyses of diverse watermelon cultivars reveal the role of fruit associated microbiome in carbohydrate metabolism and ripening of mature fruits. Frontiers in Plant Science 9:4.

Sánchez-López, A., Pintelon, I., Stevens, V.et al. (2018). Seed endophyte microbiome of Crotalaria pumila unpeeled: identification of plant-beneficial Methylobacteria. Inter- national Journal of Molecular Sciences 19 (1): 291. Sessitsch, A., Hardoim, P., Döring, J. et al. (2012). Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Molecular Plant-Microbe Interactions 25 (1): 28–36. Shade, A., McManus, P.S., and Handelsman, J. (2013). Unexpected diversity during community succession in the apple flower microbiome. American Society for Micro- biology 4 (2): e00602-12. Shakya, M., Gottel, N., Castro, H. et al. (2013). A multifactor analysis of fungal and bacterial community structure in the root microbiome of mature Populus deltoides trees. PLoS One 8 (10): e76382. Shetty, K.G., Rivadeneira, D.V., Jayachandran, K. et al. (2016). Isolation and molecular characterization of the fungal endophytic microbiome from conventionally and organically grown avocado trees in South Florida. Mycological Progress 15 (9): 987–989. Steinrucken, T.V., Raghavendra, A.K.H., Powell, J.R. et al. (2017). Triggering dieback in an invasive plant: endophyte diversity and pathogenicity. Australasian Plant Pathology 46 (2): 157–170. Su, Y.Y., Guo, L.D., and Hyde, K.D. (2010). Response of endophytic fungi of Stipa grandis to experimental plant function group removal in Inner Mongolia steppe, China. Fungal Diversity 43 (1): 93–101. Torres-Cortés, G., Bonneau, S., Bouchez, O. et al. (2018). Functional microbial features driving community assembly during seed germination and emergence. Frontiers in Plant Science 9: 902. Vieira, M.L., Johann, S., Hughes, F.M. et al. (2014). The diversity and antimicrobial activity of endophytic fungi associated with medicinal plant Baccharis trimera (Asteraceae) from the Brazilian savannah. Canadian Journal of Microbiology 60 (12): 847–856. Wang, W., Zhai, Y., Cao, L. et al. (2016a). Endophytic bacterial and fungal microbiota in sprouts, roots and stems of rice (Oryza sativa L.). Microbiological Research 188–189 (1–8). Wang, W., Zhai, Y., Cao, L. et al. (2016b). Illumina-based analysis of core actinobacte- riome in roots, stems, and grains of rice. Microbiological Research 190: 12–18. Wemheuer, F., Kaiser, K., Karlovsky, P. et al. (2017). Bacterial endophyte communities of three agricultural important grass species differ in their response towards management regimes. Scientific Reports 7: 40914. West, E.R., Cother, E.J., Steel, C.C. et al. (2010). The characterization and diversity of bacterial endophytes of grapevine. Canadian Journal of Microbiology 56 (3): 209–216. Winston, M.E., Hampton-Marcell, J., Zarraonaindia, I. et al. (2014). Understanding cultivar-specificity and soil determinants of the Cannabis microbiome. PLoS One 9 (6): e99641. Younginger, B.S. and Ballhorn, D.J. (2017). Fungal endophyte communities in the temperate fern Polystichum munitum show early colonization and extensive temporal turnover. American Journal of Botany 104 (8): 1188–1194. Yuan, Z., Druzhinina, I.S., Labbé, J. et al. (2016). Specialized microbiome of a halophyte and its role in helping non-host plants to withstand salinity. Scientific Reports 6: 32467.

Zhao, K., Li, J., Shen, M. et al. (2018). Actinobacteria associated with Chinaberry tree are diverse and show antimicrobial activity. Scientific Reports 8 (1): 11103. Zhao, Y., Gao, Z., Tian, B. et al. (2017). Endosphere microbiome comparison between symptomatic and asymptomatic roots of Brassica napus infected with Plasmodiophora brassicae. PLoS One 12 (10): e0185907.