The plant endosphere world – bacterial life within plants

Abstract

The plant endosphere is colonized by complex microbial communities and microorganisms, which colonize the plant interior at least part of their lifetime and are termed endophytes. Their functions range from mutualism to pathogenicity. All plant organs and tissues are generally colonized by bacterial endophytes and their diversity and composition depend on the plant, the plant organ and its physiological conditions, the plant growth stage as well as on the environment. Plant-associated microorganisms, and in particular endophytes, have lately received high attention, because of the increasing awareness of the importance of host-associated microbiota for the functioning and performance of their host. Some endophyte functions are known from mostly lab assays, genome prediction and few metagenome analyses, however, we have limited understanding on in planta activities, particularly considering the diversity of micro-environments and the dynamics of conditions. In our review, we present recent findings on endosphere environments, their physiological conditions and endophyte colonization. Furthermore, we discuss microbial functions, the interaction between endophytes and plants as well as methodological limitations of endophyte research. We also provide an outlook on needs of future research to improve our understanding on the role of microbiota colonizing the endosphere on plant traits and ecosystem functioning. This article is protected by copyright. All rights reserved.

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The plant endosphere world –bacterial life within plants
Stéphane Compant ,
1
Marine C. Cambon ,
2
Corinne Vacher ,
2
Birgit Mitter ,
1
Abdul Samad
3
and Angela Sessitsch
1
*
1
Center for Health and Bioresources, Bioresources Unit,
Konrad Lorenz Straße 24, AIT Austrian Institute of
Technology, Tulln, A-3430, Austria.
2
INRAE, Univ. Bordeaux, BIOGECO, Pessac, F-33600,
France.
3
Natural Resources Canada, Canadian Forest Service,
Laurentian Forestry Centre, Québec, G1V4C7, Canada.
Summary
The plant endosphere is colonized by complex micro-
bial communities and microorganisms, which colo-
nize the plant interior at least part of their lifetime
and are termed endophytes. Their functions range
from mutualism to pathogenicity. All plant organs
and tissues are generally colonized by bacterial
endophytes and their diversity and composition
depend on the plant, the plant organ and its physio-
logical conditions, the plant growth stage as well as
on the environment. Plant-associated microorgan-
isms, and in particular endophytes, have lately
received high attention, because of the increasing
awareness of the importance of host-associated
microbiota for the functioning and performance of
their host. Some endophyte functions are known
from mostly lab assays, genome prediction and few
metagenome analyses; however, we have limited
understanding on in planta activities, particularly
considering the diversity of micro-environments and
the dynamics of conditions. In our review, we present
recent findings on endosphere environments, their
physiological conditions and endophyte colonization.
Furthermore, we discuss microbial functions, the
interaction between endophytes and plants as well
as methodological limitations of endophyte research.
We also provide an outlook on needs of future
research to improve our understanding on the role of
microbiota colonizing the endosphere on plant traits
and ecosystem functioning.
Introduction
For a long time, the scientific community thought that
plants that do not show symptoms of diseases are free of
microorganisms, particularly from bacteria. There were
few early reports on bacterial colonization of the plant
endosphere (Galippe, 1887; Laurent, 1889); however, in
the 19th century, the general belief was that healthy
plants are free of microorganisms, following the postu-
lates of Louis Pasteur (Compant et al., 2012). Bacteria
occupying root nodules of leguminous plants, nowadays
well known as rhizobia being responsible for fixing atmo-
spheric nitrogen, were discovered by Martinus Willem
Beijerinck in 1888 (Beijerinck, 1888). In the same year,
Hellriegel and Wilfarth (1888) reported that leguminous
plants are independent on mineral N, further indicating
the importance of the N-fixing symbiosis between plants
and rhizobia.
Active research on the plant endosphere as a habitat
for non-pathogenic bacteria started in the 1990s, trig-
gered by the increasing number of reports on the benefi-
cial effects of plant growth-promoting rhizobacteria
(PGPR). The pioneering work of Johanna Döbereiner on
specific bacteria which, like Herbaspirillum seropedicae,
colonize the endosphere of sugarcane and fix nitrogen
(Baldani et al., 1986; Boddey and Döbereiner, 1988)
stimulated, given their importance for the Brazilian econ-
omy, further research on bacteria colonizing the plant
endosphere. The increasing interest in studying microbial
communities in the environment together with the devel-
opment of molecular, cultivation-independent tools (like
the DNA fingerprinting tools 16S rRNA gene-based
denaturating gradient gel electrophoresis or terminal
restriction fragment length polymorphism analysis) to
study their community structure also triggered research
on endosphere microbiota.
In the early 1990s, definitions came up on the term
‘endophyte’, mostly referring to microorganisms that
inhabit internal plant tissues, at least some time of their
Received 3 July, 2020; revised 11 September, 2020; accepted 16
September, 2020. *For correspondence. E-mail angela.sessitsch@ait.
ac.at; Tel. +43(0) 50550-3509; Fax +43(0) 50550-3666.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd
Environmental Microbiology (2020) 00(00), 00–00 doi:10.1111/1462-2920.15240

lifecycle, without causing apparent harm or disease to
their host (Petrini, 1991; Wilson, 1995). Although this def-
inition has been used in many studies and represents a
pragmatic distinction between endophytes and patho-
genic colonizers, it has been recently revised by Hardoim
et al. (2015). A revised definition was needed due to the
understanding obtained in the last years showing that
pathogenicity or mutualism of microorganisms may
depend on many factors including the plant genotype, the
environment and the co-colonizing microbiota (Brader
et al., 2017). Therefore, a clear distinction between non-
pathogenic microorganisms (i.e. endophytes) and patho-
gens is often not feasible without detailed functional
analysis. Also, functional assignment of endophytes stud-
ied purely by molecular tools, e.g. by microbiome analy-
sis based only on phylogenetic markers, is usually not
possible. Therefore, Hardoim et al. (2015) suggested that
the term endophyte should refer to the habitat only and
include all microorganisms, which for all or part of their
lifetime colonize internal plant tissues. In the present
review, the term endophyte refers to any microorganism
that can colonize internal tissues of plants, including
pathogens.
In the last decade, host-associated microbiota have
gained increasing attention, triggered by spectacular find-
ings on the role of the human microbiome for human health,
behaviour and well-being. Already in 1994, Jefferson postu-
lated that the evolutionary selection unit is not the macro-
organism (e.g. the plant) but the macro-organism and all its
associated microorganisms that act in concert as a
holobiont (Jefferson, 1994). This hypothesis was further
elaborated in the highly debated hologenome theory of evo-
lution (Zilber-Rosenberg and Rosenberg, 2008; Theis
et al., 2016). The rhizosphere is considered as an important
component of the plant holobiont, but endophytes have
received increasing attention due to their intimate interac-
tion with plants. Despite this increasing awareness of micro-
bial life within plants, the endosphere is often recognized as
one habitat without considering the variety of microenviron-
ments and dynamics of microenvironment conditions. We
aim here to review the multiple facets of the plant endo-
sphere environment for bacterial colonization and life and
pinpoint to the methodological limitations and knowledge
gaps, which need to be addressed to further elucidate the
role of bacterial endophytes in plant physiology and ecosys-
tem functioning.
Microbiota in different plant compartments
Plant compartments and physiological conditions
Plants host diverse microbiota in different compartments
and tissues, i.e. vegetative organs like roots, stems and
leaves and also reproductive/disseminating organs
(flowers, fruits/seeds). Bacterial densities inside plant tis-
sues typically range from 10
5
to 10
7
of cultivable cells per
gram of root to 10
3
–10
4
in leaves and stems. In flowers,
fruits and seeds typically 10
2
–10
3
cells per gram tissue
are found (Compant et al., 2010). These numbers pin-
point to the soil environment as a major reservoir of
potential endophytes. However, the plant immune system
may control the abundance of endophytes and maintain
the most ‘plant-favorable’bacterial density in the different
organs (Liu et al., 2017). Pathogens can overcome plant
defense and can therefore reach higher cell numbers
than non-pathogenic strains (Brader et al., 2017). High
bacterial cell density can be detrimental for the host
organs. For instance, high cell density is known to induce
quorum sensing (i.e. cell-density dependent) regulated
processes such as virulence and pathogenicity but are
also important for beneficial functions (Braeken
et al., 2008; Hartmann et al., 2014). Seeds usually show
low bacterial numbers and do not provide suitable condi-
tions for microbial growth. When seeds germinate, the
bacterial cell density increases, seed-derived endophytes
colonize the different plant tissues of the emerging plant
(Mitter et al., 2017).
The occurrence, abundance and activities of individual
bacterial taxa depend on the micro-environment provided
by the plant compartment (Compant et al., 2010; Brader
et al., 2017), the plant genotype and physiology as well
as the surrounding environment (Turner et al., 2013;
Barret et al., 2015; Hardoim et al., 2015; Afzal et al.,
2019). Roots, stems, leaves, flowers, fruits and seeds
show different chemical conditions, in terms of organic
acids, carbohydrates, vitamins, sugars, but also hormones,
amino acids, fatty acids, flavonoids, glucosinolates, as well
as phenolic compounds, pH and water, which are essential
for plant growth, development, stress adaptation and
defense (Hounsome et al., 2008). Each chemical environ-
ment enables the growth of specific microorganisms show-
ing appropriate metabolic activities to colonize and inhabit
plant organs and tissues, leading to different microbial
assemblages (Compant et al., 2010; Vorholt, 2012; Sasse
et al., 2018).
From the soil environment, microorganisms reach the
rhizosphere, which is known to be directly influenced by
root exudates and constitutes a hotspot for the establish-
ment and development of microbial communities (Hiltner,
1904; Lemanceau et al., 2017). Roots can release about
10%–40% of their total photosynthetically fixed carbon
through organic and inorganic forms (Newman, 1985).
These exudates include secretions and diffusates that
are chemically very diverse, including mucilage, cellu-
lose, organic acids, amino acids, fatty acids, phenolics,
plant growth regulators, nucleotides, sugars, putrescine,
sterols and vitamins (Jones et al., 2009; Sasse
et al., 2018). Root environment, root morphology and root
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
2S. Compant et al.

exudates have a tremendous influence on shaping the
soil microbiome, but also on the establishment of bacte-
rial endophytes in roots (Pfeiffer et al., 2017; Sasse
et al., 2018) as well as in flowers, fruits (Compant
et al., 2011) and seeds (Escobar Rodríguez
et al., 2018a). However, exudates and leachates of other
plant organs than roots can also facilitate establishment
and colonization of bacterial endophytes, from the outer
to inner tissues, as shown, for instance, with the leaf
environment (Vorholt, 2012; Vacher et al., 2016), but also
with flower and fruit environments showing specific exu-
date composition (Compant et al., 2010).
In organs like seeds, not only the chemical environ-
ment is important. Seeds are desiccated tissues that
enable only specific microorganisms to thrive as endo-
phytes (Hardoim et al., 2015; Truyens et al., 2015) albeit
pathogenic strains can also destroy seeds. Further,
starch accumulation and drying during seed maturation
only allows primarily bacterial endophytes to establish
and survive if they can tolerate high osmotic pressure
(Mano et al., 2006; Rana et al., 2020). Plant emergence,
cultivar and pathogens further shape the seed microbial
community (Barret et al., 2015, 2016). These seed endo-
phytes can be acquired from the soil environment, but
also transmitted from birds or other animals to plant tis-
sues (Berg and Raaijmakers, 2018; Escobar Rodríguez
et al., 2018a). Furthermore, vertical transmission of endo-
phytes to the next generation may be possible, e.g. when
microorganisms colonize flowers and then migrate
into the developing seed (Mitter et al., 2017). Cultivation-
independent analysis also suggests that some
endophytes may be transferred from seed to seed
(Johnston-Monje and Raizada, 2011; Escobar Rodríguez
et al., 2018b). The composition of seed microbiota can
impact seed quality and ultimately plant fitness (Shade
et al., 2017; Escobar Rodríguez et al., 2020).
Plant compartments and bacterial microbiota
composition
In comparison to other plant organs, root bacterial micro-
biota often (but not always) contain the highest diversity
of microorganisms (Amend et al., 2019). Mostly found
taxa are Acidobacteria, Verrucomicrobia, Bacteroidetes,
Proteobacteria, Planctomycetes and Actinobacteria and
most of them can be also found in the rhizosphere
(Hardoim et al., 2015). However, a subpopulation of rhi-
zosphere microbiota can enter roots. The most abundant
phyla often found were, for instance, in grapevine roots,
Proteobacteria, Acidobacteria, Actinobacteria, Bacte-
roidetes, Verrucomicrobia, Planctomycetes, Chloroflexi,
Firmicutes and Gemmatimonatedes (Samad et al., 2017).
For maize, Proteobacteria, Firmicutes, and Bacteroidetes
have been also found as predominant phyla inside roots
(Correa-Galeote et al., 2018). Different plant species usu-
ally host different microbiota composition, but also the
plant genotype and developmental stage affect diversity
and shape community structure. Furthermore, the soil
environment, its history, as well as biotic or abiotic
stresses lead to a shift in the diversity and abundance of
the different taxa inhabiting roots (Brader et al., 2017;
Correa-Galeote et al., 2018; Sasse et al., 2018; Xu
et al., 2018). The presence of different other microorgan-
isms like fungi or mycorrhizae-like fungi and microbial
interactions can additionally influence the composition of
root endophytic microbiota (Deveau et al., 2018).
Above-ground organ microbiota such as in stems, fruits
or seeds have been shown to be influenced by several
factors, including the soil environment (Rasche
et al., 2006; Klaedtke et al., 2015; Zarraonaindia et al.,
2015; Escobar Rodríguez et al., 2020). Interestingly, Har-
rison and Griffin (2020) recently reported that variation in
endophyte assemblages between below- and above-
ground tissues varied with the host growth habit. The
authors showed that in woody plants stems hosted the
richest endophyte communities, whereas in graminoids
roots had the richest communities (Harrison and
Griffin, 2020). Inside leaves, different endophytic assem-
blages have been found depending on external factors,
such as climate and microclimate, as well as plant spe-
cies and genotypes (Turner et al., 2013; Robinson
et al., 2016; Vacher et al., 2016; Mina et al., 2020). Prote-
obacteria, Firmicutes, Actinobacteria and Bacteroidetes
have been isolated from maize seeds (Johnston-Monje
and Raizada, 2011; Rana et al., 2020). Also, molecular
community analysis evidenced these taxa within seeds,
e.g. of Brassicaceae (Barret et al., 2015) or of Setaria
(Escobar Rodríguez et al., 2018a).
Colonization pattern of endophytes
Colonization patterns and niches of plant-associated
microorganisms from the soil to aerial organs have been
studied for decades for both pathogenic and non-
pathogenic strains. Roots have been widely studied
where microorganisms could be endophytic crossing
from the rhizosphere to the rhizodermis before reaching
the cortical cell layers, the central cylinder (stele) and the
xylem vessels (Compant et al., 2010; Brader et al., 2017)
(Fig. 1). Different pathways have been described to
explain crossing from the rhizosphere to root internal tis-
sues (Kandel et al., 2017). Microorganisms can enter
roots at the root tip and root hair level (Kandel
et al., 2017), and root hair inner colonization has been
demonstrated (Mercado-Blanco and Prieto, 2012;
Mercado-Blanco, 2015). This is, for instance, the case for
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
The bacterial endosphere world 3

Fig. 1. Confocal microscopy pictures of reproductive/disseminating or vegetative plant organs and bacteria.
A. Cucumis melo L. fruit.
B and C. Caryopsis of Triticum aestivum L.
D. Anther of Vitis vinifera L.
E. Pollen of Vitis vinifera L.
F and G. Developing fruit and seed of Vitis vinifera L.
H and I. Leaf of Vitis vinifera L.
J. Leaf of Triticum aestivum L.
K, L and M. Root of Vitis vinifera L. Transversal section (A, H-M), longitudinal section (B and C, F and G), surface (D, E). Bacteria (arrows)
appear as green, orange, white (A) by fluorescence in situ hybridization using general and specific probes targeting bacterial taxa or green (C, E,
G, I, J, L, M) due to general staining with Syto9
®
. Scale of a bacterium: 1–2μm.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
4S. Compant et al.

some plant beneficial endophytes like strains belonging
to Pseudomonas spp. in olive roots (Mercado-Blanco
and Prieto, 2012; Mercado-Blanco, 2015). Root crack col-
onization has been further described. Microorganisms
can enter root tissues at cracks of the secondary root
emergence zone enabling a non-active process for bac-
teria to be present inside root tissues (Compant
et al., 2010; Hardoim et al., 2015; Compant et al., 2019).
Bacteria enter roots at the junctions of rhizodermal
cells through plant cell wall degradation using plant cell
wall-degrading enzymes, including polymer-degrading
cellulases, cellobiohydrolases, endoglucanases and
xylanases (Liu et al., 2017). Furthermore, pathogens and
insects may open a path for non-pathogenic bacteria to
enter roots (Compant et al., 2005, 2010). All these routes
of penetration have been demonstrated with inoculated
non-pathogenic or pathogenic microbial strains, but evi-
dence has also been obtained by microscopy analysis of
samples obtained from natural environments (Compant
et al., 2016) (Fig. 1). Comparative analysis of root coloni-
zation has been performed for beneficial and pathogenic
Pseudomonas syringae strains on pepper plants
(Passera et al., 2019). Both strains showed a preference
for colonization at the secondary root emergence site.
The beneficial strain was also found in high density on
the surface of primary roots, while the pathogenic strain
was found more often on secondary roots. The latter
strain also colonizes strongly damaged roots and locates
over the xylem zones, whereas the beneficial strain did
not (Passera et al., 2019).
Albeit most endophytic bacteria colonize intercellular
spaces, also intracellular colonization as well as the
combination of both colonization types have been
observed. This has been described for instance with the
beneficial endophyte Paraburkholderia phytofirmans
strain PsJN in grapevine roots, where the strain colo-
nizes mostly intercellular spaces, but it was also found
to colonize intracellularly in the endodermis as well as in
some cortical cells (Compant et al., 2008). Intracellular
colonization has been also demonstrated with other
model endophytes such as strains of Azoarcus,Glu-
conacetobacter,Herbaspirillum and Klebsiella spp.
(Turner et al., 2013). Inside stems, several bacterial taxa
have been described to colonize mostly intercellular
spaces but also intracellular spaces, with presence in
the vascular system, in the parenchyma layer as well as
inside the epidermis. The cells inside the vascular sys-
tem such as xylem vessels derive mostly from the roots,
although translocation to the vascular system has been
shown additionally for microorganisms deriving from
insects (Hardoim et al., 2015) such as in the case of
Phytoplasma (Brader et al., 2017). For the stem part
residing in soil, such as tubers and stolons, several bac-
terial taxa were shown to inhabit these organs and to
derive from the surrounding soil as it has been
described for roots (Kõiv et al., 2015).
In leaves, several reviews have focused further on the
distribution, range and bacterial taxa with presence inside
substomatal chambers, parenchyma and the vascular
system. As for stems, microorganisms have different
routes of colonization, the ones inside the vascular sys-
tem mostly derive from root xylem vessels, while those
close to the surface derive from the external environment
of leaves (Vacher et al., 2016). Sabaratnam and Beattie
(2003) compared colonization characteristics of the path-
ogen Pseudomonas syringae pv. syringae B728a and a
non-pathogenic strain, Pantoea agglomerans BRT98, on
and inside leaves of bean and maize. P. syringae B728a
could colonize the leaf interior, while the Pantoea strain
could not. On pear leaves, P. syringae pv. syringae multi-
plied on the leaf surface, before colonizing internal leaf
tissues through the trichome base and fissures in the
cuticular cell layer (Mansvelt and Hattingh, 1987). This
pathogen also showed to colonize xylem vessels and the
leaf apoplast by using syringolin A that suppresses plant
resistance by blocking SA signalling (Misas-Villamil
et al., 2013). However, also non-pathogenic strains can
colonize leaves endophytically as seen, for instance, with
Methylobacterium PA1 (Peredo and Simmons, 2018).
In flowers, various bacteria were found to colonize the
epidermis, the xylem vessels, the ovary, the ovules and
stigma as well as other parts such as the flower recepta-
cle, the petal, sepal and anthers with their filaments as
well as pollen (Compant et al., 2011). Firmicutes,
Actinobacteria and Proteobacteria strains have been, for
instance, isolated from these organs (Shi et al., 2010;
Compant et al., 2011; Fürnkranz et al., 2012). Coloniza-
tion is related to the surface of the flowers for bacteria
inhabiting niches close to the surfaces of the organs,
while other routes involve colonization from roots to the
aerial plant parts as discussed with pathogenic
(Maude, 1996) or non-pathogenic strains (Compant
et al., 2011). In fruits, bacteria have been also visualized
as colonizing the exocarp, the mesocarp and the endo-
carp in grapevine or melon as well several plants
(Compant et al., 2011; Glassner et al., 2015) and in the
parenchyma or xylem vessels. Inside fruits, seed tissues
inhabiting microorganisms are the outer and inner seed
coat parts, the embryo hypocotyl root-axis, the plumule
and the cotyledon as well as the endosperm (Escobar
Rodríguez et al., 2018b; Glassner et al., 2018) (Fig. 1).
Seeds may also host pathogens, which may enter via the
floral pathway as seen for instance with Xanthomonas
citri subsp. fuscans in bean seeds (Darsonval
et al., 2008). This pathogen was observed entering seeds
through vascular elements and parenchyma of funiculus,
but also via the micropyle and testa. The same pathogen
was also found inside seeds on radicle surfaces, in
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
The bacterial endosphere world 5

cotyledons and plumules (Darrasse et al., 2018). Also,
the beneficial endophyte P. phytofirmans strain PsJN can
colonize the seed embryo of pepper, tomato, wheat and
maize, using the floral pathway (Mitter et al., 2017).
Bacterial traits enabling life in the endosphere
Several bacterial traits enable life in the plant endosphere
(Compant et al., 2010; Pinski et al., 2019). Lipopolysac-
charides, flagella, pili, and twitching motility (Duijff
et al., 1997, Dörr et al., 1998, Böhm et al., 2007; Tadra-
Sfeir et al., 2011) have been linked to endophytic coloniza-
tion and are also important for rhizosphere competence
(Compant et al., 2010). Bacteria have also to enter inside
plants and this requires uptake and degradation of plant-
derived compounds. Pathogens are known for using multi-
ple tools including carbohydrate active enzymes to enter
plants (Brader et al., 2017), and the same type of
enzymes are utilized by non-pathogenic strains. For exam-
ple, a mutant of Azoarcus sp. BH72 devoid of
endoglucanase activity had a decreased ability to colonize
rice (Reinhold-Hurek et al., 2006). Comparative genomics
of endophytic bacterial strains revealed that genes related
to motility, chemotaxis, signal transduction, transcriptional
regulators, stress-related enzymes, transporters and
secretion systems are important for colonization of host
plant internal tissues (Hardoim et al., 2015). Furthermore,
expansins could be also associated with endophytic colo-
nization as seen with a mutant of Bacillus subtilis
168 (Ampomah et al. 2013). Once inside plants chemo-
taxis towards L-arabinose present in xylem vessels of
cucumber has been shown for strains of Pseudomonas
spp. (Malfanova et al., 2013). Bacteria also have to adapt
to the rather stressful environment inside plants and detox-
ification mechanisms have been shown in several endo-
phytes (Compant et al., 2010). Furthermore, siderophore
and biocontrol metabolites may have a role for endophytic
colonization as well as secretion systems. For instance, it
has been shown that a knock-out mutant of Kosakonia
lacking the type 6 secretion system (T6SS) showed signifi-
cantly reduced rhizosphere and endosphere colonization
(Mosquito et al., 2020). Herbaspirillum rubrisubalbicans
M1 T3SS mutants were also less successful in endophytic
colonization (Schmidt et al., 2012).
Functions exhibited by non-pathogenic endophytes
in the plant endosphere
Plant-associated microorganisms are known for various
functions, which are particularly important for the plant
host and which have been reviewed recently (Hardoim
et al., 2015). These functions involve N cycling, phos-
phate mobilization, plant defense induction, antibiotic pro-
duction, out-competition of pathogens, as well as
improving plant tolerance to biotic and abiotic stresses
(Turner et al., 2013). These activities have been widely
demonstrated, either by lab assays or genome analysis
of individual strains or by metagenome analysis
(Sessitsch et al., 2012; Saminathan et al., 2018; Carrión
et al., 2019). However, most of these analyses indicate
potential functional activities or show activity in lab
assays, but there is only scarce information on activities
in planta, especially when plants are grown in the field.
The benefits of plant-associated microorganisms, particu-
larly of non-pathogenic endophytes, for the health and
growth of their host have been demonstrated in numer-
ous studies inoculating plants with individual microbial
strains. However, inoculation experiments or application
also often fails under field or more natural conditions,
which may be due to limited colonization and/or due to
no or low activity. In some studies, in situ activity has
been analyzed by expression or transcriptome analysis,
which can indicate functional activities (Schenk
et al., 2012). Sessitsch et al. (2012) showed, for example,
that different N-cycling genes are expressed in rice roots
indicating N fixation, nitrification and denitrification pro-
cesses in planta. More recently, Carrión et al. (2019)
showed the transcriptional and functional analysis of
disease-suppressive bacterial consortia demonstrating
the role of secondary metabolites produced by the endo-
phytic root microbiome. Furthermore, Xu et al. (2018)
demonstrated that drought increases Actinobacteria
populations in sorghum roots and has a significant effect
on the transcriptional activity of the root-associated
microbiome. Genes associated with carbohydrate and
amino acid metabolism and transport showed an
increased expression under drought, and the shift was
largely due to actinobacterial activity and function. Along
these lines, Sheibani-Tezerji et al. (2015) showed by
transcriptome analysis that the endophyte Para-
burkholderia phytofirmans PsJN senses and responds to
plants under osmotic stress. Also, soil contamination was
shown to influence the transcriptome of willow associated
microbiome (Yergeau et al., 2018). Saminathan et al.
(2018) demonstrated by metagenomic and meta-
transcriptomic analysis the role of the endophytic fruit
microbiome in carbohydrate metabolism and ripening of
watermelon fruits.
Effects of endophytes on plants and effects of plants
on endophytes
Many efforts have been put on studying the possible
effects of endophytes on plants and following the current
definition of endophytes (Hardoim et al., 2015), the inter-
action ranges from mutualism to pathogenicity. While the
elucidation of the effects of endophytes on plants is the
aim of an ever faster growing number of studies focusing
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
6S. Compant et al.

on stimulation of plant growth through the production of
plant hormones, increasing the availability of nutrients
and minerals, fighting or outcompeting plant pathogens,
and eliciting resilience to biotic and abiotic stresses
(reviewed, e.g. in the study by Bulgarelli et al., 2013;
Mitter et al., 2013; Hardoim et al., 2015), we still have lim-
ited understanding on the effects of plants on their micro-
bial inhabitants. The plant is an environment for
microorganisms with different ecological niches, inter-
connected at the spatial and temporal scale. Moreover,
plants are under constant influence from external (envi-
ronmental) and internal (developmental) factors, resulting
in a dynamic system of adaptive responses of physiologi-
cal processes. Thus, existence in the plant may require
microorganisms to be highly adaptable, both on commu-
nity and cellular level. From studies employing commu-
nity sequencing, we know that the endophytic
communities are dynamic and change during plant devel-
opment (Shi et al., 2014; Ren et al., 2015; Borruso
et al., 2018), and in response to biotic (Bulgari
et al., 2012; Kõiv et al., 2015; Wemheuer et al., 2019)
and abiotic stimuli (Kandalepas et al., 2015; Ren
et al., 2015) of plants. The observed shifts in the endo-
phyte communities, which may be explained by different
abilities of individual species to adapt to changes in the
plant environment as well as by interactions between
species within the community. In this context, we may
speculate that the ecological concept of species-sorting
(Leibold et al., 2004; Holyoak et al., 2005) applies also to
microbial communities inside plants. This implies that
each plant compartment with its specific environmental
conditions is colonized by a metacommunity of microor-
ganisms and the different compartments with their meta-
communities are connected through bacterial dispersal in
xylem vessels. However, changes in the composition of
endophytic bacterial communities must begin with a reac-
tion of microorganisms on a cellular level to specific stim-
uli in the plant.
In plants, the response to changing environmental con-
ditions and development processes often cause a shift in
the cellular redox state. In a previous study, we analyzed
the genetic response of the endophyte P. phytofirmans
PsJN to osmotic stress of the host plant (Sheibani-Tezerji
et al., 2015). The gene expression pattern indicated that
the bacterium noticed and reacted to the changed redox
conditions in the plant. Among other genes involved in
the defense against oxidative stress, a cell surface sig-
nalling system was activated, which is involved in
adjusting the iron acquisition to the redox status
(Sheibani-Tezerji et al., 2015). Similarly, the endophyte
Gluconacetobacter diazotrophicus PAL5 showed
increase expression of genes encoding for ROS-
detoxifying enzymes such as superoxide dismutase and
glutathione reductase during the colonization of rice roots
(Alquéres et al., 2013). It also has been shown that H
2
O
2
breakdown by endophytes might also be involved in the
adaption of bacteria to the redox conditions during germi-
nation in seed and seedling (Gerna et al., 2020). Hydro-
gen peroxide might also shape the microbial community
in floral nectar. In principle, nectar contains everything
microorganisms need for growth, i.e. different sugars,
amino acids and organic acids. However, growth of
microorganisms is controlled by the activity of specialized
nectar proteins (nectarins), which maintains a redox
cycle, allowing for high levels of peroxide in nectar
(Carter and Thornburg, 2004). Taking all these findings
into consideration, it is not surprising that based on meta-
genomics data genes for the detoxification of reactive
oxygen species are prevalent among endophyte commu-
nities (Sessitsch et al., 2012). Apart from this rather gen-
eral response to the plant environment, which is
comparable to oxidative stress response in any other
habitat, endophytes adjust their behavior also specifically
in response to plant signals. This might be particularly
important during plant colonization.
Most endophytes derive from the soil and the root is
the initial contact point with the plant, and the detection of
signal molecules in the root exudates often initiate the
symbiosis between plants and endophytes. For example,
plant flavonoids activate a series of bacterial genes,
which results in production and secretion of the nod-
factors in rhizobia necessary for successful interaction
between plant and bacterial cells (Geurts and
Bisseling, 2002). Similarly, rice root exudates induced the
expression of genes involved in adherence and signal
transduction, while flagella synthesis was downregulated
in the endophyte Azoarcus sp. BH72, indicating that the
bacterium was primed for the switch from the rhizosphere
to the plant endosphere (Shidore et al., 2012). Further-
more, chemoattraction by plant-released oxalate is
involved in the early steps of colonization of lupin and
maize by P. phytofirmans PsJN (Kost et al., 2014). Inter-
estingly, only plant-associated members of the ‘Bur-
kholderia complex’were found to grow on oxalate while
plant pathogenic and human opportunistic species could
not use it as carbon source, indicating a role of this sugar
in communication between plant and beneficial endo-
phytes (Kost et al., 2014). Volatile organic compounds
(VOCs) play a similar role in the specific selection of
microbial colonizers by plants in the rhizosphere,
phyllosphere as well as anthosphere by either
suppressing the growth of microorganisms or serving as
carbon source for the growth of others (reviewed by
Junker and Tholl, 2013). Although there is already some
indication that endophytic bacteria actively sense the
plant environment and adjust their behavior in response
to changing conditions and plant signals, we still are at
the very beginning to understand the interplay between
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
The bacterial endosphere world 7

plants and endophytes on a molecular level. Given the
growing evidence, that the composition of endophytic
communities may influence plant phenotypic traits
(Li et al., 2019a), obtaining comprehensive understanding
of the selective forces in the plant environment and the
mechanisms of adaptation to this environment by endo-
phytes and endophytic communities may open new ave-
nues to optimize plant productivity and performance via
modulating the endophytic communities.
Improving the performance of endophytes
in promoting plant growth and health
Adaptation of strains to environmental conditions
Due to the many beneficial functions of endophytes for
plant growth and health, there is a huge interest in
applying them for a more sustainable crop production,
e.g. as biofertilizers of crop protection agents. Bacterial
endophytes have a great capability to adapt to changing
environments and can express different phenotypes in
different conditions such as pH, temperature, and host
species. Rapid adaptation of endophytes may be a pre-
requisite for the plant holobiont to better adapt to stress
conditions. Many studies have shown that plants grow-
ing in unfavorable conditions like hot, drought, saline,
and heavy metal contaminated environments can
develop different adaptation capacities to stresses,
which is partially due to their associated microorganisms
(Vandenkoornhuyse et al., 2015; Li et al., 2019a). As
discussed above, endophytes need to show a number
of characteristics to colonize plants internally such as
motility and chemotaxis functions or degradation of reac-
tive oxygen species. Moreover, transcriptome analysis of
the endophyte Bacillus mycoides EC18 showed that an
upregulation of genes involved in amino acids metabo-
lism, transcriptional regulators, and signal transduction
can play an important role in the adaptation of endophytic
strains to their ecological niche (Yi et al., 2017). Scheuerl
et al. (2020) addressed also the question on how the sur-
rounding bacterial community affects evolutionary trajec-
tories of strain adaptation to new niches by studying
environmental samples in artificial micro-ecosystems.
They found that the diversity of surrounding bacterial
communities as well as characteristics of the strain
such as genome size are important factors for strain
adaptation to new environmental conditions (Scheuerl
et al., 2020). Although this study addressed environmen-
tal microorganisms (and not specifically endophytes), this
kind of experimental evolutionary approach can help to
better understand the required traits for successful pene-
tration and internal plant colonization by endophytes.
In endophytes, horizontal gene transfer (HGT) can be
an important natural evolutionary mechanism for host
adaptation and acquisition of novel genes. Sufficient
experimental evidence has proven the implication of HGT
for ecological behavior and biotechnological application.
For example, novel functions acquired by endophytic
strains during HGT events played role in toluene biodeg-
radation and disease control in corn and wheat (Wang
et al., 2010). Moreover, HGT events conferred novel
traits in endophytes, which are important for the degrada-
tion of volatile organic contaminants (Weyens et al.,
2009) and for the resistance against heavy metals
(He et al., 2020). In particular, bacteriophage-mediated
HGT may be a powerful pathway for adaptation and
acquisition of new traits (Obeng et al., 2016; Harrison
and Brockhurst, 2017). Lately, the role of phages has
been primarily addressed in the human gut and a healthy
human gut phageome has been proposed (Manrique
et al., 2016). The role of phage-mediated HGT in the
plant environment is under-investigated but is likely to
play an important role for the adaptation of endophytes to
different plant compartments or physiological conditions
(Pratama et al., 2020). Understanding the key functions
involved in bacterial adaptation including the role of HGT
will be of further importance to benefit from endophytes
to enable holobiont adaptation to stressful conditions.
Genetic improvement of endophytes
Genetic manipulation of non-pathogenic endophytic
strains could be a useful method as an alternative to
genetic modification of the plant (Li et al., 2017). Genes
related to endophytic colonization and adaptation, plant
growth promotion, biocontrol of plant pathogens and
insects can be introduced into strains to confer new traits.
For instance, the Bacillus thuringiensis Bt gene was
introduced to the endosphere colonizer Clavibacter xyli
subsp. cynodontis to produce the endotoxin for insect
control (Tomasino et al., 1995). Similarly, silkworms were
controlled by an insecticidal protein, which was produced
by the endophytic Burkholderia pyrrocinia JK-SH007
transformed with the Bt endotoxin gene (Li et al., 2017).
Moreover, an antifungal gene was incorporated into the
genome of the endophyte Pseudomonas putida
WCS358r for biocontrol of pathogenic Fusarium spp.
(Glandorf et al., 2001). The capability of endophytic colo-
nization of B. thuringiensis has also received special
attention for the development of new type of insect-
resistant crops (Sauka, 2017). Although genetic engi-
neering of endophytes is promising to improve the perfor-
mance of individual microbial strains, the release of
genetically engineered microorganisms is not permitted
in many countries.
Genome editing tools, i.e. the use of the CRISPR-Cas9
system has been shown to be a rapid and efficient tool
for genome engineering and has been utilized efficiently
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
8S. Compant et al.

for genome editing in various organisms including plants,
fungi and bacteria (Deng et al., 2017). However, optimi-
zation is still needed to be used in most microbial taxa.
More recently, Li et al. (2019b) presented an advanced
multiplex site-specific genome engineering (aMSGE) tool
for incorporating multi-locus biosynthetic gene clusters
encoding for natural products in actinomycetes. This
highly efficient approach may be applied to a wide range
of bacteria to enhance synthesis of important
biomolecules.
Design of endophyte consortia
The design and application of microbial consortia are a
new and promising approach to enhance the positive
effects of inoculation on plants. Well-selected microbial
consortia may adapt more rapidly to diverse conditions
by making positive population-level associations, such as
biofilms and microbial mats. Several studies have proven
the advantages of consortia over single strain inoculation
in several agronomic crops (reviewed in the study by
Compant et al., 2019). Multiple strains in endophyte con-
sortia have also been shown to promote plant growth and
to mitigate abiotic stresses in tree species (Aghai
et al., 2019). For many years, microbial strains have been
combined in a non-targeted manner on a trial and error
basis obtaining variable results. However, recently, differ-
ent, knowledge-based approaches have been proposed
to develop microbial consortia showing improved benefit
for their host, either by colonizing more efficiently and/or
by showing enhanced plant growth-promoting activities.
One approach, which has been suggested is to base the
design of microbial consortia on microbial network analy-
sis, as also outlined by Vannier et al. (2019). Particularly,
core microbiome taxa, i.e. a subset of the microbiota
associated with a given host irrespective of the mac-
roenvironment (Lemanceau et al., 2017), are considered
to be highly important for plant fitness and to have
established through evolutionary mechanisms for selec-
tion and enrichment of taxa containing key functional
traits (Lemanceau et al., 2017). Along these lines, Kong
et al. (2018) suggested simplified microbial consortia
(SMC) comprising core microbial strains identified by
microbial community sequencing and network analysis. It
has been further demonstrated that the development
of SMC from complex microbial communities can be
feasible by combining enrichment with the adapted
dilution-to-extinction approach to obtain stable and func-
tional microbial consortia (Kang et al., 2019). However,
also satellite taxa and rare microbiome taxa have been
reported to have important functions (Hol et al., 2015).
Therefore, a more detailed understanding on the role of
core and satellite taxa to different microbiome functions
is required to further improve a microbiome-based design
of microbial consortia.
A second approach for the design of microbial consor-
tia is to make use of microbial consortia with synergistic
or complementary functions including high persistence in
the target environment. Here, a deep functional under-
standing of microbial genomes will help to design such
consortia. Secondary metabolites produced by endo-
phytes, for example, play an important role in biofilm for-
mation, plant colonization and in suppressing plant
diseases or by activating plant defense (Brader
et al., 2017). The rapid evolution of bacterial genomes
and metagenomes data has led increasingly to genome
mining to identify mechanisms and novel bioactive com-
pounds. For instance, Belbahri et al. (2017) used various
genome mining tools to predict core and accessory gene
clusters in all sequenced Bacillus amyloliquefaciens
genomes to evaluate their potential to synthesize sec-
ondary metabolites and to promote plant growth. They
found that particularly the accessory part of the analyzed
genomes harbor gene clusters largely related to second-
ary metabolite production, whereas genes in the core
genome were suggested to play an important role in
strain adaptation to plant-associated habitats (Belhabri
et al., 2017). Improved genome prediction of microbial
functions in relation to plant performance but also in rela-
tion to survival and activity under different, ecological
conditions will lead to the improved design of microbial
consortia.
A third approach, which has been proposed, is based
on an ecological framework (Hu et al., 2016, 2017). The
authors showed that the survival of randomly combined,
closely related Pseudomonas strains increased with
increasing consortium diversity. Increased Pseudomonas
diversity applied onto potato plants also led to increased
out-competition and antagonism of the plant pathogen
Ralstonia solancearum (Hu et al., 2016) as well as to a
higher production of plant hormones, siderophores and
assimilation of nutrients (Hu et al., 2017). This approach
needs to be further tested and validated for other taxa
and applications. Nevertheless, irrespective of the
approach how to design microbial consortia, appropriate
formulations and carrier/delivery systems to the crops
should be considered to overcome the environmental
constraints, which might affect the functioning of consor-
tia in the field conditions (Sessitsch et al., 2019).
Methodological challenges and limitations to study
endophytes
Endophyte research has received increasing attention in
the last years. However, due to the presence of the plant
host organelles resembling bacterial cells as well as gen-
erally rather low bacterial densities, methodological
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
The bacterial endosphere world 9

considerations are important to address. An overview on
the experimental flow for hypothesis-driven and cross-
disciplinary endophyte research is shown in Fig. 2.
The first step of endophyte studies, surface-sterilization,
should be standardized
Endophytes can be studied at various biological organi-
zation levels, from the strain to the community. Regard-
less of the level, the first step is to remove epiphytes
from the surface of plant organs, without altering the
endophytic community. This step is important, but there
is no consensus on how to remove epiphytic bacteria
and no standardized protocols for each plant organ.
There is also less quality control at this stage than at
stages that require more advanced techniques, such as
next generation sequencing (NGS) techniques. Recently,
Chen et al. (2020a) surface-sterilized leaves of Ara-
bidopsis thaliana with 75% ethanol for 1 min before
isolating endophytes, or with 5% bleach (NaClO) for
1 min before metabarcoding analysis. Morella et al. (2019)
surface-sterilized tomato fruits by soaking them in 75%
ethanol for 20 min, and surface-sterilized tomato seeds
by first sonicating seeds to remove epiphytes, then steril-
ized them with 2.7% bleach for 20 min. They verified, by
plating, that no culturable bacteria were present in post-
sterilization washes with sterile water. Bergna et al. (2018)
surface-sterilized tomato roots by soaking them in 3%
bleach for 5 min and imprinted them on agar plates as a
sterility check. Wemheuer et al. (2019) used serial
washes of ethanol and bleach to surface-sterilize maple
tree leaves and checked the effectiveness of sterilization
by PCR amplification of the 16S rRNA gene in final
washes. Tween surfactants are sometimes used to
improve the detachment of epiphytes (Rodríguez et al.,
2019), and combinations of cultivation and PCR have
been used for checking epiphyte removal (Wemheuer
et al., 2020). Surface-sterilization by flaming has also
Fig. 2. Experimental flowchart for hypothesis-driven and cross-disciplinary endophyte research. To unravel plant endophyte functions, 1) the
study goal needs to be defined, including the hypothesis to test. 2) The sampling design should allow to test the hypothesis or to collect the endo-
phytic strains that will be necessary to test the hypothesis. 3) Plant tissue samples should be surface-sterilized to remove epiphytic microbes and
their DNA, and the effectiveness of sterilization should be checked. This step, which relies on low-cost and low-tech methods, is often over-
looked. 4.1) Taxonomic and functional information about endophytes are usually required to test the hypothesis. Meta-omic approaches are part
of the multitude of methods available, but they are not the only ones. Their use cannot by itself justify a study. 4.2) Culture-dependent
approaches are complementary to meta-omic approaches. Isolated strains or synthetic communities of endophytes can be inoculated to assess
their influence on plant physiology. 5) The data should be analyzed statistically to test the hypothesis. This step requires advanced bioinformatic
and statistical methods because meta-omic approaches provide heterogeneous and high-dimensional datasets that need to be reduced and inte-
grated to make sense.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
10 S. Compant et al.

been used for studying the stem endosphere in grape-
vine (Deyett and Rolshausen, 2020). Recently, Binetruy
et al. (2019) showed that the choice of sterilization proto-
col has a major impact on the measure of microbial diver-
sity associated with insects and even suggested a
reconsideration of results of previous studies. We should,
therefore, not underestimate the variability triggered by
protocols of surface-sterilization and sterility check
(or bacterial DNA absence check) in the studies of plant
endophytes. This variability will induce biases in meta-
analyses of sequence datasets, which will increasingly
be used in the future to get generic results on the plant
microbiome (Rocca et al., 2018). A standardization of this
first step (Fig. 2), at least for major crops and model plant
species, therefore seems highly important, also because
the following steps are generally costly and time-consum-
ing, especially if they require NGS. Recently, Saldierna
Guzmán et al. (2020) performed a comparison of leaf
sterilization protocols for two species, an angiosperm and
a gymnosperm. They found that complete removal of the
cuticle was required to achieve the sterilization of leaf
surface in both species, but that the most effective
reagents to remove the cuticle without damaging the
integrity of leaf tissues differed between species. They
also demonstrated that scanning electron microscopy
(SEM) is more efficient than PCR and imprints in check-
ing sterility. Further studies, along the lines of this first
study, are needed to determine whether the results are
generalizable to other species in the angiosperm and
gymnosperm groups.
Traditional methods have still a role to play in endophyte
studies
NGS techniques have revolutionized our understanding
of the plant microbiome over the past decade and their
benefits no longer need to be demonstrated.
Metabarcoding approaches, in particular, have become
standard in studying the structure of plant endophytic
bacterial communities (e.g. Gdanetz and Trail, 2017;
Donald et al., 2019; Vergine et al., 2019; Wemheuer
et al., 2019; Chen et al., 2020a, 2020b; Deyett and
Rolshausen, 2020; Kuźniar et al., 2020), because they
encompass both the cultivable and non-cultivable fraction
of the bacterial diversity. However, these approaches
have biases and limitations (Beckers et al., 2016; Lucaciu
et al., 2019; Zinger et al., 2019). A major issue, in the
specific case of bacterial endophytes, is the presence of
plant chloroplasts. Specific primers and blocking primers
were developed to avoid their amplification (Chelius and
Triplett, 2001; Redford et al., 2010; Fitzpatrick
et al., 2018) but despite these developments,
amplification of genes from bacterial endophytes is still
challenging. Therefore, more traditional methods, such
as isolation, culture and fingerprinting, have been
maintained and are becoming popular again. For
instance, traditional fingerprinting methods, such as ter-
minal restriction fragment length polymorphism (T-RLFP),
can yield results comparable to recent metabarcoding
approaches regarding the structure of plant bacterial
endophytic communities, but their cost is much lower,
making them accessible to a larger scientific community
(Johnston-Monje and Mejia, 2020). Compared to second-
generation metabarcoding approaches, isolation and
culture of bacterial endophytes permit more accurate tax-
onomic identification through the sequencing of longer
portions of marker genes (Asghari et al., 2019) or whole-
genome sequencing (WGS) (López-Fernández
et al., 2015; Chaudhry et al., 2017; Jauri et al., 2019;
Eida et al., 2020). They also permit the phenotypic char-
acterization of isolates and the identification of the genes
and metabolic pathways governing the phenotype. More-
over, culture-dependent approaches pave the way for
experimentation on plant–endophyte interactions. For
example, isolated strains can be reassembled to form
synthetic communities (SynCom) (Paredes et al., 2018;
Carlström et al., 2019; Liu et al., 2019; Vannier
et al., 2019), which can then be re-inoculated to assess
their role on plant growth and health. Chen et al. (2020a)
used such experimental approach, in A. thaliana, to dem-
onstrate the causal role of the foliar endophytic bacterial
community in plant health. Several recent studies com-
bined metabarcoding approaches with culture-dependent
approaches to go beyond the simple taxonomic descrip-
tion of endophytic communities associated with plant
organs. Donald et al. (2019) combined metabarcoding
data with co-occurrence analyses and co-cultures, to
decipher interactions between foliar endophytes of tropi-
cal palms. Gdanetz and Trail (2017) also combined
metabarcoding approaches with microbiological assays
to identify antagonists of a major fungal pathogen in
wheat. However, those culture-dependent methods are
still challenging as many plant endophytes are yet
unculturable or need specific plant-based culture media
(see Sarhan et al., 2019 for a review). In the future, it
may be useful to invest in the development of plant-
based culture media to isolate and characterize more
endophyte species, and to re-invest in low-throughput
culture-dependent methods and traditional fingerprinting
methods to promote the study of endophytes of a larger
range of plant species, including those from low-income
countries. In parallel, where possible, we should of
course continue to use and develop -omics technologies
to better understand the role of bacterial endophytes for
the plant response to biotic and abiotic stresses.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
The bacterial endosphere world 11

Multi-omic approaches are the future the field but
conceptual frameworks should develop in parallel
A multitude of -omics tools –including (meta)genomics,
(meta)transcriptomics, (meta)proteomics, metabolomics,
and also culturomics –are now available to understand
the functional role of endophytic communities (Kaul
et al., 2016; Levy et al., 2018; Sarhan et al., 2019). Levy
et al. (2018) recently reviewed the objectives, strengths
and limitations of each of these tools. A shared feature of
all these tools is that they evolve extremely fast and pro-
vide high dimension and heterogeneous datasets that
need to be reduced and integrated to elucidate valuable
information, using advanced bioinformatics and statistical
approaches (Rohart et al., 2017). All these tools give
complementary insights into the function of bacterial
endophytes. For instance, Sessitsch et al. (2012)
pioneered in the field of metagenomics, also called shot-
gun metagenomics, by identifying the genes, traits and
metabolic processes that are important for the endophytic
lifestyle. Recently, Terra et al. (2019) combined a trans-
criptomic and proteomic approach, to identify the meta-
bolic pathways and functions that are activated, in an
endophytic bacterial strain that promotes growth in sugar-
cane, when it is exposed to the apoplast fluid. These
-omics methods can be complemented by other
approaches, such as confocal microscopy, which, after
inoculation of a strain into an axenic plant, makes it pos-
sible to verify that the strain is indeed an endophyte and
to identify the tissues it colonizes (Bünger et al., 2020).
Agtuca et al. (2020) combined confocal and fluorescence
imaging with in situ metabalomics to discover the metab-
olites that are over-expressed in plant tissues colonized
by an endophytic bacterium. The future challenge in plant
endophyte research is, however, not only to master and
combine advanced tools, but also to link research ques-
tions to conceptual frameworks (Saikkonen et al., 2020).
Multi-omics approaches are undoubtedly the future of the
field and will provide microbiology-based innovations in
the field of agriculture (Compant et al., 2019). However,
endophyte research should not only be driven by techni-
cal developments, fluxes of data and bioeconomy, but
also by hypotheses in the fields of ecology and evolution
(Saikkonen et al., 2020). To this end, we need to develop
conceptual frameworks that consider the effects of the
plant microbial community on the plant phenotype (and
vice et versa), and upscale these results across space
and time. Such a framework has to consider the whole
system, i.e. the host plant, the surrounding microbiome,
other organisms in the plant environment as well as the
environment with all structural and functional aspects and
multitrophic interactions. The development of these
frameworks will require interdisciplinary approaches that
go beyond -omics methods, by integrating evolutionary
ecology, plant physiology and pathology, plant genetics
and epigenetics, and Earth and ecosystem sciences.
They will allow us to better understand the functioning
and evolution of plant holobionts and to predict their
response and feedback effects on environmental
changes, at the global scale (Saikkonen et al., 2020; Zhu
and Penuelas, 2020).
Conclusions and future prospects
The plant endosphere represents a highly diverse and
dynamic environment for bacteria, providing multiple
niches in different plant compartments for proliferation.
Due to plant development as well as due to fluctuating
environmental conditions endophytes are exposed to
highly dynamic conditions as plants respond to changing
conditions associated with physiological changes. In the
last years, we have accumulated knowledge on microbial
community dynamics in the various plant micro-environ-
ments, however, full understanding on which physiologi-
cal conditions favor which microorganisms is still lacking.
Here, also advanced knowledge on the identity and
dynamics of plant metabolites will be needed as well as
the linkage between microbiomes, plant and microbial
metabolites and environmental/physiological conditions.
Similarly, we have obtained knowledge on the potential
activities on plant-associated microorganisms including
endophytes, either by lab assays, genome or
metagenome analysis. However, the activities performed
in planta are poorly understood, particularly considering
the diversity of micro-environments and conditions endo-
phytes are exposed to. To fully understand the role and
contribution of endophytes to plant performance and
functioning we need to address in planta activities, linking
colonization and cell numbers in various plant tissues
and at various plant growth stages with activity levels.
Activity levels may be assessed, e.g. by transcriptome
analysis, however, other complementary -omics
approaches like metabolomics will be further useful to
understand microbial functions and activities in the endo-
sphere. Furthermore, a better understanding of the plant
response to endophytes, particularly in context of envi-
ronmental conditions, will be required as well as a better
understanding on the interplay between plants and endo-
sphere microbiota at a molecular level. Also, microbiome
research has to move from analyzing structural composi-
tion of microbiota to understanding functional aspects,
e.g. of core taxa being consistently associated with a
given plant species and satellite microbiota.
Understanding microbial communities and their activi-
ties in the plant endosphere is challenged by methodo-
logical constraints. The presence of transcriptomes and
genomes from the plant host or from microorganisms in
the phyllosphere and rhizosphere challenges any in
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
12 S. Compant et al.

planta analysis of endophytes. These constrains com-
bined with huge data amounts starting to arise from
-omics studies lead to a real need for the standardization
of protocols, e.g. for surface sterilization or sample prepa-
ration. Hypothesis-driven concepts and research
approaches combined with multi-disciplinary analysis
(Fig. 2) will lead to a better understanding of endophyte
activity and adaptation to their host plant, plant–
endophyte interactions, and overall, the evolution of the
plant holobiont. This knowledge will play a key role in the
design of microbial consortia showing synergistic activi-
ties to contribute to a more sustainable, microbe-based
crop production.
Acknowledgements
M.C.C. post-doctoral grant is funded by the Consortium Bio-
contrôle (BCMicrobiome) and the ANR NGB (ANR-
17-CE32-0011). C.V. has received funding from the LABEX
COTE (ANR-10-LABX-45, MicroMic) and the LABEX CEBA
(ANR-10-LABX-25-01, Drought and Vertige).
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