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CHAPTER 11
Natural Products from Endophytic Microorganisms
DAVID C. NWOBODO
1
2
3
1,2,*
and PETER M. EZE
3
*Corresponding author
ABSTRACT
Endophytic microorganisms that live in plant tissues without inflicting harm have been
broadly studied over the past few decades, and their potential for natural product discovery
and development has been widely recognized. The diversity of endophytic microorganisms
and their capacity to synthesize a variety of bioactive molecules make them an attractive
source for both the agricultural and pharmaceutical industries. The isolation, identification,
and characterization of natural products from endophytic microorganisms involve various
techniques, which have been refined over the years to work on the productivity of the
cycle and increase the yield of natural products. Researchers have also used molecular
biology techniques to identify and classify endophytic microorganisms and their natural
products, allowing for more targeted screening of microorganisms for specific compounds
of interest. Continued research in this field is critical to the improvement of new treatment
options for human and animal diseases, as well as sustainable solutions for agricultural
practices. In this chapter, we examine current data on endophytes as a wellspring of novel
and natural bioactive compounds, their diversity, and the various techniques used for the
isolation, identification, and characterization of these microorganisms and their natural
products.
11.1 INTRODUCTION
Plants have for some time been utilized as a wellspring of natural molecules for the
treatment of different diseases and different applications. Unexpectedly, lately, microorganisms related to plants, as opposed to plants themselves, have demonstrated to offer
molecules and compounds with high therapeutic potential (Subbulakshmi et al., 2012).
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Endophytic microorganisms are a collection of different fungi, bacteria, and actinomycetes that live inside the tissues of plants without inflicting harm (Petrini, 1991). These
microorganisms are ubiquitous in nature and can be found in different plant species,
such as crops, medicinal plants, and trees. Natural products from endophytic microorganisms comprise a vast wellspring of undiscovered bioactive compounds with the
potential to revolutionize the fields of medicine, agriculture, and industry. Throughout
recent many years, endophytic microorganisms definitely stand out because of their
capacity to create a large number of bioactive compounds with possible applications
in agriculture, medicine, and industry (Manganyi and Ateba, 2020). These mixtures
incorporate antimicrobials, anticancer specialists, cancer prevention agents, and other
normal items.
The term endophyte (Gr. endo, inside; phyton, plant) was coined rst by De Bary in
1866 and has become profoundly implanted in literature ever since (Xiang and LiangDong, 2012). De Bary previously presented the expression “epiphyte” for organisms that
live on the outer layer of their host and “endophyte” for those living inside the plant tissue.
Presently, endophytic organisms are dened as microbes that reside inward plant
tissues, comprising various groups of microorganisms (fungi, bacteria, viruses, protozoa)
and even microalgae, and do not cause harmful effects in their host (Vinu et al., 2021).
Initially, the term endophyte was broadly used to include everything from harmful foliar
microbes to mycorrhizal root symbionts (Rezwana, 2007). Although all pathogenic fungi
penetrate the host tissue and exist endophytically, fungi that create apparent symptoms
of disease are excluded from the endophyte category (Caroll, 1986). Nonetheless, a few
mycologists likewise incorporate in the endophyte category fungi that inhabit plant organs
at some stage in their life cycle without inicting obvious harm to their host, as well as
latent internal infections (Petrini, 1986). Therefore, in its most conservative denition, the
term endophyte now includes only organisms that reside within plant tissues at some time
during their life cycle without causing any symptoms.
With recent advances in drug research and development, the bioprospecting of endo-
phytic microbes for useful natural products has become one of the prime focuses. The
study of endophytic microorganisms and their natural products has become a signicant
area of exploration as they offer a promising alternative to synthetic compounds, which
are often associated with toxicity and resistance issues. The objective of this chapter is
to provide an overview of the natural products produced by endophytic microorganisms,
their applications, and future perspectives. The chapter will also discuss techniques for the
isolation and identication of endophytes and the challenges associated with their study.
11.1.1 RATIONAL/WHY ENDOPHYTES?
From time immemorial, man has been troubled by diseases that have inconspicuously
necessitated an effort to maintain good health by man. On the other hand, the emergence
of new diseases, the evolution of disease-causing agents, and what’s more, the enormous
evolvement of general health problems in the total populace have also necessitated an
evolution in man’s approach to tackling these challenges. One such approach to tackling
237
diseases is the use of plants and plant products. The use of plants and herbs in the treatment of disease conditions is as old as man himself. However, not only man has been
placed under the pressure and necessity for survival but plants themselves too have been
placed under such necessity, and evolved naturally to defend themselves. This cycle is
very interesting as it points to a special group of microorganisms, the “endophytes” at the
center. These endophytic microorganisms astonishingly have some innate ability to solve
some crucial plant natural problems and have necessitated their coexistence in a mutual
relationship with the host plant. The mutual relationships between the endophytes and
their hosts are believed to result in survival benefits for both partners (Khare et al., 2018).
Microbial endophytes might protect and enhance the survival of their host plants by the
production of a myriad of biologically active compounds (Strobel, 2018), which can be
harnessed for drug discovery.
Endophytic organisms gained prominence in 1981 after it was discovered that they
could defend their hosts from insects, pests, diseases, and even domestic herbivorous
animals (Webber, 1981). However, according to a report by Stelmasiewicz et al. (2023),
compounds produced by endophytes have gained increasingly signicant attention over
the last 22 years. Due to poverty and the cost of pharmaceutical drugs, people living in
developing countries, and a large number of the world’s population rely on traditional
herbs and plant-based medicines (W.H.O., 2019). However, one will wonder whether the
biological properties or effects derived from these plant-based medicines are produced
by the plants themselves or due to a mutualistic relationship with one or more benecial
microorganisms that reside intercellularly within the plant tissues. In a plant–microbe
relationship, endophytes have attracted much research interest because of their ability
to provide not only novel sources of antimicrobial substances (Nwobodo et al., 2020a)
and cytotoxic compounds, such as anticarcinogenic molecules (Uzma et al., 2018) but
also bio-stimulants for essential oil biosynthesis (El Enshasy et al., 2019; Nwobodo et al.,
2022a). They have also been proven to stimulate plant growth, facilitate the solubility
of nutrients in the plant rhizosphere, and act as biological control agents (Poveda and
Baptista, 2021; Poveda et al., 2020), or enhance plant immunity from biotic stresses (Cui
et al., 2021). Hence, there has been increased interest in endophytic microorganisms as
producers of novel bioactive compounds in recent years.
Second, as the quest for less harmful and more efcient bioactive compounds from
natural sources persists, products originating from plant sources will likely necessitate
enormous-scale harvesting, resulting in the mass destruction of such plants, leading to
climate-related imbalance and ecological disruption (Lawrence and Vandecar, 2015).
Several investigations have led to nding essential plant secondary metabolites (SMs) from
endophytic microbes (Nwobodo et al., 2022a; Bielecka et al., 2022; Ebada et al., 2016),
implying that such organisms could be used as alternate suppliers of these compounds. As
a result, studying plant-associated endophytes may give an alternate method of discovering
novel active metabolites of pharmaceutical importance, while maintaining climatic and
ecological balance.
Until recently, these groups of microorganisms were often neglected as ecosystem
components, which is the reason they are currently viewed as a treasure mine of untapped
biodiversity (Nicoletti and Fiorentino, 2015). Several investigations have documented a
238
vast number of bioactive chemicals isolated from endophytes, including phenols, quinines,
alkaloids, avonoids, peptides, terpenoids, and steroids (Manganyi and Ateba, 2020).
Almost all plant species have one or more endophytic organisms, but only a few are studied
for their endophytic biodiversity and capacity to synthesize bioactive SMs (Strobel, 2018).
Endophytes are thought to be more metabolically active than their free-living counterparts
because of their distinct activities in nature and activation of multiple pathways of metabolism for survival in host tissues (Fadiji and Babalola, 2020).
11.2 DIVERSITY OF ENDOPHYTIC MICROORGANISMS
Endophytes are currently regarded as an excellent source of bioactive natural products due
to the fact that many of them occupy millions of peculiar biological niches and develop in
a variety of strange conditions (Nwobodo et al., 2020b). Endophytes are microbes that are
found in a wide variety of plants, from tropical rainforests to dry deserts and are highly
diverse. Although it is believed that a single host may contain up to a million species of
endophytes, only a few of these have been identified so far (Gakuubi et al., 2021). This
suggests that there is a potential to discover new natural products from the multitude of
endophytes inhabiting different plants in various ecosystems. The diversity of endophytes
is influenced by a few factors, for example, the host plant particularity , geological area, and
environmental conditions. Endophytes exhibit host specificity, which means that they are
only found in certain plant species or families (Kim et al., 2013).
Geographic location also greatly contributes to the diversity of endophytic microorganisms. Different regions of the world have unique plant species that harbor diverse
endophytic microorganisms. Temperature, pH, and nutrition availability are all environ-
mental factors that inuence the variety of endophytic microorganisms (Ali et al., 2021).
Endophytic microorganisms living in plants growing in extreme environments, such as
deserts or thermal springs, are metabolically adapted to survive under such harsh conditions (Kochhar et al., 2022). It has also been demonstrated that the diversity of endophytes
in the tropical and subtropical regions is substantially greater than in other parts of the
world (Meshram and Gupta, 2019). Additionally, variations in seasons and weather have
also been identied as a major driver of the distribution and variety of fungal endophytes
(Materatski et al., 2019). The most common types of endophytes are bacteria, actinomycetes, and fungi (Gouda et al., 2016).
11.2.1 ENDOPHYTIC BACTERIA AND ENDOPHYTIC ACTINOMYCETES
The endophytic environment provides protection to bacteria that can survive within plants.
Recent reports have shown that endophytic bacteria and actinomycetes are highly diverse
and play significant roles in plant growth, stress tolerance, and the synthesis of natural
products with potential applications in biotechnology and medicine (T shikhudo et al., 2023;
Paul and Pratim, 2022). The composition and diversity of endophytic bacteria isolated
from various regions of the same plant may differ (Afzal et al., 2019). The rhizosphere
239
is the most prevalent route for bacterial endophytes to colonize their hosts. The most
common phyla of bacterial endophytes are Proteobacteria, Firmicutes, and Actinobacteria,
with the most common genera being Bacillus, Pseudomonas, Burkholderia, Micrococcus,
Stenotrophomonas, Microbacterium, and Pantoea being the most frequently occurring
genera (Burragoni and Jeon, 2021). Endophytic actinobacteria are the most prevalent type
of bacterial endophytes in practical applications as they are most abundant in roots, followed
by stems, and least abundant in leaves (Ganapathy and Natesan, 2018). The most prevalent
endophytic actinobacterial genera are Streptomyces, Microbacterium, Mycobacterium,
Arthrobacter, and Curtobacterium (Hardoim et al., 2015). Generally, bacterial endophytes
are highly diverse in nature and are reported to synthesize a large variety of useful bioactive
metabolites, with over 76% of these compounds identified specifically from the genus
Streptomyces (Gouda et al., 2016). Mangrove endophytes are extremely diverse, and
according to Azman et al. (2015), it is assumed that mangrove ecosystems are excellent
breeders of novel actinobacterial species.
Endophytic actinomycetes are lamentous bacteria that have been shown to synthesize a large variety of bioactive compounds, including antibiotics, antitumor agents, and
enzymes (Barka et al., 2016). Endophytic actinomycetes are known to possess a larger
genome than endophytic bacteria, with many genes encoding for SM biosynthesis (Azman
et al., 2015). This difference in genome size and metabolic capacity may contribute to the
ability of endophytic actinomycetes to synthesize a wider group of natural products than
endophytic bacteria.
Recent developments in techniques for high-throughput sequencing have made
it possible to identify novel endophytic bacteria and actinomycetes from various plant
species and environments. These technologies have also facilitated the understanding of
the functional roles of these endophytic microbes in the plant–microbe relationship leading
to the synthesis of useful natural products.
11.2.2 ENDOPHYTIC FUNGI
Endophytic fungi are one of the most diversified populations of endophytic microorganisms,
with an estimation of over 1 million species (Sagita et al., 2021). The abundance and variety
of fungal endophytes in numerous plants have been reported, with respect to different
environmental factors such as drought, pH, temperature, salinity, and other extreme
conditions in extreme environments (Rigobelo and Baron, 2021). The majority of endophytic
fungi are Ascomycetes, Deuteromycetes, and Basidiomycetes (Gakuubi et al., 2021). Based
on characteristics such as taxonomy, host range, transmission, and plant fitness benefits,
fungal endophytes are divided into two groups: clavicipitaceous and nonclavicipitaceous
(Rodriguez et al., 2009). Clavicipitaceous fungi are phylogenetically linked and are
transmitted vertically by seeds, whereas nonclavicipitaceous endophytes, which are
predominantly Ascomycota, are polyphyletic and found in a variety of plant species. Many
of these endophytic fungi can live in both endophytic and free-living environments. The
amount of SMs produced by fungal endophytes has been found to be greater than that of any
other endophytic microbe (Zhang et al., 2006).
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Fungi have been widely studied as a source of bioactive substances. Alexander Fleming’s
1928 discovery of penicillin from Penicillium notatum was the rst milestone in this eld
(Fleming, 1945). Other organisms’ selection pressure on fungal growth, such as predators,
competitors, and viruses, causes the synthesis of metabolites by fungi to defend their growth.
However, these metabolites frequently have additional bioactivities. Endophytic fungi
colonization of host plants is thought to contribute to host plant adaptation to biotic and
abiotic stress factors, which has been linked to fungal natural product synthesis (Zhang et al.,
2006). Endophytic fungi have a variety of effects on their host plants and their survival
and are normal to exert these effects via varied pathways or methods. One such method is
via the production of SMs. Fungal endophytes are recognized as one of the most inventive
producers of SMs that play signicant biological roles and are potential sources of innovative natural products (Selim et al., 2012). Some endophytic fungi isolated from plants by
several researchers include members of the genera Fusarium, Acremonium, Cladosporium,
Aspergillus, Penicillium, Curvularia, Stemphylium, Pseudofusicoccum, and Piriformospora
(Eze et al., 2019; Abba et al., 2018; Abonyi et al., 2018; Wang et al., 2018; Karunai and
Balagengatharathilagam, 2014), among others.
11.3 METHODS FOR ISOLATION AND IDENTIFICATION OF ENDOPHYTIC
MICROORGANISMS
The isolation and identification of endophytic microorganisms can be challenging, as
these microorganisms live within the plant tissues and are often present in low numbers.
Nonetheless, it is a crucial step in understanding their potential roles in plant–microbe
interactions and in exploring their potential applications. Plant endophytic microbe isolation
and characterization require several methods that aim to eliminate surface contaminants
while preserving the microorganisms within the plant tissues. In this section, we will
describe the most common methods used for the isolation and identification of endophytic
microorganisms.
11.3.1 ISOLATION METHODS
There are several methods that have been used for the isolation of endophytic microorganisms. The major methods include the following.
Culture-dependent methods involve the use of different media for the isolation and cultivation of endophytic microorganisms, based on the principle that different microbial species
have diverse nutritional and environmental requirements for growth. Examples of media
used in these methods include potato dextrose agar (PDA), nutrient agar (NA), and tryptic
soy agar (TSA). This method involves the following.
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11.3.1.1.1 Dilution Plating
This method involves the dilution of plant tissue extracts in a suitable nutrient-rich growth
medium, followed by the plating of the dilutions on agar plates (Singh et al., 2022). The
method allows for the isolation of individual colonies from the diluted plant tissue homogenates. Endophytic microorganisms have been isolated from diverse plant tissues including
stems, leaves, and roots using the dilution plating technique.
11.3.1.1.2 Direct Plating
This method involves the direct plating of plant tissue sections on agar plates (Singh et al.,
2022). The method eliminates the need for homogenization and dilution of plant tissues, but
it is less commonly used due to the difficulty in obtaining uniform sections of plant tissues.
Direct plating has been shown to be effective in isolating endophytic microorganisms from
the roots and stems of various plant species.
Despite their usefulness in isolating endophytic microorganisms, culture-dependent
methods have limitations, such as the underrepresentation of microbial diversity due to the
inability of some microorganisms to grow under laboratory conditions.
Culture-independent methods, on the other hand, are based on the extraction and amplification of microbial DNA from plant tissue, followed by the sequencing and analysis of the
DNA without the need for cultivation. These methods are more comprehensive in revealing
the microbial diversity present in the plant tissue (Hardoim et al., 2015). Polymerase chain
reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and terminal restriction
fragment length polymorphism (T-RFLP) are examples of culture-independent technologies.
Culture-independent methods also have limitations such as the risk of DNA sample contamination and the difficulty in interpreting the results.
11.3.2 IDENTIFICATION METHODS
Once the endophytic microorganisms have been isolated, various methods can be used to
identify them, which include the following.
One of the most common methods is the morphological or phenotypic identification
method, which involves the characterization of endophytic microorganisms based on their
observable characteristics, such as colony morphology, pigmentation, and growth rate
on different growth media (Ambikapathy et al., 2023). This is a traditional method of
242
identifying microorganisms but has limitations as many endophytic microorganisms have
similar characteristics.
The application of molecular techniques for the characterization of endophytic microorganisms is increasingly being adopted. These techniques involve the sequencing of conserved
genes such as the 16S rRNA gene for bacteria (Tiwari and Bae, 2022) and the internal
transcribed spacer (ITS) region for fungi (Nwobodo et al., 2022a) to identify the microorganisms at the species or genus level. Examples of molecular identification techniques for
endophytic microorganisms include PCR and DNA sequencing. PCR amplifies specific
regions of microbial DNA, allowing for the identification of specific microbial groups.
DNA sequencing provides more accurate identification of microbial species based on the
comparison of DNA sequences with reference sequences in databases. Molecular identification has been shown to be a reliable and accurate method for identifying endophytic
microorganisms.
It is vital to note that the method of isolation and identication chosen is dependent on
a number of criteria, such as the type of microorganism being studied, the availability of
resources, and the research objectives. The combination of different methods can provide a
deeper comprehension of the diversity and features of endophytic microorganisms.
11.4 BIOACTIVE COMPOUNDS FROM ENDOPHYTIC MICROORGANISMS
Endophytic microorganisms including fungi and bacteria have been discovered to be
producers of potential bioactive agents with diverse applications. Particularly, endophytic
microorganisms, mostly fungi, have gained popularity as a source of pharmacological
metabolites with various therapeutic uses. In recent years, there has been an upsurge in the
production and commercialization of these metabolites from endophytes. Tiwari and Bae
(2022) have reported several such metabolites, and Figure 11.1 provides a few selected
examples. Based on their activity , these compounds can be categorized into several categories including antibiotics, anticancer, antiviral, antifungal agents, and antioxidants.
11.4.1 ANTIBIOTICS
Endophytic microorganisms are a promising source of antibiotics, which are important for
treating bacterial infections. The production of antibiotics by endophytes is believed to be
a mechanism of competition with other microorganisms in the host plant. The polyketide
citrinin produced by the endophytic fungus Penicillium janthinellum isolated from Melia
azedarach fruits displayed 100% antibacterial action against Leishmania
sp. (Marinho et al.,
2005). Liu et al. (2008) found a bioactive substance called 7-amino-4-methyl coumarin in the
culture extracts of the endophytic fungus Xylaria sp. YX-28 obtained from Ginkgo biloba L.