REVIEW

Open Access https://doi.org/10.48130/GR-2023-0018

Grass Research 2023, 3:18

Endophyte symbiosis: evolutionary development, and impacts of

plant agriculture
Xiaoqian Chang1*, Blair Young1,2, Nicole Vaccaro2, Raquele Strickland1, Walter Goldstein3, Lena Struwe1,2 and
James F White1*
1 Department of Plant Biology, Rutgers University, New Brunswick, NJ 08901, USA
2 Department of Ecology, Evolution, and Natural Resources, Rutgers University, New Brunswick, NJ 08901, USA
3 Mandaamin Institute, Lake Geneva, WI 53121, USA

* Corresponding authors, E-mail: [email protected]; [email protected]

Abstract
Land plants can absorb soil microbes (bacterial, fungal and algal) into their cells and tissues. Plant endophytes enhance plant growth, stimulate
elongation of root hairs, increase branching of roots, allow plants access to more nutrients, and stimulate oxidative stress tolerance. In the
rhizophagy cycle, microbes are absorbed from soil directly into plant root cells where nutrients are extracted oxidatively, which provides
nutrients to support plant growth. Early land plants lacked true roots, but possessed non-photosynthetic filaments (e.g., caulonemata, rhizoids)
that may have cultivated diazotrophic bacteria within their cells as a source of nitrogen, just as bryophyte and pteridophyte rhizoids do today.
Extant land plant lineages, such as bryophytes, pteridophytes, gymnosperms, and flowering plants, often produce epidermal structures (e.g.,
trichomes, papillae, paraphyllia, scales) on their roots, leaves, stems, or thalli; these often contain symbiotic nitrogen-fixing bacteria. Little is
understood about how plants interact with soil and plant microbiomes. In this article we present novel endophytic phenomena in diverse
lineages of land plants (liverworts, ferns, monocots, and eudicots) and explain how such symbiotic systems might have evolved over hundreds of
millions of years. Due to these endophytic and symbiotic systems, land plants have the capability to obtain nutrients from the environment.
Cultivation practices used in commercial agriculture can detract from the innate capabilities of plants to use microbes as a source of nutrients and
might be harmful to plant health.

Citation: Chang X, Young B, Vaccaro N, Strickland R, Goldstein W, et al. 2023. Endophyte symbiosis: evolutionary development, and impacts of plant
agriculture. Grass Research 3:18 https://doi.org/10.48130/GR-2023-0018

The first land plants of ferns produce filamentous rhizoids that contain clusters of
bacteria internally and show production of ammonium around
When land plants first evolved on Earth in the early Paleo- bacteria in plant filaments (Fig. 2).
zoic era (likely over 500 million years ago), they gained the
capacity to grow, survive, and reproduce in comparatively drier
Evolution of oxidative nutrient extraction from
environments than their algal ancestors, with initially little
organic soil, limited nitrogenous nutrients, and not much soil microbes
protection against harsh UV-rich sunrays[1]. Early land plant The earliest land plants were small, poikilohydric, and non-
relationships with fungal and bacterial endophytes helped vascular, most similar to modern bryophytes[2−7]. They lacked
plants to supplement their limited access to nitrogen from their roots but instead had filaments such as rhizoids and other cells
substrate[2−7]. Plants interface with microbes in the soil through into which they likely absorbed and cultivated soil microbes
their roots, and plants become an integral part of the microbial similar to bryophytes[4−7]. In modern plants, and likely in early
community by using exudates to energize soil microbes and by land plants, plant cells interact chemically with their internal
absorbing nutrients from the microbial community[8]. Plants bacteria to coax nitrogen and other nutrients from microbe
thus share in the nutritional bounty of the microbial commu- cells[9]. This has been proposed to be a simple interaction
nity in the soil[8]. Early land plants like the fossil vascular plant whereby bacteria secrete ethylene that triggers plants to
Rhynia lacked true roots and instead used rhizoids to interface release sugars then used by the bacteria, while simultaneously,
with the substrate[6]. Extant bryophytes (mosses, liverworts, and the plant cell produces superoxide (formed from oxygen in the
hornworts) lack roots and seeds, but possess non-photosyn- air) and covers the bacterial cells with it[9,10]. Exposure to super-
thetic filaments that have bacterial endophytes internally and oxide causes the bacteria to secrete reduced forms of nitrogen
extract nitrogen from them based on histochemical analyses (from bacterial nitrogen fixation) as antioxidants to protect
(Fig. 1). Pteridophytes (ferns and relatives) have roots to host themselves from oxidative damage stemming from the super-
endophytic bacteria but lack seeds to readily pass microbes to oxide exposure[9,10]. This simple but likely ancient interaction
offspring (ferns release spores, like bryophytes). Ferns in the with microbes has permitted plants to obtain nutrients within
sporophyte stage possess bacteria within root cells that have their own cells from bacteria, especially nitrogen. Most plants
been shown to foster the rhizophagy cycle. The gametophytes today engage in this symbiotic interaction with bacteria
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 Evolution of endophyte symbiosis

a b a b

Fig. 1 Thallus surface and pegged rhizoid of a liverwort (Riccia Fig. 2 Boston fern (Nephrolepis exaltata; Nephrolepidaceae)
fluitans). (a) Rhizoid stained with nitro blue tetrazolium (NBT) to gametophyte rhizoids stained with a saturated solution of
visualize superoxide (blue color) around bacterial clusters within bromothymol blue to show ammonium (purple) around bacteria.
the filament (arrow; bar = 10 µ). (b) Rhizoid containing multiple (a) Rhizoid tip showing aggregation of bacteria in the tip (arrow;
clusters of bacteria, stained blue using NBT due to superoxide bar = 10 µ). (b) Rhizoid tip showing bacterial clusters in filament
around bacteria (arrows; bar = 10 µ). (arrow; bar = 10 µ).

absorbed into their cells and tissues[8−16]. We refer to the nitrogen fixing bacteria are absorbed, then their host plant may
microbes that were internalized into plant tissues and cells as obtain nitrogen from these bacteria[8,9]. Plant cells produce
endophytes [endo = internal; phyte = plant][8]. Endophytes are superoxide in response to microbe-produced ethylene within
defined as mostly nonpathogenic microbes that inhabit plant the plant cell[9]. Superoxide is a powerful oxidant that oxidizes
tissues and often provide beneficial outcomes for the plant[8]. the cell walls off the microbe cells—resulting in formation of
Plants in the wild absorb soil microbes to the extent that some bacterial protoplasts without cell walls[8]. The microbe cell walls
of their cells may become filled with microbes[8−16]. Land plants that are oxidized thereby provide nutrients directly to the plant
are also associated with fungi in a symbiotic interaction called within its cells[9−12]. Soil microbe cell walls contain many nutri-
mycorrhizae[17]. Mycorrhizal fungi attach to roots and extend ents within them, such as metals, phosphorus, nitrogen and
into the soil, improving nutrient absorption from soils[17]. other compounds and elements[12]. The plant continues to
Mycorrhizal fungi also internalize soil microbes in their subject the endophytic microbes, in this case as wall-less proto-
hyphae[17]. Plants associate with and contain communities of plasts, to superoxide, and some of these protoplasts eventually
microbes (bacteria and fungi) to such a large extent that they become fully degraded releasing all their nutrients[8−12]. Other
may be considered 'farmers of soil microbes'[8]. bacteria will not be fully degraded; some bacteria are not capa-
ble of nitrogen fixation and some produce antioxidants that
can counteract superoxide and similar oxidants[8,12].
Biological processes plants employ to obtain soil
nutrients Root hairs as sites for nutrient acquisition
Modern plants have evolved sophisticated systems for
One of the important functions of root hairs (in addition to
attracting, internalizing, and managing soil microbes. They
uptake of water and solubilized nutrients) is the processing and
have developed structures, including root hairs and leaf hairs,
extraction of nutrients from soil microbes—this includes stimu-
to foster, host, and process soil microbes. One of the processes
lating nitrogen fixation in bacteria and coaxing them to secrete
these plants use to acquire microbes from the soil is called the their nitrogen—or rather reduced forms of nitrogen that serve
rhizophagy cycle [rhizo = root; phagy = eating] (see Fig. 3)[8−16]. to neutralize plant-produced superoxide[9,10]. The function of
In the rhizophagy cycle, plants attract soil microbes to root tips root hairs is tied intimately to the microbiome of the plant and
by secretion of root exudates[8]. These exudates are composed the soil. Bacteria that may produce and secrete nitrogen are
of sugars produced during photosynthesis, organic acids, and hosted within the root hairs, and in tandem with hormones,
sometimes other nutrients that signal to microbes that nutri- this stimulates development of longer root hairs. These endo-
ents are available at the plant root tips[8]. Microbes from the soil phytic bacteria accumulate in the root hair initial, which
are then attracted to the root tips. appears as a bump on root epidermal cells[9]. The bacteria in
Scientists are still trying to decipher how plants are signaling the root hair initials have been found to secrete ethylene and
soil microbes for absorption and influencing the environment nitric oxide[9]. Both of these compounds are growth hormones
in the soil to favor solubilization of soil nutrients[18]. These close for plants that then result in a growth spurt resulting in the
interactions between plants and soil microbes are areas of development of a long root hair[9].
active study by plant biologists and agronomists. It has been During the growing of a root hair, bacterial protoplasts accu-
hypothesized that plants can obtain particular nutrients by ab- mulated in the tips of the root hairs are ejected from the root
sorbing microbes that specifically carry nutrients by changing tip through pores in the plant cell wall into the soil[12]. The plant
their exudate composition[18]. For example, if a plant requires also releases small amounts of exudate through the tip of the
additional nitrogen in its tissues, it will secrete exudates that root hair to enable the bacteria that were recently ejected to
lack nitrogen but are rich in sugars[18,19]. Absence of nitrogen in reform their cell walls and their soil-capable mode of growth[8].
plant root exudates then selects for soil bacteria that produce The remaining bacteria inside the root hairs reproduce by divi-
nitrogen through nitrogen fixation from the air[18]. Once the sion or budding of the protoplast, and continue to fix

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Evolution of endophyte symbiosis

Microbe Expulsion at Hair Tips

Zone
e Entry Cells
Microb rbed into Root
a re A bso So ft )
es are
one (Microb ere Cell Walls
e Exit Z n Wh
Microb late Elongatio irs)
ic ro bes S ti m u
th e Tips of Ha
(M d E xit at
Hairs an
of Root Nutrients Extracted Microbes Enter Root
from Microbes Cells Carrying Nutrients
By Reactive Oxygen From Soil
Microbes Exit Root
Hairs Exhanusted of RHIZOPHAGY
Nutrients CYCLE

Microbes Recharge
With Nutrients in Rhizosphere

Fig. 3 Diagram showing the rhizophagy cycle, with a schematic of a root, with root cap to the right and developing root hairs to the left. In
the rhizophagy cycle microbes alternate between a free-living phase in soil and an intracellular phase in root cells. The microbes obtain
nutrients in the soil phase, and nutrients are extracted by the plant from microbes oxidatively in the endophytic phase inside the root.
Microbes are expelled from root hairs at tips where they reform cell walls and reenter soil to acquire additional nutrients[12].

nitrogen[9] (Fig. 4). Root hairs move endophytic bacteria around a b

the periphery of hairs at a speed of approximately 9-11
micrometers/second in a process that is similar to cyclosis[8],
although precisely how this movement happens is still a
mystery. This movement of bacteria around the root hair stimu-
lates replication of the microbes[9]. It is hypothesized that
plants facilitate reproduction of microbes in root cells that help
it acquire nutrients through the rhizophagy process[10]. A few
bacteria enter a root cell at the root tip, but later many more
cells of those bacteria are ejected back into the soil from the
root hair tip[8]. Thus, the plant facilitates endophytic replication
and increases the numbers of bacteria within its cells and in the
Fig. 4 Root hairs containing bacteria internally[9]. (a) Love-lies-
soil for optimal nutrient uptake. Root hairs are one of the
bleeding (Amaranthus caudatus; Amaranthaceae) root hairs
hypothesized sites in plants that accumulate microbes that
(arrow) stained with diaminobenzidine tetrahydrochloride to stain
sustain nitrogen fixation and transfer to plant cells. hydrogen peroxide (purple-brown color) around bacteria
internally (bar = 10 µ). (b) Root hair of tall fescue (Festuca
Trichomes as nitrogen-fixing structures in plants arundinacea; Poaceae) stained with nitro blue tetrazolium to
visualize superoxide (blue color) around the bacteria in the tip of
Above ground plant hairs also function to replicate microbes the hair (arrow; bar = 10 µ).
and foster plant growth.
Trichomes (epidermal hairs) are also symbiotic nitrogen trichomes. Microbes appear at the pits and there are multiple
fixing structures on plants that function like tiny root
interactions between plant trichome cell and groups of
nodules[10]. Trichomes (Fig. 5) are commonly found on leaves
microbes distributed beneath the lateral pits. Thus, a single
but may also be present on stems (including rhizomes), flowers,
trichome with lateral pits interacts with many clusters of bacte-
fruits, and seeds. Most extant plants have some type of hairs on
them, and their morphology varies considerably. The simplest ria, while non-pitted filamentous trichomes interact with a
hair forms are unbranched, filamentous (sometimes with pores single cluster of bacteria at the hair tip. This suggests greater
along their sides) and often possess microbes inside them[10]. efficiency in extraction of nutrients from microbes inside pitted
Microbes tend to accumulate at the tips of these hairs, similar trichomes than in non-pitted trichomes where clusters of
to root hairs. Filamentous trichomes with pits along their sides bacteria accumulate only inside the tips[10]. A third kind of
are also quite common in plants[10]. Microbes appear in the pits trichome, the glandular trichome type, has been shown to be
in the pores in the trichome cell. In the pitted trichome the very efficient at absorbing nitrogen from the air. Glandular
pores appear to somehow relate to the function of those trichomes are present in tomato, hemp, hops, and many other

Chang et al. Grass Research 2023, 3:18 Page 3 of 8

 Evolution of endophyte symbiosis

a b a b

Fig. 5 Trichomes on seed plants stained using acidified Fig. 6 Leaves of portia tree (Thespesia populnea; Malvaceae) with
diphenylamine stain to visualize nitrate (purple; arrows) around bacterial leaf nodules[10]. (a) Leaf stained with acidified
clusters of bacteria in hairs[10]. (a) Two trichomes (at arrows) on diphenylamine to show nitrate (blue color) in leaves around
petals of Canadian goldenrod (Solidago canadensis; Asteraceae) bacterial masses (arrows point to leaf nodules) (bar = 50 µm). (b)
(bar = 10 µ). (b) Trichome on young leaf of common mullein Arrow points to mass of bacteria from a leaf nodule stained with
(Verbascum thapsus; Scrophulariaceae) showing accumulation of acidified diphenylamine to show nitrate (blue color) around
nitrate around bacterial mass in head of trichome (bar = 10 µ). bacteria (bar = 50 µm).

plants. Their leaves and flowering structures bear glandular a b

trichomes that are filled with various chemicals including
antioxidants[10]. We speculate here that the antioxidants scav-
enge oxygen around bacteria to increase the efficiency of nitro-
gen fixation by bacteria inside the glandular trichome cells.
Scavenging oxygen around the bacteria is needed because
oxygen inhibits nitrogen fixation in bacteria[19].

Leaf nodules for nitrogen fixation

It's widely known that legumes (Fabaceae) and some other
plant families may form root nodules that contain nitrogen
fixing bacteria. People frequently cultivate legumes to increase Fig. 7 Oblong-leaved vanilla (Vanilla phaeantha; Orchidaceae)
nitrogen content in soil. It is less known that plants in many showing bacteria within nucleus and emergence from nuclei[10]. (a)
families may produce structures in leaves that contain nitrogen Nucleus stained with acidified diphenylamine to show nitrate
fixing bacteria. These structures are called leaf nodules[10] (Fig. (blue-purple color) (bar = 10 µm). (b) Nucleus stained with nitro
6). Our experiments with isotopic nitrogen showed that leaf blue tetrazolium to show superoxide (blue color) around bacteria
(arrows) in cytoplasm (bar = 10 µm).
nodules are efficient at acquiring nitrogen from the air[10]. Many
plants that do not produce root nodules but possess endo-
phytes in leaf nodules, hairs or other structures, are known to
be effective at increasing nitrogen content in their tissues and
grow under nutrient-poor soil conditions[10,20,21]. Leaf nodules
are likely therefore another way to obtain the nutrients that
plants require[10].
Nucleus

Nuclear envelope symbiosis

In non-photosynthetic cells of leaves there is a different kind
of symbiosis, called nuclear envelope symbiosis, that appears to
result in nitrogen formation and nitrogen transfer to the devel-
oping plant cell (Figs 7 & 8). This is when bacteria colonize into
the nuclear envelope and replicate there[10,22,23]. Generally, Fig. 8 Bacterial tracks (arrows) from nucleus through cytoplasm
microbes entering the nuclear envelope produce proteins to periplasmic space of mint (Mentha sp.; Lamiaceae) (bar = 15
called nucleomodulins that affect plant gene expression[22,23]. µm).
Nuclear colonization could therefore play a role in control of
cell development, however, very little is understood about the plant, which results in nitrogen being released from the
nuclear envelope colonization phenomenon. A previous study bacteria[10]. These nuclear envelope symbioses are often
suggested that the nitrogen fixation phase may be happening located in epidermal cells of meristems of roots, stems and
in the nuclear envelope when the bacterial cells are replicating leaves where chloroplasts are lacking. We hypothesize that the
using plant cell provided sugars[10]. As the plant cell develops, bacteria enter, or may already be present, in meristematic cells
the bacteria emerge from the nuclear envelope into the cyto- during chromosomal replication while the nuclear envelope is
plasm where they are exposed to superoxide produced by the not present, then after cell division, they become trapped

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Evolution of endophyte symbiosis

between the two membranes of the nuclear envelope[10]. The The seed microbiome
bacteria replicate in the nuclear envelope then escape from the
nuclear envelope, perhaps through pores in the outer Seed plants transport symbiotic microbes from soils through-
membrane of the nuclear envelope, and then move through out the plant and onto seed surfaces and into seeds[32−35].
the cytoplasm to the periplasmic space just underneath the cell Microbes are moved into the developing ovary of a plant and
wall where nitrogen extraction continues[10]. Use of copper the fertilized ovule inside the ovary develops into a seed[33,34].
sulfate to visualize reducing sugars suggests that the plant cell The microbes are often located within the interior of seeds in
supplies sugars to the bacteria within the nuclear envelope[10]. the nucellus tissues and perhaps within the embryo itself[32,33].
Nuclear envelope colonization is a means whereby bacteria Plants also carry microbes on the surfaces of seeds or their
may be readily transmitted reliably from parent cell to daugh- associated tissues, like dry leaf or husk material[33,35]. It is
ter cells in the plant meristem and can thereby colonize the thought that some of the microbes that a plant has acquired
entire plant if present early in development. are located in the seeds, and when those seeds germinate, they
already have a small community of microbes nearby to use for
rhizophagy cycle activity, nitrogen fixation, and mineral solubi-
Nitrogen-use efficiency and nitrogen fixation in lization in soils[8,32−35]. These 'early' microbes are extremely
plants important for plant seedling health. Many experiments have
Plants often have multiple types of structures and processes shown that seedlings with microbes perform better than
in which they cultivate microbes and extract nutrients from seedlings from seeds that have lost or have reduced numbers
them. We refer to the presence of multiple types of nutrient of symbiotic microbes[8,25,31]. A healthy seed microbiome
transfer symbioses in a plant as 'symbiosis stacking'[10]. For requires time to develop on maturing seeds. To obtain a
example, a plant may possess rhizophagy cycle activity in roots healthy seed microbiome, seeds should have sufficient time to
mature in their natural setting[35]. In the wild, seeds may remain
where plants acquire a broad set of soil nutrients through
on the plant for weeks, fall to soil, or may be vectored by
oxidative extraction in root cells just behind the apical meris-
animals internally or in some other manner, while the micro-
tem, nuclear envelope symbiosis in epidermal cells, and the
biome community develops on and within the seeds[35]. Failure
same plant may also possess other nitrogen transfer symbiosis
to permit such seed microbiome maturation results in seeds
in trichomes and leaf nodules[10,15,24,25]. Plants that engage in
that may have fewer endophytic microbes, and are of reduced
nitrogen transfer symbiosis stacking are more efficient in terms
hardiness[35]. This period of development of the microbiome on
of nitrogen use; they require less added nitrogen than plants
seeds may be referred to as the 'microbiome ripening
that do not have these symbioses. High nitrogen use efficiency
process'[8,35]. Failure for microbiome ripening results in seeds
in crops is indicative of crops that have greater access to soil
that have incompletely developed microbiomes[35].
microbes and have acquired or contain more endophytic
microbes active in nitrogen fixation[10,12,13,15,24,26,27].
The microbiome immune system of plants
Nutritional content of crop plants Healthy plant seeds come with their microbiomes, and this
microbiome is part of the immune system of plants[8,34−38].
There has been a discussion extending back decades regard-
Many of the microbes that vector on and within seeds and colo-
ing declining food nutritional content that is thought to corre-
nize plants also have the capacity to grow out away from the
late with the advent of industrial (conventional) agriculture and seedling or the mature plant, into the soil[8,37]. Such symbiotic
abundant use of inorganic (synthetic) fertilizers[28−30]. This asso- microbes may colonize potentially pathogenic fungi, for exam-
ciation of reduced food nutrient quality with the use of agro- ple Fusarium species, which may cause a damping off
chemicals, particularly fertilizers, has been difficult to confirm. disease[37]. Instead, once the fungus is colonized by the bacte-
Wang et al.[29] showed that antioxidant capacity and flavonoid ria the behavior of the fungus changes[37]. The fungus may
content of organically grown blueberries were notably higher show reduced virulence, be less destructive to plants, or be
than those of conventionally grown blueberries. Often experi- non-destructive, causing no disease at all[8,37]. In some cases the
ments do not include sufficient control data sets or sample size fungus may then actually grow inside the plants as an endo-
to get statistically significant results on nutrient density. phyte[8]. When seeds have fully established endophytic micro-
However, in experiments that we have conducted, we have biomes, they are at least partially resistant to fungal pathogen
found that many plants with microbial endophytes accumulate attacks[8,37]. However, when the microbiome on seeds is absent,
higher amounts of nutrients (particularly including, manga- seedlings become vulnerable to attacks by many species of
nese, iron, magnesium, zinc, nitrogen, etc.) than plants without fungi[36].
symbiotic microbes or with reduced amounts of microbes[8]. Endophytic microbes also stimulate plants to upregulate
From a perspective of nutrient density, it is clear that more their oxidative stress traits[8,39−42]. Plants produce antioxidants
symbiotic microbes result in higher rhizophagy cycle activity, and other stress-reducing traits that help them cope with reac-
and more nutrients available to plants[8,12]. In addition, the tive oxygen generated during stress periods[39,40]. Through this
oxidative interactions between plant cells and endophytic process, plants become more resistant to oxidative stress and
microbes result in formation of more phenolics, carotenoids, to superoxide and other oxidants that the plant is producing to
and other kinds of antioxidant compounds that accumulate in manage microbes in the rhizophagy cycle or to coax nitrogen
plants[8,24,25,31]. Thus, it is reasonable to expect that plants with from microbes throughout the plant[8,12,13,16]. This increased
greater symbiosis with microbes have greater accumulation of stress tolerance makes plants with endophytic microbes more
nutrients than plants without microbes or with reduced resistant to many kinds of environmental stresses and
amounts of microbes. diseases[40−42].

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 Evolution of endophyte symbiosis

Agricultural practices that degrade benefits of Other agronomic practices that affect diaspore
microbial endophyte symbiosis vectored microbiomes
Most plants in nature have symbiotic nutrient acquisition Tissue-culturing of plants has a negative impact on the plant
systems that help them to acquire nutrients that they need to microbiome and on any endophytes that engage in fixation
grow and reproduce. Plants in nature don't require our fertiliz- and transfer of nitrogen to plants and in rhizophagy cycle
ers or other agrochemicals[8−12]. The process of domestication activity[8,16]. When plants are tissue-cultured, they are
and modern cultural practices negatively impact symbiotic frequently treated with antibiotics to control bacteria or fungi.
systems within plants, and as a consequence crop plants have The plant tissues that form in these tissue cultures are often
become reliant on chemical inputs that further degrade activi- free of microbes or show reduced microbe populations[16], and
ties of plants with regard to endophytic microbes[16,43−49]. Some therefore may lack a healthy microbiome.
of the modern practices that degrade symbiotic systems in Seed treatments with acids to remove surface tissues as in
crop plants are discussed below. delinting in cotton, or with fungicides to protect plants from
diseases such as damping off diseases, also negatively affects
seed microbiomes. In cotton, seeds are often treated with
Losses of microbes during domestication
powerful acids to digest away cotton fibers so that seeds flow
Many of our food crops have come through a process of better in mechanical field planters, however, the beneficial
domestication and cultivation that has resulted in loss of micro- microbes are transmitted within and on the seed fibers[32,41].
biomes from seeds or fruits[43−46]. An example here is corn. The Loss of those microbes results in plants that are vulnerable to
wild corn ancestor teosinte possessed a thick and hard husk fungal diseases[36−38]. Repeated use of antifungals on crop
(outer layer)[44]. The microbiome of teosinte is in part carried on seeds has the long-term effect of degrading the seed micro-
the husk of the kernels[44]. In the development of modern corn, biome. Many plants contain fungal endophytes that are elimi-
one of the changes that happened was that the husk became nated when antifungal agrichemicals are applied to seeds and
soft and thin on the kernels[44]. This means that the natural way seedlings. Thus, antifungal applications to the surfaces of seeds
of carrying some of the microbes to the next generation results in more damage to the seed microbiome. The preferred
changed. The indigenous Americans discovered ways to culti- solution to the problem of reduced population numbers of
vate corn while promoting endophytic microbes[14]. The beneficial microbes on seeds is to add such microbes to seeds.
Iroquois used what they referred to as 'corn medicine' to It seems counterintuitive that the solution to a fungal disease
reestablish the microbiome on depleted corn seedlings[47]. problem is to add microbes to seeds, however, that is what has
They would gather wild grasses, including grasses like Canada been found to protect seeds and seedlings[37,38].
wild rye (Elymus canadensis), Virginia wild rye (Elymus virgini- Tillage that results in deep turning of soils may interfere with
ana), and common reed (Phragmites australis), then they took the soil microbiome, burying microbial communities deep in
the wild grass roots and ground them up in water. In some the soil column. Deep turning is sometimes carried out to kill
cases they would heat up the water, which had the effect of insects and weeds that may accumulate in the surface layers of
reducing the amount of some microbes but triggered some soils; however, it is in those surface inches that most of the
spore-forming bacteria to increase in number since heat acti- microbes live and grow. Displacement of the microbial layer at
vates their endospores to germinate and grow[47]. They would lower layers may result in reductions in the soil microbiome.
take corn seeds from the previous harvest and place them in Under these conditions when plants begin to grow, their roots
enter the soil, but because there are few microbes there, they
this water and let the seed germinate slightly so that the root
extract fewer nutrients from that soil[8,12].
emerged, thereby permitting colonization of the roots by
Many wild plants utilize bacteria that fix nitrogen as one
bacteria from wild grasses[47]. The Iroquois farmers then
source of nitrogen[8−14,20,21]. Overuse of synthetic nitrogen
planted the endophyte-colonized seedlings in their fields.
fertilizer suppresses nitrogen fixation in endophytic and soil
These historical agricultural practices are instances of 'micro-
microbes[48,49]. This result in plants not getting their nitrogen
biome transference', moving microbes from wild plants to corn
from symbiotic microbes even if such bacteria are present in
to improve the growth and health of their corn crops.
plant tissues or the soil[49]. Humans commonly cultivate plants
An experiment conducted a few years ago involved cultivat-
that thrive due to fertilizer inputs and therefore do not need to
ing the annual coyote tobacco (Nicotiana attenuata,
invest in endophytic systems, i.e., our commercial agriculture is
Solanaceae)[37]. Investigators gathered wild seeds of the species
largely based on plants that are dependent on synthetic fertil-
and brought it into cultivation, planting it each spring, and izer. There are many things we still do not know about endo-
then harvesting seeds in the fall for storage in a cool dry place phyte-plant relationships. We do not know how much nitrogen
until the following spring. After approximately 7 years of prevents or reduces endophytic nutrient transfer. What is clear
continuous cultivation, investigators found that a wilt disease is that synthetic/commercial fertilizers negatively affect plant
developed in the cultivated crop. They went back to the wild use of endophytic and soil microbes.
population and isolated microbes from the wild plants. They
found that continuous cultivation resulted in loss of endo-
phytic microbes from the plants. Reinfection of the endophytic
Conclusions
microbes back into the cultivated plants from the wild popula- Plants have evolved with microbes since they conquered the
tion resulted in elimination of the wilt disease from the culti- terrestrial environment. In the harsh new terrestrial environ-
vated crop. Thus, simply the process of repeated cultivation ment, plants relied on symbiotic microbes to get the nutrients
may result in loss of components of the microbiome[37]. they needed to survive. We still understand little about how

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Evolution of endophyte symbiosis

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evolved in plants, how they function, and how they are regu- 9. Chang X, Kingsley KL, White JF. 2021. Chemical interactions at the
lated. It is imperative to develop data on how we can manage interface of plant root hair cells and intracellular bacteria. Microor-
and manipulate endophytic associations in crop plants to maxi- ganisms 9:1041
mize their benefit to crops (resulting in better plant health and 10. Micci A, Zhang Q, Chang X, Kingsley K, Park L, et al. 2022. Histo-
higher crop yields) and minimize agronomic practices that chemical evidence for nitrogen-transfer endosymbiosis in non-
negatively impact the environment and plant health. photosynthetic cells of leaves and inflorescence bracts of
angiosperms. Biology 11:876
11. Paungfoo-Lonhienne C, Rentsch D, Robatzek S, Webb RI, Sagu-
Author contributions lenko E, et al. 2010. Turning the table: plants consume microbes as
a source of nutrients. PLoS One 5:e11915
All authors contributed equally to research and writing of
12. White JF, Kingsley KL, Verma SK, Kowalski KP. 2018. Rhizophagy
this review article.
cycle: an oxidative process in plants for nutrient extraction from
symbiotic microbes. Microorganisms 6:95
Data availability 13. Macedo-Raygoza GM, Valdez-Salas B, Prado FM, Prieto KR,
Yamaguchi LF, et al. 2019. Enterobacter cloacae, an endophyte that
All data will be provided upon request to the corresponding
establishes a nutrient-transfer symbiosis with banana plants and
authors. protects against the black sigatoka pathogen. Frontiers in Microbi-
ology 10:804
Acknowledgments 14. Beltran-Garcia MJ, Martinez-Rodriguez A, Olmos-Arriaga I, Valdez-
Salas B, Chavez-Castrillon YY, et al. 2021. Probiotic endophytes for
The authors are grateful for support from USDA Multi-state more sustainable banana production. Microorganisms 9:1805
project 4147 (Protecting Plants from Diseases and Stress Using 15. Thomas P, Agrawal M, Bharathkumar CB. 2019. Diverse cellular
Endophytic Microbes) and USDA NIFA (Corn/Endophyte Part- colonizing endophytic bacteria in field shoots and in vitro cultured
nerships for Organic Farmers). papaya with physiological and functional implications. Physiologia
Plantarum 166:729−47
Conflict of interest 16. White JF, Chang X, Kingsley KL, Zhang Q, Chiaranunt P, et al. 2021.
Endophytic bacteria in grass crop growth promotion and biostim-
The authors declare that they have no conflict of interest. ulation. Grass Research 1:5
17. Sangwan S, Prasanna R. 2022. Mycorrhizae helper bacteria: unlock-
ing their potential as bioenhancers of plant–arbuscular mycor-
Dates rhizal fungal associations. Microbial Ecology 84:1−10
Received 27 June 2023; Accepted 17 August 2023; Published 18. Carvalhais LC, Dennis PG, Fedoseyenko D, Hajirezaei MR, Borriss R,
online 11 September 2023 et al. 2011. Root exudation of sugars, amino acids, and organic
acids by maize as affected by nitrogen, phosphorus, potassium,
and iron deficiency. Journal of Plant Nutrition and Soil Science
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