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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 ,
Marine C. Cambon ,
Corinne Vacher ,
Birgit Mitter ,
Abdul Samad
and Angela Sessitsch
Center for Health and Bioresources, Bioresources Unit,
Konrad Lorenz Straße 24, AIT Austrian Institute of
Technology, Tulln, A-3430, Austria.
INRAE, Univ. Bordeaux, BIOGECO, Pessac, F-33600,
Natural Resources Canada, Canadian Forest Service,
Laurentian Forestry Centre, Québec, G1V4C7, Canada.
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 ndings 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.
For a long time, the scientic 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 xing 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-xing 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 bene-
cial effects of plant growth-promoting rhizobacteria
(PGPR). The pioneering work of Johanna Döbereiner on
specic bacteria which, like Herbaspirillum seropedicae,
colonize the endosphere of sugarcane and x 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 ngerprinting 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, denitions 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.; 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), 0000 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 denition 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
In the last decade, host-associated microbiota have
gained increasing attention, triggered by spectacular nd-
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
(owers, fruits/seeds). Bacterial densities inside plant tis-
sues typically range from 10
to 10
of cultivable cells per
gram of root to 10
in leaves and stems. In owers,
fruits and seeds typically 10
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-favorablebacterial 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 benecial 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, owers, fruits and seeds
show different chemical conditions, in terms of organic
acids, carbohydrates, vitamins, sugars, but also hormones,
amino acids, fatty acids, avonoids, 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 specic 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 inuenced 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 xed 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 inuence 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 owers, 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 ower and fruit environments showing specic 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 specic 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 owers 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 tness (Shade
et al., 2017; Escobar Rodríguez et al., 2020).
Plant compartments and bacterial microbiota
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, Chloroexi,
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 inuence 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 inuenced 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 Grifn (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
Grifn, 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 uorescence in situ hybridization using general and specic probes targeting bacterial taxa or green (C, E,
G, I, J, L, M) due to general staining with Syto9
. Scale of a bacterium: 12μm.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
4S. Compant et al.
some plant benecial 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 benecial 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 benecial 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 benecial 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
benecial endophyte Paraburkholderia phytormans
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 ssures 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 owers, 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 ower recepta-
cle, the petal, sepal and anthers with their laments 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 owers 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
oral 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 benecial endophyte P. phytormans strain PsJN can
colonize the seed embryo of pepper, tomato, wheat and
maize, using the oral 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, agella, 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-
ication 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 signi-
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 eld.
The benets 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 eld 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 xation, nitrication and denitrication 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 signicant 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 phytormans PsJN senses and responds to
plants under osmotic stress. Also, soil contamination was
shown to inuence 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
denition 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, ghting 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 inuence 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 specic 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 specic 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. phytormans
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
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 oral 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 ndings
into consideration, it is not surprising that based on meta-
genomics data genes for the detoxication 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 specically
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 avonoids 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 agella 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. phytormans PsJN (Kost et al., 2014). Inter-
estingly, only plant-associated members of the Bur-
kholderia complexwere 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 benecial endo-
phytes (Kost et al., 2014). Volatile organic compounds
(VOCs) play a similar role in the specic 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 inuence 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 benecial 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 articial 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 specically 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. Sufcient
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 benet 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 modication 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 efcient tool
for genome engineering and has been utilized efciently
© 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-specic genome engineering (aMSGE) tool
for incorporating multi-locus biosynthetic gene clusters
encoding for natural products in actinomycetes. This
highly efcient approach may be applied to a wide range
of bacteria to enhance synthesis of important
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
biolms 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 benet
for their host, either by colonizing more efciently 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 tness 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 simplied microbial consortia
(SMC) comprising core microbial strains identied 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 biolm 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
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 eld conditions (Sessitsch et al., 2019).
Methodological challenges and limitations to study
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 ow for hypothesis-driven and cross-
disciplinary endophyte research is shown in Fig. 2.
The rst 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 rst 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 rst sonicating seeds to remove epiphytes, then steril-
ized them with 2.7% bleach for 20 min. They veried, 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 amplication of the 16S rRNA gene in nal
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 aming has also
Fig. 2. Experimental owchart for hypothesis-driven and cross-disciplinary endophyte research. To unravel plant endophyte functions, 1) the
study goal needs to be dened, 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 inuence 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
rst 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 efcient than PCR and imprints in check-
ing sterility. Further studies, along the lines of this rst
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
NGS techniques have revolutionized our understanding
of the plant microbiome over the past decade and their
benets 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
specic case of bacterial endophytes, is the presence of
plant chloroplasts. Specic primers and blocking primers
were developed to avoid their amplication (Chelius and
Triplett, 2001; Redford et al., 2010; Fitzpatrick
et al., 2018) but despite these developments,
amplication of genes from bacterial endophytes is still
challenging. Therefore, more traditional methods, such
as isolation, culture and ngerprinting, have been
maintained and are becoming popular again. For
instance, traditional ngerprinting 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 scientic community
(Johnston-Monje and Mejia, 2020). Compared to second-
generation metabarcoding approaches, isolation and
culture of bacterial endophytes permit more accurate tax-
onomic identication 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 identication of the genes
and metabolic pathways governing the phenotype. More-
over, culture-dependent approaches pave the way for
experimentation on plantendophyte 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 specic 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 ngerprinting
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 eld 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 eld 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 uid. 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 uorescence
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
eld and will provide microbiology-based innovations in
the eld of agriculture (Compant et al., 2019). However,
endophyte research should not only be driven by techni-
cal developments, uxes of data and bioeconomy, but
also by hypotheses in the elds 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 uctuating
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.
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).
Afzal, I., Shinwari, Z.K., Sikandar, S., and Shahzad, S.
(2019) Plant benecial endophytic bacteria: mechanisms,
diversity, host range and genetic determinants. Microbiol
Res 221:3649.
Aghai, M.M., Khan, Z., Joseph, M.R., Stoda, A.M., Sher, A.W.,
Ettl, G.J., and Doty, S.L. (2019) The effect of microbial
endophyte consortia on Pseudotsuga menziesii and Thuja
plicata survival, growth, and physiology across edaphic gra-
dients. Front Microbiol 10: 1353.
Agtuca, B.J., Stopka, S.A., Tuleski, T.R., do Amaral, F.P.,
Evans, S., Liu, Y., et al. (2020) In-situ metabolomic analy-
sis of Setaria viridis roots colonized by benecial endo-
phytic bacteria. Mol Plant Microbe Interact 33: 272283.
Alquéres, S., Meneses, C., Rouws, L., Rothballer, M.,
Baldani, I., Schmid, M., and Hartmann, A. (2013) The bac-
terial superoxide dismutase and glutathione reductase are
crucial for endophytic colonization of rice roots by Glu-
conacetobacter diazotrophicus PAL5. Mol Plant Microbe
Interact 26: 937945.
Amend, A.S., Cobian, G.M., Laruson, A.J., Remple, K.,
Tucker, S.J., Poff, K.E., et al. (2019) Phytobiomes are
compositionally nested from the ground up. PeerJ 7:
Ampomah, O.Y., Avetisyan, A., Hansen, E., Svenson, J.,
Huser, T., Jensen, J.B., and Bhuvaneswari, T. (2013) The
thuEFGKAB operon of rhizobia and Agrobacterium
tumefaciens code for transport of trehalose, maltitol and
isomers of sucrose and their assimilation through the for-
mation of their 3-ketoderivatives. J Bacteriol 195:
Asghari, S., Harighi, B., Mozafari, A.A., Esmaeel, Q., and Ait
Barka, E. (2019) Screening of endophytic bacteria isolated
from domesticated and wild growing grapevines as poten-
tial biological control agents against crown gall disease.
BioControl 64: 723735.
Baldani, J.I., Baldani, V.L.D., Seldin, L., and Doöbereiner, J.
(1986) Characterization of Herbaspirillum seropedicae
gen. Nov., sp. nov., a root-associated nitrogen-xing bac-
terium. Int J Syst Bacteriol 36:8693.
Barret, M., Briand, M., Bonneau, S., Préveaux, A.,
Valière, S., Bouchez, O., et al. (2015) Emergence shapes
the structure of the seed microbiota. Appl Environ
Microbiol 81: 12571266.
Barret, M., Guimbaud, J.F., Darrasse, A., and Jacques, M.A.
(2016) Plant microbiota affects seed transmission of phy-
topathogenic micro-organisms. Mol Plant Pathol 17:
Beckers, B., Beeck, M.O.D., Thijs, S., Truyens, S.,
Weyens, N., Boerjan, W., and Vangronsveld, J. (2016)
Performance of 16S rDNA primer pairs in the study of rhi-
zosphere and endosphere bacterial microbiomes in
metabarcoding studies. Front Microbiol 7: 650.
Beijerinck, M.W. (1888) Cultur des Bacillus radicola aus den
Knöllchen. Bot Ztg 46: 740750.
Belbahri, L., Chenari Bouket, A., Rekik, I., Alenezi, F.N.,
Vallat, A., Luptakova, L., et al. (2017) Comparative genomics
of Bacillus amyloliquefaciens strains reveals a core genome
with traits for habitat adaptation and a secondary metabolites
rich accessory genome. Front Microbiol 8: 1438.
Berg, G., and Raaijmakers, J.M. (2018) Saving seed
microbiomes. ISME J 12: 11671170.
Bergna, A., Cernava, T., Rändler, M., Grosch, R.,
Zachow, C., and Berg, G. (2018) Tomato seeds preferably
transmit plant benecial endophytes. Phytobiomes J 2:
Binetruy, F., Dupraz, M., Buysse, M., and Duron, O. (2019)
Surface sterilization methods impact measures of internal
microbial diversity in ticks. Parasites Vectors 12: 268.
Boddey, R.M., and Döbereiner, J. (1988) Nitrogen xation
associated with grasses and cereals: recent results and
perspectives for future research. Plant Soil 108:5365.
Böhm, M., Hurek, T., and Reinhold-Hurek, B. (2007)
Twitching motility is essential for endophytic rice coloniza-
tion by the N
-xing endophyte Azoarcus sp. strain BH72.
Mol Plant Microbe Interact 20: 526533.
Borruso, L., Wellstein, C., Bani, A., Casagrande, S.,
Margoni, A., Tonin, R., et al. (2018) Temporal shifts in
endophyte bacterial community composition of sessile oak
(Quercus petraea) are linked to foliar nitrogen, stomatal
length, and herbivory. PeerJ 6: e5769.
Brader, G., Compant, S., Vescio, K., Mitter, B., Trognitz, F.,
Ma, L.-J., and Sessitsch, A. (2017) Ecology and genomic
insights into plant-pathogenic and plant-nonpathogenic
endophytes. Annu Rev Phytopathol 55:6183.
Braeken, K., Daniels, R., Ndayizeye, M., Vanderleyden, J.,
and Michiels, J. (2008) Quorum sensing in bacteria-plant
interactions. In Molecular Mechanisms of Plant and
Microbe Coexistence. Soil Biology Vol. 15, Nautiyal, C.S.,
and Dion, P. (eds). Berlin, Heidelberg: Springer, pp. 265289.
Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van
Themaat, E., and Schulze-Lefert, P. (2013) Structure and
functions of the bacterial microbiota of plants. Annu Rev
Plant Biol 64: 807838.
Bulgari, D., Bozkurt, A.I., Casati, P., Ca
glayan, K.,
Quaglino, F., and Bianco, P.A. (2012) Endophytic bacterial
community living in roots of healthy and Candidatus
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
The bacterial endosphere world 13
Phytoplasma Mali-infected apple (Malus domestica,
Borkh.) trees. Antonie Van Leeuwenhoek 102: 677687.
Bünger, W., Jiang, X., Müller, J., Hurek, T., and Reinhold-
Hurek, B. (2020) Novel cultivated endophytic Verrucomicrobia
reveal deep-rooting traits of bacteria to associate with plants.
Sci Rep 10: 8692.
Carlström, C.I., Field, C.M., Bortfeld-Miller, M., Müller, B.,
Sunagawa, S., and Vorholt, J.A. (2019) Synthetic micro-
biota reveal priority effects and keystone strains in the
Arabidopsis phyllosphere. Nat Ecol Evol 3: 14451454.
Carrión, V.J., Perez-Jaramillo, J., Cordovez, V., Tracanna, V.,
de Hollander, M., Ruiz-Buck, D., et al. (2019) Pathogen-
induced activation of disease-suppressive functions in the
endophytic root microbiome. Science 366:606612.
Carter, C., and Thornburg, R.W. (2004) Is the nectar redox
cycle a oral defense against microbial attack? Trends
Plant Sci 9: 320324.
Chaudhry, V., Sharma, S., Bansal, K., and Patil, P.B. (2017)
Glimpse into the genomes of rice endophytic bacteria:
diversity and distribution of Firmicutes. Front Microbiol 7:
Chelius, M.K., and Triplett, E.W. (2001) The diversity of
archaea and bacteria in association with the roots of Zea
mays L. Microb Ecol 41: 252263.
Chen, T., Nomura, K., Wang, X., Sohrabi, R., Xu, J., Yao, L.,
et al. (2020a) A plant genetic network for preventing
dysbiosis in the phyllosphere. Nature 580: 653657.
Chen, X., Krug, L., Yang, H., Li, H., Yang, M., Berg, G., and
Cernava, T. (2020b) Nicotiana tabacum seed endophytic
communities share a common core structure and
genotype-specic signatures in diverging cultivars. Com-
put Struct Biotechnol J 18: 287295.
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 Biol Biochem 42: 669678.
Compant, S., Kaplan, H., Sessitsch, A., Nowak, J., Ait
Barka, E., and Clément, C. (2008) Endophytic colonization
of Vitis vinifera L. by Burkholderia phytormans strain
PsJN: from the rhizosphere to inorescence tissues.
FEMS Microbiol Ecol 63:8493.
Compant, S., Mitter, B., Colli-Mull, J.G., Gangl, H., and
Sessitsch, A. (2011) Endophytes of grapevine owers,
berries, and seeds: identication of cultivable bacteria,
comparison with other plant parts, and visualization of
niches of colonization. Microb Ecol 62: 188197.
Compant, S., Reiter, B., Sessitsch, A., Nowak, J.,
Clément, C., and Ait Barka, E. (2005) Endophytic coloni-
zation of Vitis vinifera L. by plant growth-promoting bacte-
rium Burkholderia sp. strain PsJN. Appl Environ Microbiol
71: 16851693.
Compant, S., Saikkonen, K., Mitter, B., Campisano, A., and
Mercado-Blanco, J. (2016) Editorial special issue: soil,
plants and endophytes. Plant Soil 405:111.
Compant, S., Samad, A., Faist, H., and Sessitsch, A. (2019)
A review on the plant microbiome: ecology, functions, and
emerging trends in microbial application. J Adv Res 19:
Compant, S., Sessitsch, A., and Mathieu, F. (2012) The
anniversary of the rst postulation of the soil origin
of endophytic bacteria a tribute to M.L.V. Galippe. Plant
Soil 356: 299301.
Correa-Galeote, D., Bedmar, E.J., and Arone, G.J. (2018)
Maize endophytic bacterial diversity as affected by soil
cultivation history. Front Microbiol 9: 484.
Darrasse, A., Barret, M., Cesbron, S., Compant, S., and
Jacques, M.A. (2018) Niches and routes of transmission
of Xanthomonas citri pv. fuscans to bean seeds. Plant Soil
422: 115128.
Darsonval, A., Darrasse, A., Meyer, D., Demarty, M.,
Durand, K., Bureau, C., et al. (2008) The type III secretion
system of Xanthomonas fuscans subsp. fuscans is
involved in the phyllosphere colonization process and in
transmission to seeds of susceptible beans. Appl Environ
Microbiol 74: 26692678.
Deng, H., Gao, R., Liao, X., and Cai, Y. (2017) CRISPR sys-
tem in lamentous fungi: current achievements and future
directions. Gene 627: 212221.
Deveau, A., Bonito, G., Uehling, J., Paoletti, M., Becker, M.,
Bindschedler, S., et al. (2018) Bacterial-fungal interac-
tions: ecology, mechanisms and challenges. FEMS
Microbiol Rev 42: 335352.
Deyett, E., and Rolshausen, P.E. (2020) Endophytic microbial
assemblage in grapevine. FEMS Microbiol Ecol 96:aa053.
Dörr, J., Hurek, T., and Reinhold-Hurek, B. (1998) Type IV
pili are involved in plantmicrobe and fungusmicrobe
interactions. Mol Microbiol 30:717.
Donald, J., Barthélemy, M., Gazal, N., Eveno, Y., Manzi, S.,
Eparvier, V., et al. (2019) Tropical palm endophytes
exhibit low competitive structuring when assessed using
co-occurrence and antipathogen activity analysis. Front
For Glob Change 2: 86.
Duijff, B.J., Gianinazzi-Pearson, V., and Lemanceau, P.
(1997) Involvement of the outer membrane lipopolysac-
charides in the endophytic colonization of tomato roots by
biocontrol Pseudomonas uorescens strain WCS417r.
New Phytol 135: 325334.
Eida, A.A., Bougouffa, S., Alam, I., Hirt, H., and Saad, M.M.
(2020) Complete genome sequence of Paenibacillus
sp. JZ16 a plant growth promoting root endophytic bacte-
rium of the desert halophyte Zygophyllum simplex.Curr
Microbiol 77: 10971103.
Escobar Rodríguez, C., Antonielli, L., Mitter, B., Trognitz, F.,
and Sessitsch, A. (2020) Heritability and functional impor-
tance of the Setaria viridis bacterial seed microbiome.
Phytobiomes J 4:4052.
Escobar Rodríguez, C., Mitter, B., Antonielli, L., Trognitz, F.,
Compant, S., and Sessitsch, A. (2018a) Roots and pani-
cles of the C4 model grasses Setaria viridis (L). And
S. pumila host distinct bacterial assemblages with core
taxa conserved across host genotypes and sampling
sites. Front Microbiol 9: 2708.
Escobar Rodríguez, C., Mitter, B., Barret, M., Sessitsch, A., and
Compant, S. (2018b) Commentary: seed bacterial inhabitants
and their routes of colonization. Plant Soil 422: 129134.
Fitzpatrick, C.R., Lu-Irving, P., Copeland, J., Guttmann, D.S.,
Wang, P.W., Baltrus, D.A., et al. (2018) Chloroplast
sequence variation and the efcacy of peptide nucleic
acids for blocking host amplication in plant microbiome
studies. Microbiome 6: 144.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
14 S. Compant et al.
Fürnkranz, M., Lukesch, B., Müller, H., Huss, H., Grube, M.,
and Berg, G. (2012) Microbial diversity inside pumpkins:
microhabitat-specic communities display a high antago-
nistic potential against phytopathogens. Microb Ecol 63:
Galippe, V. (1887) Note sur la presence de micro-
organismes dans les tissues végétaux. C R Hebd Sci
Mem Soc Biol 39: 410416.
Gdanetz, K., and Trail, F. (2017) The wheat microbiome
under four management strategies and potential for endo-
phytes in disease protection. Phytobiomes J 1: 158168.
Gerna, D., Roach, T., Mitter, B., Stöggl, W., and Kranner, I.
(2020) Hydrogen peroxide metabolism in interkingdom
interaction between bacteria and wheat seeds and seed-
lings. Mol Plant Microbe Interact 33: 336348.
Geurts, R., and Bisseling, T. (2002) Rhizobium nod factor
perception and signalling. Plant Cell 14: S239S249.
Glandorf, D.C., Verheggen, P., Jansen, T., Jorritsma, J.-W.,
Smit, E., Leeang, P., et al. (2001) Effect of genetically
modied Pseudomonas putida WCS358r on the fungal
rhizosphere microora of eld-grown wheat. Appl Environ
Microbiol 67: 33713378.
Glassner, H., Zchori-Fein, E., Compant, S., Sessitsch, A.,
Katzir, N., Portnoy, V., and Yaron, S. (2015) Characteriza-
tion of endophytic bacteria from cucurbit fruits with poten-
tial benets to agriculture in melons (Cucumis melo L.).
FEMS Microbiol Ecol 91:v074.
Glassner, H., Zchori-Fein, E., Yaron, S., Sessitsch, A.,
Sauer, U., and Compant, S. (2018) Bacterial niches inside
seeds of Cucumis melo L. Plant and Soil 422: 101113.
Hardoim, P.R., Van Overbeek, L.S., Berg, G., Pirttilä, A.M.,
Compant, S., Campisano, A., et al. (2015) The hidden
world within plants: ecological and evolutionary consider-
ations for dening functioning of microbial endophytes.
Microbiol Mol Biol Rev 79: 293320.
Harrison, E., and Brockhurst, M.A. (2017) Ecological and
evolutionary benets of temperate phage: what does or
doesnt kill you makes you stronger. Bioessays 39:
Harrison, J.G., and Grifn, E.A. (2020) The diversity and dis-
tribution of endophytes across biomes, plant phylogeny
and host tissues: how far have we come and where do we
go from here? Environ Microbiol 22: 21072123. https://
Hartmann, A., Rothballer, M., Hense, B.A., and Schröder, P.
(2014) Bacterial quorum sensing compounds are impor-
tant modulators of microbe-plant interactions. Front Plant
Sci 5: 131.
He, W., Megharaj, M., Wu, C.-Y., Subashchandrabose, S.R.,
and Dai, C.-C. (2020) Endophyte-assisted
phytoremediation: mechanisms and current application
strategies for soil mixed pollutants. Crit Rev Biotechnol
Hellriegel, H., and Wilfarth, H. (1888) Untersuchung über die
Stickstoffnahrung der Gramineen und Leguminosen.
Berlin, Germany: Buchdruckerei der "Post" Kayssler.
Hiltner, L. (1904) Über neuere Erfahrungen und Probleme
auf dem Gebiete der Bodenbakteriologie unter besonderer
Berücksichtigung der Gründüngung und Brache. Arb DLG
Hol, W.H.G., de Boer, W., Hollander, M.D., Kuramae, E.E.,
Meisner, A., and van der Putten, W.H. (2015) Context
dependency and saturating effects of loss of rare soil
microbes on plant productivity. Front Plant Sci 6: 485.
Holyoak, M., Leibold, M.A., and Holt, R.D. (2005) Meta-
communities: Spatial Dynamics and Ecological Communi-
ties. Chicago, IL; London: The University of Chicago Press.
Hounsome, N., Hounsome, B., Tomos, D., and Edwards-
Jones, G. (2008) Plant metabolites and nutritional quality
of vegetables. J Food Sci 73: R48R65.
Hu, J., Wei, Z., Friman, V.-P., Gu, S.-H., Wang, X.-F.,
Eisenhauer, N., et al. (2016) Probiotic diversity enhances
rhizosphere microbiome function and plant disease sup-
pression. MBio 7: e01790e01816.
Hu, J., Wei, Z., Weidner, S., Friman, V.-P., Xu, Y.-C.,
Shen, Q.-R., and Jousset, A. (2017) Probiotic Pseudomo-
nas communities enhance plant growth and nutrient
assimilation via diversity-mediated ecosystem functioning.
Soil Biol Biochem 113: 122129.
Jauri, P.V., Beracochea, M., Fernández, B., and
Battistoni, F. (2019) Whole-genome sequencing of Strep-
tomyces sp. strain uyfa156 a cultivar-specic plant
growth-promoting endophyte of Festuca arundinacea.
Microbiol Resour Announc 8: e00722e00719.
Jefferson, R. (1994) The Hologenome. Agriculture, Environ-
ment and the Developing World: A Future of PCR. NY:
Cold Spring Harbor.
Johnston-Monje, D., and Mejia, J.L. (2020) Botanical
microbiomes on the cheap: inexpensive molecular nger-
printing methods to study plant-associated communities of
bacteria and fungi. Appl Plant Sci 8: e11334.
Johnston-Monje, D., and Raizada, M.N. (2011) Conservation
and diversity of seed associated endophytes in Zea
across boundaries of evolution, ethnography and ecology.
PLoS One 6: e20396.
Jones, D.L., Nguyen, C., and Finlay, R.D. (2009) Carbon
ow in the rhizosphere: carbon trading at the soilroot
interface. Plant Soil 321:533.
Junker, R.R., and Tholl, D. (2013) Volatile organic com-
pound mediated interactions at the plant-microbe inter-
face. J Chem Ecol 39: 810825.
Kandalepas, D., Blum, M.J., and Van Bael, S.A. (2015)
Shifts in symbiotic endophyte communities of a founda-
tional salt marsh grass following oil exposure from the
Deepwater horizon oil spill. PLoS One 10: e0122378.
Kandel, S.L., Joubert, P.M., and Doty, S.L. (2017) Bacterial
endophyte colonization and distribution within plants.
Microorganisms 5: 77.
Kang, D., Jacquiod, S., Herschend, J., Wei, S., Nesme, J.,
and Sørensen, S.J. (2019) Construction of simplied
microbial consortia to degrade recalcitrant materials based
on enrichment and dilution-to-extinction cultures. Front
Microbiol 10: 3010.
Kaul, S., Sharma, T., and Dhar, M.K. (2016) Omics tools for
better understanding the plant-endophyte interactions.
Front Plant Sci 7: 955.
Klaedtke, S., Jacques, M.-A., Raggi, L., Préveaux, A.,
Bonneau, S., Negri, V., et al. (2015) Terroir is a key driver
of seed-associated microbial assemblages. Environ
Microbiol 18: 17921804.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
The bacterial endosphere world 15
Kõiv, V., Roosaare, M., Vedler, E., Kivistik, P.A., Toppi, K.,
Schryer, D.W., et al. (2015) Microbial population dynamics
in response to Pectobacterium atrosepticum infection in
potato tubers. Sci Rep 5: 11606.
Kong, Z., Hart, M., and Liu, H. (2018) Paving the way from
the lab to the eld: using synthetic microbial consortia to
produce high-quality crops. Front Plant Sci 9: 1467.
Kost, T., Stopnisek, N., Agnoli, K., Eberl, L., and
Weisskopf, L. (2014) Oxalotrophy, a widespread trait of
plant-associated Burkholderia species, is involved in suc-
cessful root colonization of lupin and maize by Bur-
kholderia phytormans.Front Microbiol 4: 421.
Kuáźniar, A., Włodarczyk, K., Grzdziel, J., Goraj, W.,
Gałzka, A., and Woli
nska, A. (2020) Culture-independent
analysis of an endophytic core microbiome in two species
of wheat: Triticum aestivum L. (cv. Hondia) and the rst
report of microbiota in Triticum spelta L. (cv. Rokosz).
Syst Appl Microbiol 43: 126025.
Laurent, É. (1889) Sur lexistence de microbes dans les
tissus des plantes supérieures. Bull Soc Roy Bot Bel-
gique 1889: 233244.
Leibold, M.A., Holyoak, M., Mouquet, N., Amarasekare, P.,
Chase, J.M., Hoopes, M.F., et al. (2004) The met-
acommunity concept: a framework for multi-scale commu-
nity ecology. Ecol Lett 7: 601613.
Lemanceau, P., Blouin, M., Muller, D., and Moënne-
Loccoz, Y. (2017) Let the core microbiota be functional.
Trends Plant Sci 22: 583595.
Levy, A., Conway, J.M., Dangl, J.L., and Woyke, T. (2018)
Elucidating bacterial gene functions in the plant micro-
biome. Cell Host Microbe 24: 475485.
Li, F., He, X., Sun, Y., Zhang, X., Tang, X., Li, Y., and Yi, Y.
(2019a) Distinct endophytes are used by diverse plants
for adaptation to karst regions. Sci Rep 9:19.
Li, L., Wei, K., Liu, X., Wu, Y., Zheng, G., Chen, S., et al.
(2019b) aMSGE: advanced multiplex site-specic genome
engineering with orthogonal modular recombinases in acti-
nomycetes. Metab Eng 52: 153167.
Li, Y., Wu, C., Xing, Z., Gao, B., and Zhang, L. (2017) Engi-
neering the bacterial endophyte Burkholderia pyrrocinia
JK-SH007 for the control of lepidoptera larvae by introduc-
ing the cry218 genes of Bacillus thuringiensis.Biotechnol
Biotechnol Equip 31: 11671172.
Liu, H., Carvalhais, L.C., Crawford, M., Singh, E., Dennis, P.
G., Pieterse, C.M.J., and Schenk, P.M. (2017) Inner plant
values: diversity, colonization and benets from endo-
phytic bacteria. Front Microbiol 8: 2552.
Liu, Y.-X., Qin, Y., and Bai, Y. (2019) Reductionist synthetic
community approaches in root microbiome research. Curr
Opin Microbiol 49:97102.
López Fernández, S., Sonego, P., Moretto, M., Pancher, M.,
Engelen, K., Pertot, I., and Campisano, A. (2015) Whole-
genome comparative analysis of virulence genes unveils
similarities and differences between endophytes and other
symbiotic bacteria. Front Microbiol 6: 419.
Lucaciu, R., Pelikan, C., Gerner, S.M., Zioutis, C.,
Köstlbacher, S., Marx, H., et al. (2019) A bioinformatics guide
to plant microbiome analysis. Front Plant Sci 10: 1313.
Malfanova, N., Kamilova, F., Validov, S., Chebotar, V., and
Lugtenberg, B. (2013) Is L-arabinose important for the
endophytic lifestyle of Pseudomonas spp.? Arch Microbiol
Mano, H., Tanaka, F., Watanabe, A., Kaga, H., Okunishi, S.,
and Morisaki, H. (2006) Culturable surface and endophytic
bacterial ora of the maturing seeds of rice plants (Oryza
sativa) cultivated in a paddy eld. Microbes Environ 21:
Manrique, P., Bolduc, B., Walk, S.T., van der Oost, J., de
Vos, W.M., and Young, M.J. (2016) Healthy human gut
phageome. Proc Natl Acad Sci U S A 113: 1040010405.
Mansvelt, E.L., and Hattingh, M.J. (1987) Scanning electron
microscopy of colonization of pear leaves by Pseudomo-
nas syringae pv. syringae.Can J Bot 65: 25172522.
Maude, R.B. (1996) Seedborne Diseases and their Control:
Principles and Practice. Wallingford, UK: CAB Internation,
pp. 1280.
Mercado-Blanco, J. (2015) Life of microbes inside the plant.
In Principles of Plant-Microbe Interactions, Lugtenberg, B.
J.J. (ed). Berlin: Springer International Publishing,
pp. 2532.
Mercado-Blanco, J., and Prieto, P. (2012) Bacterial endo-
phytes and root hairs. Plant Soil 361: 301306.
Mina, D., Pereira, J.A., Lino-Neto, T., and Baptista, P. (2020)
Epiphytic and endophytic bacteria on olive tree
phyllosphere: exploring tissue and cultivar effect. Microb
Ecol 80: 145157.
Misas-Villamil, J.C., Kolodziejek, I., Crabill, E., Kaschani, F.,
Niessen, S., Shindo, T., et al. (2013) Pseudomonas
syringae pv. syringae uses proteasome inhibitor syringolin
a to colonize from wound infection sites. PLoS Pathog 9:
Mitter, B., Brader, G., Afzal, M., Compant, S., Naveed, M.,
Trognitz, F., and Sessitsch, A. (2013) Advances in eluci-
dating benecial interactions between plants, soil and bac-
teria. Adv Agron 121: 381445.
Mitter, B., Pfaffenbichler, N., Flavell, R., Compant, S.,
Antonielli, L., Petric, A., et al. (2017) A new approach to
modify plant microbiomes and traits by introducing bene-
cial bacteria at owering into progeny seeds. Front
Microbiol 8: 11.
Mosquito, S., Bertani, I., Licastro, D., Compant, S.,
Myers, M.P., Hinarejos, E., et al. (2020) In planta coloniza-
tion and role of t6ss in two rice Kosakonia endophytes.
Mol Plant Microbe Interact 33: 349363.
Morella, N.M., Zhang, X., and Koskella, B. (2019) Tomato
seed-associated bacteria confer protection of seedlings
against foliar disease caused by Pseudomonas syringae.
Phytobiomes J 3: 177190.
Newman, E.I. (1985) The rhizosphere: carbon sources and
microbial populations. In Ecological Interactions in Soil,
Fitter, A.H. (ed). Spec. Publ. No 4 of the British Ecological
Society. Oxford: Blackwell Scientic Publ, pp. 107121.
Obeng, N., Pratama, A.A., and van Elsas, J.D. (2016) The
signicance of mutualistic phages for bacterial ecology
and evolution. Trends Microbiol 24:6.
Paredes, S.H., Gao, T., Law, T.F., Finkel, O.M., Mucyn, T.,
Teixeira, P.J.P.L., et al. (2018) Design of synthetic bacte-
rial communities for predictable plant phenotypes. PLoS
Biol 16: e2003962.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
16 S. Compant et al.
Passera, A., Compant, S., Casati, P., Maturo, M.G.,
Battelli, G., Quaglino, F., et al. (2019) Not just a patho-
gen? Description of a plant-benecial Pseudomonas
syringae strain. Front Microbiol 10: 1409.
Peredo, E.L., and Simmons, S.L. (2018) Leaf-FISH: micro-
scale imaging of bacterial taxa on phyllosphere. Front
Microbiol 8: 2669.
Petrini, O. (1991) Fungal endophytes of tree leaves. In
Microbial Ecology of Leaves, Andrews, J.H., and
Hirano, S.S. (eds). New York, NY: Springer Verlag,
pp. 179197.
Pfeiffer, S., Mitter, B., Oswald, A., Schloter-Hai, B.,
Schloter, M., Declerck, S., and Sessitsch, A. (2017) Rhizo-
sphere microbiomes of potato cultivated in the high Andes
show stable and dynamic core microbiomes with different
responses to plant development. FEMS Microbiol Ecol 93:
pii: w242.
Pinski, A., Betekhtin, A., Hupert-Kocurek, K., Mur, L., and
Hasterok, R. (2019) Dening the genetic basis of plant-
endophytic bacteria interactions. Int J Mol Sci 20: 1947.
Pratama, A.A., Terpstra, J., Martinez de Oliveria, A.L., and
ao Salles, J. (2020) The role of rhizosphere bacterio-
phages in plant health. Trends Microbiol 28: 709718.
Rana, K.L., Kour, D., Kaur, T., Devi, R., Yadav, A.N.,
Yadav, N., et al. (2020) Endophytic microbes: biodiver-
sity, plant growth-promoting mechanisms and potential
applications for agricultural sustainability. Antonie Van
Leeuwenhoek 113: 10751107.
Rasche, F., Velvis, H., Zachow, C., Berg, G., Van Elsas, J.
D., and Sessitsch, A. (2006) Impact of transgenic potatoes
expressing anti-bacterial agents on bacterial endophytes
is comparable with the effects of plant genotype, soil type
and pathogen infection. J Appl Ecol 43: 555566.
Redford, A.J., Bowers, R.M., Knight, R., Linhart, Y., and
Fierer, N. (2010) The ecology of the phyllosphere: geo-
graphic and phylogenetic variability in the distribution of
bacteria on tree leaves. Environ Microbiol 12: 28852893.
Reinhold-Hurek, B., Maes, T., Gemmer, S., Van
Montagu, M., and Hurek, T. (2006) An endoglucanase is
involved in infection of rice roots by the not-cellulose-
metabolizing endophyte Azoarcus sp. strain BH72. Mol
Plant Microbe Interact 19: 181188.
Ren, G., Zhang, H., Lin, X., Zhu, J., and Jia, Z. (2015)
Response of leaf endophytic bacterial community to ele-
vated CO
at different growth stages of rice plant. Front
Microbiol 6: 855.
Robinson, R.J., Fraaije, B.A., Clark, I.M., Jackson, R.W.,
Hirsch, P.R., and Mauchline, T.H. (2016) Endophytic bac-
terial community composition in wheat (Triticum aestivum)
is determined by plant tissue type, developmental stage
and soil nutrient availability. Plant Soil 405: 381396.
Rocca, J.D., Simonin, M., Blaszczak, J.R., Ernakovich, J.G.,
Gibbons, S.M., Midani, F.S., and Washburne, A.D. (2018)
The microbiome stress project: toward a global meta-
analysis of environmental stressors and their effects on
microbial communities. Front Microbiol 9: 3272.
Rodriguez, P.A., Rothballer, M., Chowdhury, S.P.,
Nussbaumer, T., Gutjahr, C., and Falter-Braun, P. (2019)
Systems biology of plant-microbiome interactions. Mol
Plant 12: 804821.
Rohart, F., Gautier, B., Singh, A., and Cao, K.-A.L. (2017)
mixOmics: an R package for omics feature selection and
multiple data integration. PLoS Comput Biol 13:
Sabaratnam, S., and Beattie, G.A. (2003) Differences
between Pseudomonas syringae pv. syringae B728a
and Pantoea agglomerans BRT98 in epiphytic and endo-
phytic colonization of leaves. Appl Environ Microbiol 69:
Saikkonen, K., Nissinen, R., and Helander, M. (2020)
Toward comprehensive plant microbiome research. Front
Ecol Evol 8: 61.
Saldierna Guzmán, J.P., Nguyen, K., and Hart, S.C. (2020)
Simple methods to remove microbes from leaf surfaces.
J Basic Microbiol 60: 730734.
Samad, A., Trognitz, F., Compant, S., Antonielli, L., and
Sessitsch, A. (2017) Shared and host-specic microbiome
diversity and functioning of grapevine and accompanying
weed plants. Environ Microbiol 19: 14071424.
Saminathan, T., García, M., Ghimire, B., Lopez, C.,
Bodunrin, A., Nimmakayala, P., et al. (2018) Metagenomic
and metatranscriptomic analyses of diverse watermelon
cultivars reveal the role of fruit associated microbiome in
carbohydrate metabolism and ripening of mature fruits.
Front Plant Sci 9:4.
Sarhan, M.S., Hamza, M.A., Youssef, H.H., Patz, S.,
Becker, M., ElSawey, H., et al. (2019) Culturomics of the
plant prokaryotic microbiome and the dawn of plant-based
culture media - a review. J Adv Res 19:1527.
Sasse, J., Martinoia, E., and Northen, T. (2018) Feed your
friends: do plant exudates shape the root microbiome?
Trends Plant Sci 23:2541.
Sauka, D.H. (2017) Bacillus thuringiensis and their endo-
phytic capabilities. EC Microbiol 9: 170171.
Schenk, P.M., Carvalhais, L.C., and Kazan, K. (2012)
Unraveling plant-microbe interactions: can multi-species
transcriptomics help? Trends Biotechnol 30: 177184.
Scheuerl, T., Hopkins, M., Nowell, R.W., Rivett, D.W.,
Barraclough, T.G., and Bell, T. (2020) Bacterial adaptation
is constrained in complex communities. Nat Commun
Schmidt, M.A., Balsanelli, E., Faoro, H., Cruz, L.M.,
Wassem, R., de Baura, V.A., et al. (2012) The type III
secretion system is necessary for the development of a
pathogenic and endophytic interaction between Her-
baspirillum rubrisubalbicans and Poaceae.BMC
Microbiol 12: 98.
Sessitsch, A., Hardoim, P., Doring, J., Weilharter, A.,
Krause, A., Woyke, T., et al. (2012) Functional character-
istics of an endophyte community colonizing rice roots as
revealed by metagenomic analysis. Mol Plant Microbe
Interact 25:2836.
Sessitsch, A., Pfaffenbichler, N., and Mitter, B. (2019) Micro-
biome applications from lab to eld: facing complexity.
Trends Plant Sci 24: 194198.
Shade, A., Jacques, M.A., and Barret, M. (2017) Ecological
patterns of seed microbiome diversity, transmission, and
assembly. Curr Opin Microbiol 37:1522.
Sheibani-Tezerji, R., Rattei, T., Sessitsch, A., Trognitz, F.,
and Mitter, B. (2015) Transcriptome proling of the
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
The bacterial endosphere world 17
endophyte Burkholderia phytormans PsJN indicates
sensing of the plant environment and drought stress.
MBio 6: e00621-15.
Shi, J., Liu, A., Li, X., Feng, S., and Chen, W. (2010) Identi-
cation of endophytic bacterial strain MGP1 selected from
papaya and its biocontrol effects on pathogens infecting
harvested papaya fruits. J Sci Food Agr 90: 227232.
Shi, Y., Yang, H., Zhang, T., Sun, J., and Lou, K. (2014)
Illumina-based analysis of endophytic bacterial diversity
and space-time dynamics in sugar beet on the north slope
of Tianshan mountain. Appl Microbiol Biotechnol 98:
Shidore, T., Dinse, T., Öhrlein, J., Becker, A., and Reinhold-
Hurek, B. (2012) Transcriptomic analysis of responses to
exudates reveal genes required for rhizosphere compe-
tence of the endophyte Azoarcus sp. strain BH72. Environ
Microbiol 14: 27752787.
Tadra-Sfeir, M.Z., Souza, E.M., Faoro, H., Muller-Santos, M.,
Baura, V.A., Tuleski, T.R., et al. (2011) Naringenin regu-
lates expression of genes involved in cell wall synthesis in
Herbaspirillum seropedicae.Appl Environ Microbiol 77:
Terra, L.A., de Soares, C.P., Meneses, C.H.S.G., Sfeir, M.Z.
T., de Souza, E.M., Silveira, V., et al. (2019) Trans-
criptome and proteome proles of the diazotroph
Nitrospirillum amazonense strain CBAmC in response to
the sugarcane apoplast uid. Plant Soil 451: 145168.
Theis, K.R., Dheilly, N.M., Klassen, J.L., Brucker, R.M.,
Baines, J.F., Bosch, T.C.G., et al. (2016) Getting the
hologenome concept right: an eco-evolutionary framework
for hosts and their microbiomes. mSystems 1: e0002816.
Tomasino, S.F., Leister, R.T., Dimock, M.B., Beach, R.M.,
and Kelly, J.L. (1995) Field performance of Clavibacter
xyli subsp. cynodontis expressing the insecticidal protein
gene cryIA (c) of Bacillus thuringiensis against European
corn borer in eld corn. BioControl 5: 442448.
Truyens, S., Weyens, N., Cuypers, A., and Vangronsveld, J.
(2015) Bacterial seed endophytes: genera, vertical trans-
mission and interaction with plants. Environ Microbiol Rep
Turner, T.R., James, E.K., and Poole, P.S. (2013) The plant
microbiome. Genome Biol 14: 209219.
Vacher, C., Hampe, A., Porté, A.J., Sauer, U., Compant, S., and
Morris, C.E. (2016) The phyllosphere: microbial jungle at the
plant-climate interface. Annu Rev Ecol Evol Syst 47:124.
Vandenkoornhuyse, P., Quaiser, A., Duhamel, M., Le Van, A.,
and Dufresne, A. (2015) The importance of the microbiome
of the plant holobiont. New Phytol 206:11961206.
Vannier, N., Agler, M., and Hacquard, S. (2019) Microbiota-
mediated disease resistance in plants. PLoS Pathog 15:
Vergine, M., Meyer, J.B., Cardinale, M., Sabella, E.,
Hartmann, M., Cherubini, P., et al. (2019) The Xylella
fastidiosa-resistant olive cultivar leccino has stable endo-
phytic microbiota during the olive quick decline syndrome
(OQDS). Pathogens 9: 35.
Vorholt, J.A. (2012) Microbial life in the phyllosphere. Nat
Rev Microbiol 10: 828840.
Wang, Y., Li, H., Zhao, W., He, X., Chen, J., Geng, X., and
Xiao, M. (2010) Induction of toluene degradation and growth
promotion in corn and wheat by horizontal gene transfer
within endophytic bacteria. Soil Biol Biochem 42: 10511057.
Wemheuer, F., Berkelmann, D., Wemheuer, B., Daniel, R.,
Vidal, S., and Daghela, H.B.B. (2020) Agroforestry man-
agement systems drive the composition diversity, and
function of fungal and bacterial endophyte communities in
Theobroma cacao leaves. Microorganisms 8: 405.
Wemheuer, F., Wemheuer, B., Daniel, R., and Vidal, S.
(2019) Deciphering bacterial and fungal endophyte com-
munities in leaves of two maple trees with green islands.
Sci Rep 9: 14183.
Weyens, N., Van Der Lelie, D., Artois, T., Smeets, K.,
Taghavi, S., Newman, L., et al. (2009) Bioaugmentation
with engineered endophytic bacteria improves contami-
nant fate in phytoremediation. Environ Sci Technol 43:
Wilson, D. (1995) Endophyte the evolution of a term, and
clarication of its use and denition. Oikos 73: 274276.
Xu, L., Naylor, D., Dong, Z., Simmons, T., Pierroz, G.,
Hixson, K.K., et al. (2018) Drought delays development of
the sorghum root microbiome and enriches for monoderm
bacteria. Proc Natl Acad Sci U S A 115: E4284E4293.
Yergeau, E., Tremblay, J., Joly, S., Labrecque, M.,
Maynard, C., Pitre, F.E., et al. (2018) Soil contamination
alters the willow root and rhizosphere metatranscriptome
and the root-rhizosphere interactome. ISME J 12:
Yi, Y., de Jong, A., Frenzel, E., and Kuipers, O.P. (2017)
Comparative transcriptomics of Bacillus mycoides strains
in response to potato-root exudates reveals different
genetic adaptation of endophytic and soil isolates. Front
Microbiol 8: 1487.
Zarraonaindia, I., Owens, S.M., Weisenhorn, P., West, K.,
Hampton-Marcell, J., Lax, S., et al. (2015) The soil micro-
biome inuences grapevine-associated microbiota. MBio
6: e02527-14.
Zhu, Y.-G., and Penuelas, J. (2020) Changes in the environ-
mental microbiome in the Anthropocene. Glob Change
Biol 26: 31753177.
Zilber-Rosenberg, I., and Rosenberg, E. (2008) Role of
microorganisms in the evolution of animals and plants: the
hologenome theory of evolution. FEMS Microbiol Rev 32:
Zinger, L., Bonin, A., Alsos, I.G., Bálint, M., Bik, H.,
Boyer, F., et al. (2019) DNA metabarcoding: need for
robust experimental designs to draw sound ecological
conclusions. Mol Ecol 28: 18571862.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
18 S. Compant et al.
... Field plants show the highest population and diversity of endophytic bacteria in soil-embedded roots (Conn and Franco, 2004;Liu et al., 2017). Lower bacterial diversity commonly observed in root tissues relative to the rhizosphere has contributed to the assumption that plants selectively recruit a subset of their choice organisms from soil as endophytes (Compant et al., 2010(Compant et al., , 2021Hardoim et al., 2015). One major and initial route of plant entry of endophytic bacteria is through root hairs (Prieto et al., 2011;Compant et al., 2021). ...
... Lower bacterial diversity commonly observed in root tissues relative to the rhizosphere has contributed to the assumption that plants selectively recruit a subset of their choice organisms from soil as endophytes (Compant et al., 2010(Compant et al., , 2021Hardoim et al., 2015). One major and initial route of plant entry of endophytic bacteria is through root hairs (Prieto et al., 2011;Compant et al., 2021). This is particularly applicable for seed-associated bacteria making their entry at seed germination from the spermosphere (Nelson, 2018). ...
... Studies giving microscopic evidence on tissue colonization by bacterial endophytes have often encompassed on root tissues showing both intercellular and intracellular colonization (Compant et al., 2005(Compant et al., , 2008White et al., 2014White et al., , 2018Shehata et al., 2017). Thus, the current understanding suggests bacterial entry through root hairs or other root openings, traversing the root cortex with intracellular accommodation, xylem immigration, and subsequent vascular transmittance (Compant et al., 2011(Compant et al., , 2021Prieto et al., 2011). Microscopic studies on shoot tissue colonization by bacterial endophytes have been very few considering the large volume of literature on endophytic bacteria, which often give the impression of intercellular colonization with occasional reference to intracellular presence (Compant et al., 2005(Compant et al., , 2008(Compant et al., , 2011. ...
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We have recently described 'Cytobacts' as abundant intracellular endophytic bacteria inhabiting live plant cells based on the observations with callus and cell suspension cultures of grapevine and other plant species with the origin ascribable to field explants. In this study, we investigated the prevalence of such cytoplasmic bacterial associations in field plants across different taxa, their cultivability, and the extent of taxonomic diversity and explored the possibility of their embryo-mediated vertical transmission. Over 100 genera of field plants were surveyed for 'Cytobacts' through bright-field live-cell imaging as per our previous experience using fresh tissue sections from surface-sterilized shoot-tissues with parallel cultivation-based assessments. This revealed widespread cellular bacterial associations visualized as copious motile micro-particles in the cytoplasm with no or sparse colony forming units (CFU) from the tissue-homogenates indicating their general non-cultivability. Based on the ease of detection and the abundance of 'Cytobacts' in fresh tissue sections, the surveyed plants were empirically classified into three groups: (i) motile bacteria detected instantly in most cells; (ii) motility not so widely observed, but seen in some cells; and (iii) only occasional motile units observed, but abundant non-motile bacterial cells present. Microscopy versus 16S-rRNA V3-V4 amplicon profiling on shoot-tip tissues of four representative plants-tomato, watermelon, periwinkle, and maize-showed high bacterial abundance and taxonomic diversity (11-15 phyla) with the dominance of Proteobacteria followed by Firmicutes/Actinobacteria, and several other phyla in minor shares. The low CFU/absence of bacterial CFU from the tissue homogenates on standard bacteriological media endorsed their cultivation-recalcitrance. Intracellular bacterial colonization implied that the associated organisms are able to transmit vertically to the next generation through the seed-embryos. Microscopy and 16S-rRNA V3-V4 amplicon/metagenome profiling of mature embryos excised from fresh watermelon seeds revealed heavy embryo colonization by diverse bacteria with sparse or no CFU. Observations with grapevine fresh fruit-derived seeds and seed-embryos Frontiers in Microbiology | 1 March 2022 | Volume 13 | Article 806222 Thomas et al. Cytobacts: Ubiquitous Intracellular Endophytic Bacteria endorsed the vertical transmission by diverse cultivation-recalcitrant endophytic bacteria (CREB). By and large, Proteobacteria formed the major phylum in fresh seed-embryos with varying shares of diverse phyla. Thus, we document 'Cytobacts' comprising diverse and vertically transmissible CREBs as a ubiquitous phenomenon in vascular plants.
... Vegetative foliar tissues and floral parts that are the aerial components of the plants act as an important region to allow the growth of various types of epiphytic and endophytic microbial cells (Compant et al., 2020). The different parts of the plants like stem, leaves, and fruits comprise a systemic distribution of endophytes that is facilitated by the xylem (Compant et al., 2010). ...
The increase in population requires sustainable agriculture to meet up the rising demand of food in this present scenario. Threats and challenges to sustainable agriculture lead to the exploration of many avenues. One such way is to exploit microbial consortia, which can produce various types of biostimulants possessing plant growth-promoting properties having a significant role in managing the environmental stress and enhancing the nutrient utilization efficiency. The species diversity of microbial consortia differs intensely depending on their ecological conditions. The microbial consortium is developed for a tailored solution of soil health-linked problems. The specifically designed polymicrobial constructions would, on the one hand, provide protection against plant pathogens and, on the other hand, reduce the need of applying chemical fertilizers by making the valuable nutrients available to plants. The engineered consortium can be successfully utilized to enhance the efficacy of crop development, and the microbial associations existing at the soil rhizosphere play a vital role in determining the productivity of plants. Limited disadvantages and superior performance of the microbial consortia products (MCPs) lead to the formulation of novel microbial consortium for widespread use in agricultural practices for growth, protection, and yield of crop plants.
... In nature, plants are associated with an overwhelming number of beneficial microorganisms (e.g., endophytic or symbiotic bacteria and fungi) that play a significant role in plant health, development, and productivity, and in the modulation of metabolite synthesis (Berendsen et al. 2012;Panke-Buisse et al. 2015;Mendes et al. 2011;Castrillo et al. 2017;de Vries et al. 2020;Brader et al. 2014;Compant et al. 2021). Among these are the arbuscular mycorrhizal fungi (AMF), a ubiquitous group of soil microorganisms, forming symbiosis with more than 70% of vascular plants (Brundrett and Tedersoo 2018). ...
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Medicinal plants are an important source of therapeutic compounds used in the treatment of many diseases since ancient times. Interestingly, they form associations with numerous microorganisms developing as endophytes or symbionts in different parts of the plants. Within the soil, arbuscular mycorrhizal fungi (AMF) are the most prevalent symbiotic microorganisms forming associations with more than 70% of vascular plants. In the last decade, a number of studies have reported the positive effects of AMF on improving the production and accumulation of important active compounds in medicinal plants. In this work, we reviewed the literature on the effects of AMF on the production of secondary metabolites in medicinal plants. The major findings are as follows: AMF impact the production of secondary metabolites either directly by increasing plant biomass or indirectly by stimulating secondary metabolite biosynthetic pathways. The magnitude of the impact differs depending on the plant genotype, the AMF strain, and the environmental context (e.g., light, time of harvesting). Different methods of cultivation are used for the production of secondary metabolites by medicinal plants (e.g., greenhouse, aeroponics, hydroponics, in vitro and hairy root cultures) which also are compatible with AMF. In conclusion, the inoculation of medicinal plants with AMF is a real avenue for increasing the quantity and quality of secondary metabolites of pharmacological, medical, and cosmetic interest.
... In fact, we found that especially protists and bacteria in the rhizosphere, rather than bulk or endosphere microbes determine disease incidence and plant yield. There is a common notion that the rhizosphere is the main area where soil-borne pathogens compete with beneficial microbiota [45,46], but interestingly the tightly plant-associated root endosphere, where many mutualists and pathogens directly invade the plant [47], seems of less direct Fig. 3 Microbial functional genes and their potential interactions with pathogen density. A The random forest mean predictor importance (% increase of the MSE) of the metabolism gene categories for Fusarium oxysporum density and the relative abundance of the metabolism gene categories in different fertilization treatments. ...
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Plant health is strongly impacted by beneficial and pathogenic plant microbes, which are themselves structured by resource inputs. Organic fertilizer inputs may thus offer a means of steering soil-borne microbes, thereby affecting plant health. Concurrently, soil microbes are subject to top-down control by predators, particularly protists. However, little is known regarding the impact of microbiome predators on plant health-influencing microbes and the interactive links to plant health. Here, we aimed to decipher the importance of predator-prey interactions in influencing plant health. To achieve this goal, we investigated soil and root-associated microbiomes (bacteria, fungi and protists) over nine years of banana planting under conventional and organic fertilization regimes differing in Fusarium wilt disease incidence. We found that the reduced disease incidence and improved yield associated with organic fertilization could be best explained by higher abundances of protists and pathogen-suppressive bacteria (e.g. Bacillus spp.). The pathogen-suppressive actions of predatory protists and Bacillus spp. were mainly determined by their interactions that increased the relative abundance of secondary metabolite Q genes (e.g. nonribosomal peptide synthetase gene) within the microbiome. In a subsequent microcosm assay, we tested the interactions between predatory protists and pathogen-suppressive Bacillus spp. that showed strong improvements in plant defense. Our study shows how protistan predators stimulate disease-suppressive bacteria in the plant microbiome, ultimately enhancing plant health and yield. Thus, we suggest a new biological model useful for improving sustainable agricultural practices that is based on complex interactions between different domains of life.
... Le sol est ainsi considéré comme le réservoir primaire de microorganismes pour les plantes . A contrario, les microorganismes colonisant l'endosphère racinaire sont considérés moins nombreux et diversifiés (~10 5 -10 7 cellules par gramme de racine) mais plus spécialistes Compant et al. 2021) (Figure 1). ...
Le concept d’holobionte considère l’unité fonctionnelle composée des plantes et de ces microorganismes. Il promeut une approche holistique à la gestion des cultures et, plus généralement, à la vision du vivant. Cependant, ce concept est sujet à débat en raison du manque de preuves expérimentales de son existence. De plus, la compréhension fine des mécanismes régissant l’assemblage des microorganismes associés aux plantes reste un des enjeux majeurs de l’écologie microbienne et de l’agriculture. Le but de cette thèse était de tester la validité du concept d’holobionte, d’étudier les facteurs qui impactent l’assemblage des communautés microbiennes de l’endosphère racinaire et d’analyser la dynamique intra- et inter-annuelle du microbiote de la vigne. Grâce à une expérimentation mise en place en association avec une pépinière viticole (greffage et plantes chimériques), nous avons pu démontrer l’existence d’un recrutement actif et déterminé de microorganismes par la plante, sous l’effet dominant du porte-greffe, ce qui nous a permis d’augmenter les preuves expérimentales à l’existence du concept d’holobionte. Dans le cadre d’une seconde partie, nous nous sommes intéressés aux dynamiques d’assemblages du microbiote de la vigne, grâce à un vaste plan d’échantillonnage mis en place au sein d’un domaine viticole. Nous avons mis en avant le rôle fondamental des facteurs environnementaux, de l’âge et du cépage dans l’assemblage du microbiote endosphérique racinaire de la vigne à petite échelle géographique et l'existence de patrons temporels intra-annuels marqués dans la structuration de ces communautés. Ces travaux fournissent un ensemble de connaissances nouvelles dans le domaine de l’écologie microbienne, soutiennent l’existence d’un terroir microbiologique et soulignent l’importance de la prise en compte de ce terroir microbien dans le cadre d’une gestion durable de la vigne.
... When some of these strains were exposed to the lupin root exudates, several αand β-glucosidases were found overexpressed (four to ten-fold) (Benito, 2020). These enzymes are known to play a key role in bacterial root colonization and tissue penetration (Reinhold-Hurek et al., 2006;Liu et al., 2017;Compant et al., 2021). In addition, cellulases are not restricted to cellulose hydrolysis but could be involved in other biological functions (Medie et al., 2012); they have also been shown to be essential for root infection in rhizobia (Robledo et al., 2012). ...
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Understanding plant-microbe interactions with the possibility to modulate the plant’s microbiome is essential to design new strategies for a more productive and sustainable agriculture and to maintain natural ecosystems. Therefore, a key question is how to design bacterial consortia that will yield the desired host phenotype. This work was designed to identify the potential genomic features involved in the interaction between Micromonospora and known host plants. Seventy-four Micromonospora genomes representing diverse environments were used to generate a database of all potentially plant-related genes using a novel bioinformatic pipeline that combined screening for microbial-plant related features and comparison with available plant host proteomes. The strains were recovered in three clusters, highly correlated with several environments: plant-associated, soil/rhizosphere, and marine/mangrove. Irrespective of their isolation source, most strains shared genes coding for commonly screened plant growth promotion features, while differences in plant colonization related traits were observed. When Arabidopsis thaliana plants were inoculated with representative Micromonospora strains selected from the three environments, significant differences were in found in the corresponding plant phenotypes. Our results indicate that the identified genomic signatures help select those strains with the highest probability to successfully colonize the plant and contribute to its wellbeing. These results also suggest that plant growth promotion markers alone are not good indicators for the selection of beneficial bacteria to improve crop production and the recovery of ecosystems.
... It was demonstrated that human microbiome can affect human health and therefore likely also longevity (Biagi et al., 2016;Dato et al., 2017;Kim et al., 2018Kim et al., , 2019Badal et al., 2020). Plant microbiome play also very important role in plant health and likely longevity via symbiotic relationships with endophytic bacteria and fungi (Basit et al., 2021;Compant et al., 2021;Ghosh et al., 2021). Many plants form vast mycorrhizal fungal root networks through which they can interact with each other and even exchange different molecules (Kalia et al., 2021). ...
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Plants hold all records in longevity. Their aging is a complex process. In the presented review, we analyzed published data on various aspects of plant aging with focus on any inferences that could shed a light on aging in animals and help to fight it in human. Plant aging can be caused by many factors, such as telomere depletion, genomic instability, loss of proteostasis, changes in intercellular interaction, desynchronosis, autophagy, epigenetic changes and others. Plants have developed a number of mechanisms to increase lifespan. Among these mechanisms are gene duplication (“genetic backup”), the active work of telomerases, abundance of meristematic cells, capacity of maintaining the meristems permanently active and continuous activity of phytohormones. Plant aging usually occurs throughout the whole perennial life, but could be also seasonal senescence. Study of causes for seasonal aging can also help to uncover the mechanisms of plant longevity. The influence of different factors such as microbiome communities, glycation, alternative oxidase activity, mitochondrial dysfunction on plant longevity was also reviewed. Adaptive mechanisms of long-lived plants are considered. Further comparative study of the mechanisms underlying longevity of plants is necessary. This will allow us to reach a potentially new level of understanding of the aging process of plants.
... Therefore, the utilization of rootor soil-associated inoculants has some drawbacks, such as unsuccessful establishment in the soil/rhizosphere niches as a consequence of competition with the fluctuant rhizobiome for the limited resources, or unreliable adaptation to harsh soil conditions. As competition with other microbes for nutritional resources is limited in the endosphere (the interior of plant tissues), the utilization of endophytes for improving plant growth and stress resistance has recently been proposed as a good alternative to overcome some of these problems (Compant et al., 2021;Gonz alez-Benítez et al., 2021). Other advantages of the use of endophytic bacteria are that they are less exposed to uncontrolled changes in soil conditions, such as variations in soil pH, temperature and water content (Meena et al., 2017). ...
Salinity affects 7% of land worldwide and it is estimated that 50% of all arable land will become impacted by salinity by 2050. In this highlight, we comment the studies carried out by Choudhury and colleagues that reveal how the presence of an ACC deaminase-producing endophytic bacteria interferes with plant metabolism to improve plant survival under salt stress. They have demonstrated that the presence of Methylobacterium oryzae CBMB20, modifies the protein patterns of rice (Oryza sativa L.) under stress-free and salt-stressed conditions, revealing how endophytic bacteria produce significant modifications in plant physiology.
Endophytic studies are becoming popular with the current advancement in microbial ecology. The internal tissue of the plant represents a discreet region for diverse endophytic microbes to flourish for plant nutrition through the uptake of essential nutrient (i.e. nitrogen, phosphorus, and potassium) synthesis of phytohormones, metabolic compounds, organic acids, siderophores, and hydrolytic enzymes. Nevertheless, these microbes are less explored than expected. The mechanisms of endophytic microbes that best explain their interactions with the host plant and other microbes can unravel their functional role in agricultural biotechnology based on gene specificity and competence under biotic and abiotic stress conditions. The establishment of microbial communities in plants contributes to plant health for yield enhancement. The dominant bacterial phyla, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Chloroflexi; and fungal phyla Ascomycota, Basidiomycota, and Zygomycota previously reported from sunflower, maize, rice, and wheat using meta-omics approaches form the basis of understanding the endophytic concept in the present and future studies. Meta-omics approaches create opportunities to unravel, explore and incorporate endophytic bioproducts in developing eco-friendly agriculture. Despite the established prospects of meta-omics approaches in agricultural biotechnology and industry, providing information on the reality of endophytic microbial bioproducts in assisting stress tolerance and disease control in plants is important with the view of combating current agricultural challenges for crop production. Hence, this review focuses on the endophytic bacteria and fungi, structural diversity, meta-omics approaches, and their agricultural, biotechnological, and industrial importance.
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Despite the relevance of complex root microbial communities for plant health, growth and productivity, the molecular basis of these plant-microbe interactions is not well understood. Verrucomicrobia are cosmopolitans in the rhizosphere, nevertheless their adaptations and functions are enigmatic since the proportion of cultured members is low. Here we report four cultivated Verrucomicrobia isolated from rice, putatively representing four novel species, and a novel subdivision. The aerobic strains were isolated from roots or rhizomes of Oryza sativa and O. longistaminata. Two of them are the first cultivated endophytes of Verrucomicrobia, as validated by confocal laser scanning microscopy inside rice roots after re-infection under sterile conditions. This extended known verrucomicrobial niche spaces. Two strains were promoting root growth of rice. Discovery of root compartment-specific Verrucomicrobia permitted an across-phylum comparison of the genomic conformance to life in soil, rhizoplane or inside roots. Genome-wide protein domain comparison with niche-specific reference bacteria from distant phyla revealed signature protein domains which differentiated lifestyles in these microhabitats. Our study enabled us to shed light into the dark microbial matter of root Verrucomicrobia, to define genetic drivers for niche adaptation of bacteria to plant roots, and provides cultured strains for revealing causal relationships in plant-microbe interactions by reductionist approaches.
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Endophytic microbes are known to live asymptomatically inside their host throughout different stages of their life cycle and play crucial role in growth, development, fitness, and diversification of plant. The plant-endophyte association ranges from mutualism to pathogenicity. These microbes help the host to combat diverse arrays of both biotic and abiotic stressful conditions. Endophytic microbes play a major role in the growth promotion of their host by solubilizing of macronutrients such as phosphorous, potassium, and zinc; fixing of atmospheric nitrogen, synthesizing of phytohormones, siderophores, hydrogen cyanide, ammonia, and act as a biocontrol agent against wide array of phytopathogens. Endophytic microbes are beneficial to plant by directly promoting their growth or indirectly by inhibiting the growth of phytopathogens. Over a long period of co-evolution endophytic microbes have attained the mechanism of synthesis of various hydrolytic enzymes such as pectinase, xylanases, cellulase, and proteinase which help in the penetration of endophytic microbes into tissues of plants. The effective usages of endophytic microbes in the form of bioinoculants reduce the usage of chemical fertilizers. Endophytic microbes belong to different phyla such as Actinobacteria, Acidobacteria, Bacteroidetes, Deinococcus-thermus, Firmicutes, Proteobacteria, and Verrucomicrobia. The most predominant and studied endophytic bacteria belonged to Proteobacteria followed by Firmicutes and then by Actinobacteria. The most dominant among reported genera in most of the leguminous and non-leguminous plants are Bacillus, Pseudomonas, Fusarium, Burkholderia, Rhizobium, and Klebsiella. In future, endophytic microbes have a wide range of potential for maintaining health of plant as well as environmental conditions for agricultural sustainability. The present review is focused on endophytic microbes, their diversity in leguminous as well as non-leguminous crops, biotechnological applications, and ability to promote the growth of plant for agro-environmental sustainability
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High‐throughput sequencing technologies have revolutionized the study of plant‐associated microbial populations, but they are relatively expensive. Molecular fingerprinting techniques are more affordable, yet yield considerably less information about the microbial community. Does this mean they are no longer useful for plant microbiome research? In this paper, we review the past 10 years of studies on plant‐associated microbiomes using molecular fingerprinting methodologies, including single‐strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), amplicon length heterogeneity PCR (LH‐PCR), ribosomal intergenic spacer analysis (RISA) and automated ribosomal intergenic spacer analysis (ARISA), and terminal restriction fragment length polymorphism (TRFLP). We also present data juxtaposing results from TRFLP methods with those generated using Illumina sequencing in the comparison of rhizobacterial populations of Brazilian maize and fungal surveys in Canadian tomato roots. In both cases, the TRFLP approach yielded the desired results at a level of resolution comparable to that of the MiSeq method, but at a fraction of the cost. Community fingerprinting methods (especially TRFLP) remain relevant for the identification of dominant microbes in a population, the observation of shifts in plant microbiome community diversity, and for screening samples before their use in more sensitive and expensive approaches.
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The aboveground parts of terrestrial plants, collectively called the phyllosphere, have a key role in the global balance of atmospheric carbon dioxide and oxygen. The phyllosphere represents one of the most abundant habitats for microbiota colonization. Whether and how plants control phyllosphere microbiota to ensure plant health is not well understood. Here we show that the Arabidopsis quadruple mutant (min7 fls2 efr cerk1; hereafter, mfec)¹, simultaneously defective in pattern-triggered immunity and the MIN7 vesicle-trafficking pathway, or a constitutively activated cell death1 (cad1) mutant, carrying a S205F mutation in a membrane-attack-complex/perforin (MACPF)-domain protein, harbour altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis. The Shannon diversity index and the relative abundance of Firmicutes were markedly reduced, whereas Proteobacteria were enriched in the mfec and cad1S205F mutants, bearing cross-kingdom resemblance to some aspects of the dysbiosis that occurs in human inflammatory bowel disease. Bacterial community transplantation experiments demonstrated a causal role of a properly assembled leaf bacterial community in phyllosphere health. Pattern-triggered immune signalling, MIN7 and CAD1 are found in major land plant lineages and are probably key components of a genetic network through which terrestrial plants control the level and nurture the diversity of endophytic phyllosphere microbiota for survival and health in a microorganism-rich environment.
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The plant vascular system has remained an under-explored niche despite its potential for hosting beneficial microbes. The aim of this work was to determine the origin of the microbial endophytes inhabiting grapevine. We focused on a single commercial vineyard in California over a two-year period and used an amplicon metagenomics approach to profile the bacterial (16S -V4) and fungal (ITS) communities of the microbiome across a continuum of six grapevine compartments; bulk soil, rhizosphere, root, cordon, cane and sap. Our data supported that roots are a bottleneck to microbial richness and that they are mostly colonized with soilborne microbes, including possible plant growth promoting bacteria recruited by the host, but also saprohytic and pathogenic fungal invaders. A core group of taxa was identified throughout the vine, however there were clear partitioning of the microbiome based on host's niche. Above and below ground plant tissues displayed distinct microbial fingerprints and were intermixed in a limited capacity mostly by way of the plant sap. We discuss how cultural practices and human contact may shape the endosphere microbiome and identify potential channels for transmission of its residents.
There is a growing appreciation for the important roles microorganisms play in association with plants. Microorganisms are drawn to distinct plant surfaces by the nutrient-rich microenvironment, and in turn some of these colonizing microbes provide mutualistic benefits to their host. The development of plant probiotics to increase crop yield and provide plant resistance against biotic and abiotic stresses, while minimizing chemical inputs, would benefit from a deeper mechanistic understanding of plant-microbe interaction. Technological advances in molecular biology and high-throughput -omics provide stepping stones to the elucidation of critical microbiome gene functions that aid in improving plant performance. Here, we review -omics-based approaches that are propelling forward the current understanding of plant-associated bacterial gene functions, and describe how these technologies have helped unravel key bacterial genes and pathways that mediate pathogenic, beneficial, and commensal host interactions.
Endophytes have been defined as microorganisms living inside plant tissues without causing negative effects on their hosts. Endophytic microbes have been extensively studied for their plant growth‐promoting traits. However, analyses of endophytes require complete removal of epiphytic microorganisms. We found that the established tests to evaluate surface sterility, polymerase chain reaction, and leaf imprints, are unreliable. Therefore, we used scanning electron microscopy (SEM) as an additional assessment of epiphyte removal. We used a diverse suite of sterilization protocols to remove epiphytic microorganisms from the leaves of a gymnosperm and an angiosperm tree to test the influence of leaf morphology on the efficacy of these methods. Additionally, leaf tissue damage was also evaluated by SEM, as damaging the leaves might have an impact on endophytes and could lead to inaccurate assessment of endophytic communities. Our study indicates, that complete removal of the leaf cuticle by the sterilization technique assures loss of epiphytic microbes, and that leaves of different tree species may require different sterilization protocols. Furthermore, our study demonstrates the importance of choosing the appropriate sterilization protocol to prevent erroneous interpretation of host‐endophyte interactions. Moreover, it shows the utility of SEM for evaluating the effectiveness of surface sterilization methods and their impact on leaf tissue integrity.
Microbiomes and their hosts influence each other; for instance, the microbiome improves host fitness, whereas the host supports microbiome nutrition. Most studies on this topic have focused on the role of bacteria and fungi, although research on viruses that infect bacteria, known as ‘bacteriophages’ (phages), has gained importance due to the potential role bacteriophages play in the resilience and functionality of the gut microbiome. Like the gut microbiome, the rhizosphere harbors a complex microbiome, but little is known about the role of phages in this ecosystem. In this opinion, we extend the knowledge gained in human gut virus metagenomics (viromics) to disentangle the potential role of phages in driving the resilience and functionality of the rhizosphere microbiome. We propose that future comparative studies across environments are necessary to unravel the underlying mechanisms through which phages drive the composition and functionality of the rhizosphere microbiome and its interaction with the plant host. Importantly, such understanding might generate strategies to improve plant resistance and resilience in the context of climate change.
The Earth's surface is increasingly affected by human activity, from the loss of biodiversity to chemical pollution. Human impacts on the Earth ecosystem are so fundamental that a stratigraphic signature in sediments and ice has been produced. This has led to the proposal of a new geological epoch, the Anthropocene (Waters et al., 2016). The start of the early Anthropocene is marked by the spread of agriculture and deforestation, as well as large‐scale species exchange. Technological revolutions in the mid‐20th century fueled rapid population growth and industrialization, leading to diverse geochemical signatures, such as pollution from synthetic chemicals, and metals, and enrichment of nutrients.
Cacao (Theobroma cacao L.) is one of the most economically important crops worldwide. Despite the important role of endophytes for plant growth and health, very little is known about the effect of agroforestry management systems on the endophyte communities of T. cacao. To close this knowledge gap, we investigated the diversity, community composition, and function of bacterial and fungal endophytes in the leaves of T. cacao trees growing in five major cacao-growing regions in the central region of Cameroon using DNA metabarcoding. Fungal but not bacterial alpha diversity measures differed significantly between the agroforestry management systems. Interestingly, less managed home-garden cacao forests harbored the lowest fungal richness and diversity. Our results suggest that the composition of bacterial and fungal endophyte communities is predominantly affected by agroforestry management systems and, to a lesser extent, by environmental properties. The core microbiome detected comprised important fungal phytopathogens, such as Lasiodiplodia species. Several predicted pathways of bacterial endophytes and functional guilds of fungal endophytes differed between the agroforest systems which might be attributed to bacteria and fungi specifically associated with a single agroforest. Our results provide the basis for future studies on foliar fungal and bacterial endophytes of T. cacao and their responsiveness towards agroforestry management systems.