Isolation and Identification of Therapeutic

Metabolite Producing Endophytic

Microorganisms from Medicinal Plants

Submitted in partial fulfillment of the Requirements for the degree of

Bachelor of Technology in Biotechnology

By

ASHUTOSH PUROHIT

(Reg. No. 12BBT0044)

DEPARTMENT OF BIOTECHNOLOGY

SCHOOL OF BIO SCIENCE AND TECHNOLOGY

9th may 2016

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 DECLARATION

I hereby declare that the thesis entitled “Isolation and Identification of Therapeutic
Metabolite Producing Endophytic Microorganisms from Medicinal Plants” submitted by me, for
the award of the degree of [Link] in Biotechnology to VIT University, is a record of bonafide research
work carried out by me under the supervision of Prof. S Venkat Kumar

I further declare that the work reported in this thesis has not been submitted and will not be
submitted, either in part or full, for the award of any other degree or diploma in this institute or of any
other institute or university.

Signature of the Candidate

Place:

Date:

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 CERTIFICATE
This is to certify that the dissertation entitled “Isolation and Identification of Therapeutic
Metabolite Producing Endophytic Microorganisms from Medicinal Plants” submitted by
ASHUTOSH PUROHIT (12BBT0044), to the VIT University, for the award of the degree of [Link],
in Biotechnology, is a record of bonafide work carried out by him under my supervision, as per the VIT
code of academics and research ethics.

The contents of this report have not been submitted and will not be submitted either in part or
in full, for the award of any degree or diploma in this institute or any other institute or university. The
thesis fulfills the requirements and regulations of the University and in my opinion meets the necessary
standards for submission.

Place:

Date: Signature of the Guide(s)

Thesis Approval Form

3
 This thesis, entitled

Isolation and Identification of Therapeutic

Metabolite Producing Endophytic
Microorganism from Medicinal Plants

and authored by ASHUTOSH PUROHIT (Reg. No. 12BBT0044), is hereby accepted and approved.

External Examiner(s) Head of Department Dean SBST

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ABSTRACT

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 Acknowledgement
I sincerely thank Dr. G. Viswanathan, Founder and Chancellor of VIT University, for providing spiritual
and technical support with the best infrastructure to carry out the research meticulously.

I thank Dr Ramalingan C, Dean of the School of Biosciences and Technology for allowing us to carry
this project out to the best of our abilities.

I acknowledge with a deep sense of gratitude and happiness the guidance that Prof. Venkat kumar S has
given us. His tutelage has been an irreplaceable source of learning.

I also extend our gratitude towards Prof. Ramanathan K. for his continued support. He has been an
intellectual and moral compass throughout this endeavor.

I would be amiss if I did not thank the valuable assistance provided to us by Ms. Divya G.

Place: Vellore (ASHUTOSH PUROHIT)

Date:

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 TABLE OF CONTENTS
CHAPTER NO. TITLE PAGE NO.
ABSTRACT V
ACKNOWLEDGMENT Vi
LIST OF FIGURES Viii
LIST OF TABLES X
LIST OF SYMBOLS AND ABBREVIATIONS Xi

1. INTRODUCTION 1
2. LITERATURE REVIEW 4
3. METHODS AND MATERIALS 8
3.1 SELECTION OF PLANTS FOR ISOLATION OF 8
ENDOPHYTES
3.2 SAMPLING AREA AND COLLECTION OF PLANT 8
MATERIAL
3.3 CHARACTERIZATION OF ENDOPHYTES 10
3.4 PHYTOCHEMICAL ANALYSIS 10
4. RESULTS AND DISCUSSIONS 13
4.1 ISOLATION AND IDENTIFICATTION OF ENDOPHYTIC 13
ORGANISM
4.2 ENZYME ACTIVITY EVALUATION FOR BACTERIA 13
4.3 ANTIBACTERIAL SCREENING 17
4.4 BIOCHEMICAL ANALYSIS 20

5. CONCLUSION 44

6. REFERENCES 46

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LIST OF FIGURES

FIGURE NO. TITLE FIGURE NO.

representation of natural
product drug discovery
Figure 1.4.2 Figure 1.4.2
approach discussed in this
review.
Graphical representation of
Figure1.4.3 Figure1.4.3
taxol biosynthesis in Taxus spp
Endophytic bacterial pure
culture
Figure 3.2.3 A Figure 3.2.3 A

fungal mother culture (different Commented [m1]: Capitalize first letter of first word for all
Figure 3.2.3 B Figure 3.2.3 B figures and tables
fungi strain can be observed)
fungal culture can be obtained

Figure 3.2.3 C by doing sub culturing from the Figure 3.2.3 C

mother culture

Asparagus stem culture

Figure 4.1 A (different fungi shows different Figure 4.1 A

colors)

Penicillium sp. Identified by

Lactophenol Cotton Blue
Figure 4.1 B Figure 4.1 B
(LPCB)

Cocci shaped Gram negative

Figure 4.1 C bacteria was identified by Figure 4.1 C

Gram staining

Citrate utilization test showing

Figure 4.2 positive result Figure 4.2

Fungal crude sample with

Figure 4.3 A Figure 4.3 A
acetate and benzene show

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antibacterial susptibility test for
Staphylococcus aureus

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LIST OF TABLES
TABLE NO. TITLE PAGE NO.
Table 2.1 compound extracted from
various endophytes
Table 4.4 Preliminary qualitative
phytochemicals screening
result

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 CHAPTER 1
INTRODUCTION
1.1. Endophytes
Taken literally, the word endophyte means “in the plant” (endon Gr. = within, phyton = plant).
The usage of this term is as broad as its literal definition and spectrum of potential hosts and
inhabitants, e.g. bacteria, fungi, plants and insects in plant, but also for algae within algae. Any
organ of the host can be colonised. Equally variable is the usage of the term “endophyte” for
variable life history strategies of the symbiosis, ranging from facultatively saprobic to parasitic
to exploitive to mutualistic. The term endophyte is, for example, used for pathogenic
endophytic algae, parasitic endophytic plant, mutualistic endophytic bacteria and fungi, and
pathogenic bacteria and fungi in latent developmental phases, but also for microorganisms in
commensalistic symbioses. Some authors also designate the interactions of mycorrhizal fungi
with the roots of their hosts as being endophytic. However, we concur with Brundrett, who
distinguishes mycorrhizal from endophytic interactions; the former having synchronised plant-
fungus development and nutrient transfer at specialised interfaces. Nevertheless, as we will see
in this book, distinctions between mycorrhizal and non-mycorrhizal fungi are not always clear-
cut. Not only can mycorrhizal fungi become pathogenic, but, for example, dark septate
endophytes (DSE) can assume mycorrhizal functions. In addition, there are also cases in which
fungal root endophytes seem to be saprobes, e.g. Oidiodendron maius and Phialocephala
fortinii. Although there are diverse uses for the word endophyte, “endophytes” are most
commonly defined as those organisms whose “…infections are inconspicuous, the infected
host tissues are at least transiently symptomless, and the microbial colonisation can be
demonstrated to be internal...” Although these authors used this definition to describe fungal Commented [m2]: Please put reference

endophytes, it is equally applicable to bacterial endophytes. It is important to remember that

the definition describes a momentary status. Thus it includes an assemblage of microorganisms
with different life history strategies: those that grow saprophytically on dead or senescing
tissues following an endophytic growth phase, avirulent microorganisms as well as latent
pathogens and virulent pathogens in the early stages of infection. Unfortunately, taken literally,
it can include all pathogens at some stage of their development. Since the plant host responds
to at least some infections with mechanical defence reactions, there is merit to Petrini’s
additional characterisation of endophytic interactions as not “causing apparent harm”, which

11
presumably refers to an absence of macroscopically visible symptoms. Aware of the
determinative discrepancies, we will nevertheless use the term “endophyte” to describe those
bacteria and fungi that can be detected at a particular moment within the tissues of apparently
healthy plant hosts.
1.2. Why endophytes
The need for new and useful compounds to provide assistance and relief in all aspects of the
human condition is ever growing. Drug resistance in bacteria, the appearance of life-threading
viruses, the recurring problems with disease in persons with organ transplants, and the
tremendous increase in the incidence of fungal infections in the world's population each only
underscore our inadequacy to cope with these medical problems. Added to this are enormous
difficulties in raising enough food on certain areas of the Earth to support local human
populations. Environmental degradation, loss of biodiversity, and spoilage of land and water
also add to problems facing mankind. Endophytes, microorganisms that reside in the tissues of
living plants, are relatively unstudied and potential sources of novel natural products for
exploitation in medicine, agriculture, and industry. It is noteworthy that, of the nearly 300,000
plant species that exist on the earth, each individual plant is host to one or more endophytes.
Only a few these plants have ever been completely studied relative to their endophytic biology.
Consequently, the opportunity to find new and interesting endophytic microorganisms among
myriads of plants in different settings and ecosystems is great.
1.3. Source for endophytes
Fungal endophytes are generally from the phylum Ascomycota, though other phyla are
represented. Some specific examples of which are found in orders Hypocreales and Xylariales
of the Sordariomycetes (Pyrenomycetes) class. Additionally the class of Loculoascomycetes
includes endophytes. Although endophytes may be diverse taxonomically, Rodriguez et al.
classified fungal endophytes broadly into ecological categories or functional classes.
A number of endophytes are now known that grow within seaweeds and algae. One such
example is Ulvella leptochaete, which has recently been discovered from host algae
including cladophora and laurentia from India. Commented [m3]: Check if scientific name and genus name
then capitalize first alphabet and italicise
Bacterial endophytes may belong to a broad range of taxa, including α-Proteobacteria, β-
Proteobacteria, γ-Proteobacteria, Firmicutes, Actinobacteria, etc… Bacterial endophytes have
been found to become intracellular in root and shoot cells of many plants, with entry into cells
in the meristems. In this intracellular form bacteria lose cell walls but continue to divide and
metabolize. These wall-less intracellular forms of bacteria are called L-forms. Paungfoo-

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Lonhienne et al. observed the degradation of intracellular microbes within root cells and
hypothesized that intracellular microbes may be a source of organic nutrients or vitamins for
plants; they termed this process 'rhizophagy'.
1.4. Endophytes for medicinal and industrial application
The wide range of compounds produced by endophytes have been shown to combat pathogens
and even cancers in animals including humans. One notable endophyte with medicinal benefits
to humans was discovered by Gary Strobel: Pestalotiopsis microspora, an endophytic fungus Commented [m4]: italicize

of Taxus wallachiana (Himalayan Yew) was found to produce taxol. Endophytes are also being Commented [m5]: all scientific names italicize

investigated for roles in biofuels production. Inoculating plants with certain endophytes may
provide increased disease or parasite resistance while others may possess metabolic processes
that convert cellulose and other carbon sources into "myco-diesel" hydrocarbons and
hydrocarbon derivatives.
1.5. Natural product discovery
The need for novel chemical compounds to treat human diseases is ever increasing. The rapid
development of drug-resistant microbes, the discovery of new cases of life-threatening
infections, and the constant recurrence of diseases have pushed for advances in the field of drug
discovery. In principle, there are three pathways for discovering new pharmacologically
significant compounds: rational drug design, where the drug is purposefully tailored towards
specific targets in the microbial cell combinatorial chemistry, which involves synthesis of a
combinatorial library of compounds, which are then tested against the cellular target to
determine the most potent compounds and natural product discovery, by isolating bioactive
compounds from biological sources. Of late, pharmaceutical companies have shifted their
interest towards the first two pathways. These pathways utilize the latest advances in three-
dimensional X-ray crystallography, drug-docking tools, and other computer-aided
methodologies which significantly cut the development time of a compound, from compound
synthesis to market delivery. There are, however, downfalls with these approaches, including
the high cost to discover novel compounds and for production. Moreover, until the detailed
mechanisms of targeted cellular death and survival are comprehensively elucidated, it will
remain difficult to select potential targets for structure-guided drug design. Furthermore, being
laboratory-synthesized compounds, combinatorial compounds are often insufficiently
complex, possessing limited structural rigidity, and require extensive purification steps and
bioactivity testing to conclusively characterize the bioactive compounds . In addition, there has
been a steady increase in the negative public perception regarding the use of synthetic drugs

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due to their long-term safety and environmental concerns. Consequently, efforts are being
made to re-explore the potential of natural products as sources of novel drugs

Figure 1. Graphical representation of natural product drug discovery approach

discussed in this review.
1.6. Endophytes as sources of natural products
Endophytes refer to the microorganisms (mostly fungi and bacteria) colonizing the intercellular
and intracellular regions of healthy plant tissues at a particular time, whose presence is
unobtrusive and asymptomatic. Most plant species that have been previously studied host at
least one endophytic microbe, with plants growing in unique environmental settings generally
hosting novel endophytic microorganisms.
Endophytes form a symbiotic relationship with their plant host. It is believed that in many cases
the microbes function as the biological defense for the plant against foreign phytopathogens.
The protection mechanism of the endophytes are exerted directly, by releasing metabolites to
attack any antagonists or lyse affected cells, and indirectly, by either inducing host defence
mechanisms or promoting growth.
Antibiotics or hydrolytic enzymes can be released by endophytes to prevent colonization of
microbial plant pathogens, or prevent insect and nematodes from infecting plants. In other cases
endophytes release metabolites which activate host defense mechanism against other
pathogenic organisms, in a process known as induced systemic resistance. Similarly,
endophytes can also promote plant growth in an attempt to outcompete cell apoptosis induced
by infecting pathogens. Plant growth promotion by endophytes may be exerted by several
mechanisms, such as production of phytohormones, synthesis of siderophores, nitrogen
fixation, solubilization of minerals such as phosphorus, or via enzymatic activities, such as
ethylene suppression by 1-aminocyclopropane-1-carboxylate deaminase. The plant host also
benefits from the endophytes by their natural resistance to soil contaminants, their ability to
degrade xenobiotics, or their action as vectors to introduce degradative traits to plants, which
substantially assist phytoremediation.

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Endophytes can produce the same or similar secondary metabolites as their host. Bioactive
compounds which are co-produced by the plants as well as their associated endophytes include
the anticancer drug camptothecin, the anticancer drug lead compound podophyllotoxin, and the
natural insecticide azadirachtin . There are several mechanisms proposed for the simultaneous
production of these biological compounds. In some cases, such as that of gibberellin, the
biosynthetic mechanism of the same compound evolves independently in plants and their
microbial counterparts. On the other hand, horizontal gene transfer between the plant host and
its endophytes have long been hypothesised, although so far this process has only been shown
to occur between microbial endophytes. It has been strongly suggested, however, that
interactions between endophytes and their respective plant host contributes to the co-
production of these bioactive molecules.
Endophytes have recently generated significant interest in the microbial chemistry community
due to their immense potential to contribute to the discovery of new bioactive compounds. It
has been suggested that the close biological association between endophytes and their plant
host results in the production of a greater number and diversity of biological molecules
compared to epiphytes or soil-related microbes. Moreover, the symbiotic nature of this
relationship indicates that endophytic bioactive compounds are likely to possess reduced cell
toxicity, as these chemicals do not kill the eukaryotic host system. This is of significance to the
medical community as potential drugs may not adversely affect human cells.
One of the most successful stories of natural products from endophytes is the multibillion-
dollar anticancer drug Taxol (paclitaxel). The compound was initially isolated from the Pacific
yew tree, Taxus brevifolia , a traditional medicinal plant used by Native Americans. Since then,
several other plants from the genus Taxus have been reported to produce Taxol. Nonetheless,
these plants are slow-growing with generally isolated geographical distribution. Investigations
of endophytes from this plant revealed that some fungi, such as Taxomyces andreanae, also
produced the exact same compound. The biological production of this compound
in Taxus plants have been characterised. While horizontal gene transfer has long been proposed
for the biosynthesis of Taxol in endophytes, it has been recently showed that the endophyte
genomes did not contain any sequences with significant homology to the Taxol biosynthetic
genes from Taxus spp. Indicating Taxol biosynthesis in endophytes might have developed
independently from its plant host. Nevertheless, this example supports the rationale that
traditional medicinal plants can be used as the starting point to investigate endophytes for their
production of biologically active compounds.

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As mentioned earlier, approximately three quarters of anti-infectives are natural products or
natural-product derived structures. However, rather than using combinatorial chemistry to
synthesise these derivatives, the biosynthesis of these natural products has been elucidated at
the genetic level. As the synthesis of most of these natural products is regulated by single gene
clusters, numerous research groups have attempted to isolate these clusters and utilise genetic
engineering to biologically synthesise the native compounds as well as their derivatives. Two Commented [m6]: synthesize not synthesise

of the most studied and largest classes of secondary metabolites are the polyketides and non-
ribosomal peptides.

Figure 2. Graphical representation of taxol biosynthesis in Taxus spp. (adapted from

Walker and Croteau (2001)). Multiple arrows indicate several as yet undefined steps.
1.7. The search for endophytes
It is speculated that there may be many thousands of endophytes useful to mankind but since
there are few scientists working in this field, and since environmental contamination,
deforestation and biodiversity loss are widespread, many endophytes might be permanently
lost before their utility is explored.

Endophytic species are very diverse; only a small minority of existing endophytes have been
characterized. A single plant organ (leaf, stem or root) of a plant can harbor many different
species of endophytes, both bacterial and fungal. Additionally, some endophytic bacteria may
live within endophytic fungi.

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Endophytes can be identified in several ways, usually through amplifying and sequencing a
small piece of DNA. Some endophytes can be cultured from a piece of their host plant in an
appropriate growth medium. An important step in culturing endophytes is to surface disinfect
plant tissues prior to placement on culture media. This kills epiphytic microbes, ensuring only
growth of endophytic microbes. Not all endophytes can be cultured in this way, as shown by
discovery of cryptic, unculturable endophyte species through DNA based analysis of leaf
tissue. Some grass endophytes in genus Epichloë can be seen as intercellular sinuous strands
of hyphae under the microscope following leaf sheath or culm tissue staining with aniline
blue. Many endophytes do not sporulate when cultured. Since fungal identification
by morphology is based primarily on spore-bearing structures, this fact makes visual
identification of some endophytic cultures challenging.
1.8. Therapeutic metabolite
Metabolites are intermediate or products of metabolism. They have wide application in our
body as small molecules. They have different roles to play like supply, signaling, inhibitory
and stimulatory effect on an enzyme in our body. A major metabolites play a direct role in
growth and development. Ethylene production in the major metabolite of industrial
biotechnology is a good example. Secondary metabolite do not have direct role in the process,
but have an ecological function. For example, antibiotics and dyes etc.
Some metabolite produced by the endophytes have therapeutic value as they can be used as
drug or prodrug. endophytic fungus play significant role in physical and ecological life of their
host. In recent years, there is evidence that secondary metabolites produced by the endophyte
is not random, but is correlated with its ecological niche . Endophytes conversation with their
host’s metabolism in favor of the synthesis of biologically active secondary metabolites may
occur. Plant endophytic fungi and bacteria produce novel bioactive secondary metabolite
compound with potential application in field of agriculture, medicine and food industries, with
potential application in novel bioactive secondary metabolites are the source. Interestingly,
some of the compounds produced by the endophytic microbe’s taxol, cryptocoin, cryptocandin,
jesterone, oocydin, isopestacin, psuedomycins and ambuic acids.

1.9. Extraction of endophytes

Initial step is selection of proper and promising plant. For extraction of endophyte select mature
healthy parts of plants like leaves, stems, root etc. than surface sterilization and culture of
microorganism on growth medium. Isolation and characterization of bioactive substances from
culture filtrates is done using bioassay guided fractionation and spectroscopic methods.

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18
 OBJECTIVES

 To isolate and identify secondary metabolite producing endophytes from

medicinal plants.
 Screen out the endophytes by performing various analysis.
 Partial purification of secondary metabolites and applications in medicinal
field. Commented [m7]: objectives are absurd reframe

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 CHAPTER 2
LITERATURE REVIEW
1. The term endophyte (Gr. endon, within; phyton, plant) was first coined by De Bary (De Bary,
1866) and an endophyte is a bacterial or fungal microorganism, which spends the whole or
part of its life cycle colonizing inter- and/or intra-cellular inside the healthy tissues of the
host plant, typically causing no apparent symptoms of disease (Wilson, 1995). The presence
of endophytes was reported by Vogl in 1898 who revealed a mycelium residing in the grass
seed Lolium temulentum. In 1904 in Germany Freeman identified an endophytic fungus, in Commented [m8]: italicize

Persian darnel (annual grass).

2. Bacterial endophytes have been known for >100 years. The presence of bacteria resident
within the tissues of healthy plants was first reported as early as 1926 (Hallman et al., 1997).
In 1926, Perotti recognized endophytic growth as a particular stage in the life of bacteria,
described as an advanced stage of infection and as having a close relationship with
mutualistic symbiosis. Perotti (1926) was the first to describe the occurrence of
nonpathogenic flora in root tissues and Henning and Villforth (1940) reported the presence
of bacteria in the leaves, stems and roots of apparently healthy plants. Since then, endophytes
have been defined as microorganisms that could be isolated from surface-sterilized plant
organs. Since 1940S there have been numerous reports on endophytic bacteria in various
plant tissues (Hallmann, et al., 1997). In the 1980s endophytic bacteria having nitrogen fixing
ability were found in graminaceous plants (Reinhold- Hurek and Hurek, 1998b).
3. These endophytic relationships may have begun to evolve from the time that higher plants
first appeared on Earth hundreds of millions years ago. Evidence of plant associated microbes
has been discovered in the fossilized tissues of stems and leaves (Taylor and Taylor, 2000).
As a result of these long-held associations, it is possible that some of these endophytic
microbes may have devised genetic systems allowing for the transfer of information between
themselves and the higher plant and vice versa (Stierle, et al., 1993).
4. The term endophyte refers to interior colonization of plants by bacterial or fungal
microorganisms. Endophytes have been defined in several ways and the definitions have been
modified as the research in this field advanced. Tervet and Hollis (1948) defined endophytes
as microorganisms that are able to live inside plants without causing disease symptoms.
‘Endophytic bacteria’ are the population of bacteria that reside within the living organism
without doing substantive harm or gaining benefit other than securing residency (Kado,
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 1992). Endophytic bacteria or fungi colonize the host tissue internally, sometimes in high
numbers, without damaging the host or eliciting symptoms of plant disease according to a
widely used definition by Quispel (1992). Microorganisms living within plant tissues for all
or part of their life cycle without causing any visible symptoms of their presence are defined
as endophytes (Wilson, 1993). Bacon and White (2000) defined endophytes as ‘microbes that
colonize living, internal tissues of plants without causing any immediate, overt negative
effects’. According to Schulz & Boyle (2006) endophytic bacteria can be defined as those
bacteria that colonize the internal tissue of the plants showing no external sign of infection
or negative effect on their host.
5. Among the definitions given to endophytic bacteria the following by Hallmann et al., (1997)
seems to be the most adequate. Hallmann et al. (1997) defined endophytic bacteria as all
bacteria that can be detected inside surface sterilized plant tissues or extracted from inside
plants and having no visibly harmful effect on the host plants. This definition includes
internal colonists with apparently neutral behaviour as well as symbionts. It would also
include bacteria which migrate back and forth between the surface and inside of the plant
during their endophytic phase. The relationship between the endophyte and its host plant
may range from latent phytopathogenesis to mutualistic symbiosis (Sturz et al., 1997).
6. In accordance with their life strategies, bacterial endophytes can be classified as ‘obligate’ or
‘facultative’. Obligate endophytes are strictly dependent on the host plant for their growth
and survival and transmission to other plants occurs vertically or via vectors. Facultative
endophytes have a stage in their life cycle in which they exist outside host plants. In the
extreme view, bacterial phytopathogens might be included as (facultative or obligate)
endophytes, because they often occur in avirulent forms in plants. Avirulent forms of plant
pathogens should thus be regarded as endophytes, whereas virulent forms of these organisms
should not be included (Hardoim et al., 2008).
7. The life cycle of facultative endophytes can be characterized as biphasic, alternating between
plants and the environment (mainly soil). The vast majority of the microorganisms that can
thrive inside plants probably have a propensity to this biphasic lifestyle. In fact, the observed
microbial diversities inside plants could be explained by the ability of diverse endophytes to
enter into and persist in plants (Rosenbleuth and Martinez-Romero, 2006).
8. Soil microbial communities play an integral and often unique role in ecosystem functions
and are among the most complex, diverse, and important assemblages in the biosphere (Zhou,
et al., 2003). It seems that the bacteria best adapted for living inside plants are naturally
selected. Endophytes are recruited out of a large pool of soil or rhizospheric species and
21
 clones. Endophytic bacteria can actively or latently colonise plants locally or systemically
and both intercellularly and intracellularly. Various reports indicate that these bacteria exist
in a variety of tissue types within numerous plant species, suggesting an ubiquitous existence
in most, if not all, higher plants. Endophytic bacteria in a single plant host are not restricted
to a single species but comprise several genera and species (Ryan et al., 2008).
9. Endophytic bacteria have been isolated from a large diversity of plants was reviewed by Sturz
et al. (2000). Mundt and Hinkle (1976) reported as many as 46 different bacterial species
from 27 plant species.
10. In planta and ex planta populations of Pseudomonas species could be differentiated by
biochemical characteristics (van Peer et al., 1990). McInroy and Kloepper (1991) showed
that the endophytic bacterial populations in corn stems ranged from 1x103 initially to 1 xl0
10cfu/ml after 10 week.
11. Mavingui et al. (1992) found that there are different populations of Bacillus polymyxa in
soil, rhizosphere, and rhizoplane and that wheat roots select specific populations as
endophytes.
12. Sturz et al. (1997) characterized 15 bacterial species from red clover nodules and estimated
endophyte population densities to be in the range of 104 viable bacteria per gram of fresh
nodule.
13. Germida et al. (1998) found that the endophytic population was less diverse than the root-
surface population and the endophytes appeared to originate from the latter.
14. Bacterial endophytes are found in a variety of plants, such as sugar beet (Dent et al., 2004),
prairie plants, agronomic crops (Zinniel et al., 2002), potato varieties (Sessitsch et al., 2002),
wheat (Germida and Siciliano, 2001), and rice (Sun et al., 2008).
15. Suman et al. (2001) isolated endophytic bacteria from several cultivars of Indian sugarcane
on LGI medium. In a review by Lodewyckx et al. (2002), 81 different bacterial species were
reported to form endophytic associations with plants.
16. Endophytic microbes from medicinal plants are good source of functional metabolites
(Weber et al., 2004; Tejesvi et al., 2007; Huang et al., 2008). Endophyte infection found to
alters pattern of gene expression in the host plant (Baily et al., 2006). Interaction between
endophyte and plant is mainly controlled by the genes of both organism and host plant
modulated by the environment. Endophytes from Bioprospecting for Microbial Endophytes
and their Natural Products Ph.D Thesis: Ruby E Jalgaonwala.2013, NMU,Jalgaon. 10
angiosperms as well as gymnosperms have been studied for presence of novel secondary
metabolites. Primary metabolites are common in all living cells and are involved in the
22
 formation of biomass and generation of energy, in contrary secondary metabolites are
produced by one or few species only. These secondary metabolites are low molecular weight
compounds, they are not required for growth in pure culture and are produced as an
adaptation for the specific function in nature. Bioprospecting is most frequently used phrase
to describe the collection and screening of the biological material for commercial purposes.
The importance of natural products in the drug discovery and development has been reported
briefly. The natural products produced by endophytes have vast range of bioactivities,
representing a vast reservoir offering an enormous potential for exploitation in medicinal and
industrial uses (Zhang et al., 2006). Crude extracts from culture broth of endophytes found
to show antibacterial, antifungal, antiviral, anti-inflammatory and antitumor activities (Silva
et al., 2007). Therefore, endophytes open up new areas for the biotechnological exploitations.
17. The requirement for new antimicrobial agents generally, comes from the increasing
resistance of pathogenic microbes towards antibiotics. Many microorganisms of agricultural
concern are also known to acquired resistance to commonly used antimicrobial chemical
compounds. So the interest in natural methods of pathogen control through new, eco-friendly
agents is increased. The biologically active natural products from endophytes are excellent
resources for medicine, agriculture and industry (Guo et al., 2008). Amines and amides are
very common metabolite products from endophytes and have shown to be toxic to insect but
not to mammals. Bioactive metabolites, such as steroids, terpenoids and diterpenes also are
generated by endophytes. Endophyte also produces extracellular hydrolyases to establish a
resistance mechanism against plant invasion which includes some of the extracellular
enzymes like cellulases, proteinase, lipases and esterases. The actions of these enzymes found
to support the hypothesis of co-evolution between endophytes and their hosts. Number of
secondary metabolites produced by fungal endophytes is larger than that of any other
endophytic microorganisms (Zhang et al., 2006). Endophytic fungi are a promising source of
novel compounds. About 51% of biologically active substances from fungal endophytes were
previously unknown (Strobel, 2002a).
18. Endophyte hold main position in drug discovery as it has antibiotic, antiviral and anticancer
properties, due to their ability to produce chemical which can be used as drug. Pacli taxel was
first found in plants and later on reported from fungal endophyte. It is the first major group
of anticancer agent which is produced by endophyte and now much research has been
conducted on endophytes to determine its anticancer activity. Production of taxol was also
done from an endophytic fungus, Lasiodiplodia theobromae isolated from Morinda citrifolia Commented [m9]: italicize

with its cytotoxicity against human breast cancer cell line.

23
19. Subsequently one hundred anticancer compounds which belong to different chemical classes
with activity against 45 different cell lines have been isolated from different fungal species
belonging to different groups, out of which 57% were novel oranalogues of known
compounds. Endophytic fungi was isolated and identified from Juniperus communis L.
Horstmann, as a novel producer of deoxypodophyllotoxin (Kusari et al., 2009).
20. Chaetopyranin a benzaldehyde derivative isolated from the endophytic fungus Chaetomium
globosum associated with the marine red alga Polysiphonia urceolata. Chaetopyranin
exhibited moderate to weak cytotoxic activities against three human tumor cell lines HMEC
(human microvascular endothelial cells), SMMC-7721 (hepatocellular carcinoma cells) and
A54 (human lung epithelial cells) (Wang et al., 2004). Some of the most potent plant-derived
antitumor alkaloids have also been reported from endophytic fungi. In 2005 Camptothecin
was isolated from a fungal endophyte Entrophosphora infrequens from Nothapodytes
foetida.
21. Indole alkaloid emindole was isolated from endophyte Emericella nidulans var. acristata,
from mediterranean green alga, exhibiting antitumor activity against 36 human tumor cell
lines. Anticancer drugs, which has been given in chemotheraphy treatment for several type
of cancer including leukemia, lymphoma and lung cancer, are indole derivatives one of which
known as vincristine (Figure 4.). Vincristine available under trade names Oncovin R was
originally obtained from plant Catharanthus roseus now Chinese researchers reported
evidence that vincristine might be produced by endophytic Fusarium oxysporum from the
same plant (Zhang et al., 2006).
22. Plants with ethno botanical history are generally expected to be powerful source of
endophytes producing active natural products (Strobel et al., 2004). As more than 3000
diseases are clinically described today less than one third of these can be treated
symptomatically, and even less than that needs new therapeutic agent with infectious disease
control (Strobel and Daisy, 2003). In recent time it is found that isolated secondary
metabolites of endophyte are synthesize via variety of metabolic Bioprospecting for
Microbial Endophytes and their Natural Products Ph.D Thesis: Ruby E Jalgaonwala.2013,
NMU,Jalgaon. 28 pathways, for example polyketide, isoprenoid or amino acid derivation.
And belongs to different structural groups such as phenols, isocoumarins steroids, xanthones,
perylene derivatives, depsipetides quinines, furandiones, terpenoids, and cytochalasines.
Fungus Podospora Sp. endophytic from the plant Laggera alata (Asteraceae) shows presence
of Xanthones sterigmatocystin (Figure 5.). Also chaetoglobosin A and rhizotonic acid were

24
 reported from endophytic Chaetomium globosum in Maytenus hookeri and Rhizoctonia Sp.
in cynodon dactylon correspondingly to be active against the gastric ulcer.
23. Novel fungal genus Cinnamomum zeylanicum found to be producing extremely bioactive
volatile organic compounds (VOCs). Endophytic Muscodor albus produces a mixture of
VOCs consists primarily of various alcohols, acids, esters, ketones and lipids (Strobel et al.,
2001). Cryptocandin A an antifungal lipopeptide was isolated from endophytic
Cryptosporiopsis quercina containing a number of unusual hydroxylated amino acids and a
amino acid, 3 hydroxy-4hydroxymethylproline active against human fungal pathogens which
including Candida albicans and Trichophyton Sp. and also against some of plant pathogenic
fungi such as Sclerotinia Sclerotiorum and Botrytis cinerea. The endophytic Bioprospecting
for Microbial Endophytes and their Natural Products 29 Chloridium Sp. from [Link]
produces Javanicin (Figure 6.) which is highly activity against Pseudomonas Spp. (Kharwar
et al., 2008). A tetramic acid cryptocin (Figure 7.) was obtained from [Link] possessing
strong activity against Pyricularia oryzae plant pathogenic fungi. Ambillic acid is a highly
functionalized cyclohexenone possess strong antifungal activity found to produce by
endophytic fungus. Pestalotiopsis jester is an endophytic fungi produces the extremely
functionalized cyclohexenone epoxide jesterone and hydroxy jesterone, exhibiting excellent
antifungal activities against a variety pathogenic fungi of plants. Fungal endophytes isolated
from [Link] and Acalypha species shows potent antibacterial activity against human
pathogenic bacteria such as viz, [Link], [Link] and [Link] (Gangadevi et al.,
2008). Dual biological control reported for the fungal entomopathogens, Beauveria bassiana
(Bals.-Criv.) Vuill. (Ascomycota Hypocreales) and Lecanicillium Spp (Ascomycota
hypocreales). The mechanism of antibiosis includes production of antibiotic compounds,
bioactive volatile organic compounds and some enzymes (Ownley et al., 2010). Some
endophytic fungi were found to produce bioactive secondary metabolites not related to the
products produced by their host. Fungal endophyte Phomopsis Sp. YM 311483 produces four
new ten membered lactones active against Aspergillus niger, Botrytis cinere and Fusarium
Sp. (Huang et al., 2008). Fusarium Spendophytic from Selaginella pollescens active against
Candida albicans (Strobel and Daisy, 2003). Endophytic fungi isolated from Rhizophora
mucronata, Avicenna officialis and Avicenna marina showed maximum antibacterial activity
against bacterial pathogens.
24. An endophytic bacterial strain Bacillus subtilis ZZ120 was isolated from stem of Prunus
mume showed inhibition against Fusarium graminearum, Alternaria alternata, Rhizoctonia
solani, Cryphonectria parasitica and Glomerella glycines. Gram negative endophytic bacteria
25
 Pseudomonas Sp. have been investigated as biological control agent with respect to
production of antimicrobial metabolites (Nielson et al., 2002).
25. Endophytic Gram positive bacteria like Bacillus Sp. have also been isolated from cotton,
cucumber root and citrus plants (Mahaffee and Kloepper, 1997; Reva et al., 2002). Plant
pathogenic fungi were also control by bacterial endophytes (Berg and Hallmann, 2006).
Coronamycin characterize a complex peptide antibiotic with activities against pythiaceaus
fungi, human fungal pathogen Cryptococcus neoformans and also against the malarial
parasite Plasmodium falciparum was produced by a Verticillate Streptomyces spendophyte
from an epiphytic vine Monstera Sp. (Ezra et al., 2004,). During the isolation of endophytes,
actinomycetes generally appeared much later than endophytic bacteria and fungi and are also
able to produce various metabolites. Igarashi isolated 398 actinomycetes showing excellent
antagonistic activity against phytopathogenic fungi and bacteria. Extracts from Streptomyces
Sp. collected from Allium fistulosum suppressess infection of Alternaria brassicicola on
Chinese cabbage seedlings (Igarashi et al., 2002).They also recognized several new bioactive
compounds from endophytic actinomycetes. Two new novobiocin analogs were found to
produce by Streptomyces Sp. from Aucuba japonica, and also cedarmycins by Streptomyces
Sp. from Cryptomeria japonica. A new naphthoquinone antibiotic, alnumycin, was also
isolated from endophytic Streptomyces Sp. of Alnus glutinosa. Streptomyces
Sp.NRRL30562 from snake vine plant was found to produce new peptide antibiotic,
designated as munumbicins A-D40 with a broad spectrum of activity against several human,
phytopathogenic fungi and bacteria.
26. Streptomyces Sp. NRRL30566, from a fern-leaved grevillea (Grevillea pteridifolia) tree,
reported to produce original kakadumycin chemically related to echinomycin (Castillo et al.,
2007). Endophytic actinomycetes from Azadirachta indica [Link] were also evaluated in
response to their antimicrobial action against pathogenic fungi and bacteria. A total of 54.5%
actinomycetes were commonly recovered from roots followed by stems (23.6%) and leaves
(21.8%) dominant genus found as Streptomyces, Streptosporangium, Microbispora and
Streptoverticillium.
27. Isolates having sharp activities against Pseudomonas fluorescens, [Link] and antagonistic
activity against root pathogens Pythium and Phytophthora sp. (Verma et al., 2008).
Biologically dynamic species of Streptomyces were isolated from species of Nothofagus and
some other plants from the southern Patagonia. Having activity against plant pathogens, like
Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaere llafijiensis and Rhizoctonia solani
(Castillo et al., 2007). Endophytic actinomycetes were isolated from the Trewia nudiflora
26
 Linn with antifungal activity Bioprospecting for Microbial Endophytes and their Natural
Products against Penicillium avellaneum UC 4376. Large numbers of endophytic
actinomycetes were isolated from total of 26 medicinal plants species from Panxi plateau
showing huge spectrum of antimicrobial activities with valuable reservoirs of novel bioactive
compounds (Zha, 2010). Endophytic fungi Microdiplodia hawaiiensis CZ315 isolated from
Garcinia mangostana showing antimicrobial activity against Staphylococcus aureus, Bacillus
subtilis, Escherichia coli ,Pseudomona aeruginosa, Salmonella typhi and Micrococcus luteus,
with the minimum inhibitory concentration (MIC) in range of as S. aureus (MIC 25 µg/ml),
B. subtilis (MIC 5µg/ml), M. luteus (MIC 25 µg/ml), E. coli (MIC 20 µg/ml), S. typhi (MIC
20 µg/ml) and P. aeruginosa (MIC 10 µg/ml), respectively. Streptomyces aureofaciens found
to produce 4 Arylcoumarins with antifungal potential (Thongchai et al., 2005). Fungal
endophytes from coffee plants exposed the presence of various genera of fungi including
Acremonium, Beauveria, Cladosporium, Clonostachys and Paecilomyces and found
pathogenic against coffee berry borer, metabolites produced includes bassiani, beauvericin,
bassianolide, beauveriolide, bassiacridin, oosporein and tenellin . Commented [m10]: italicize all scientific names

Table 1. Compound extracted from various endophytes

Compound
Source Endophyte Application
Isolated
Pacific Yew (Taxus
Pestalotiopsis microspora Taxol Anti-cancer drug
brevifolia)
Rhyncholacis penicillata Serratia marcescens Oocydin A Anti – oomycete
Cryptosporiopsis cf.
Dermateaceae Cryptocandin A Anti- mycotic
quercina

Anti-fungal activity,
Rainforest Pestalotiopsis microspora Ambulic Acid
Used in solid state NMR

antifungal properties,
Hederae helicis Pestalotiopsis microspora. Pestaloside urethane degrading
property
Papua New Guinea Pestaliotiopsis jesteri Jesterone antioomycete activity
anticancer compound
Hedera Pestalotiopsis microspora Torreyanic acid

T. morobensis Pestalotiopsis microspore Pestacin Antioxidant

27
T. morobensis P. microspora Isopestacin Antioxidant
T. wilfordii F. subglutinans Subglutinol A Immunosuppressant

28
 CHAPTER 3
MATERIALS AND METHODOLOGY
3.1. Selection of plant for isolation of endophytes
Initial step for isolation of endophytes is to select a healthy plant. Among the medicinal
plants Lemon grass and Asparagus plant were found suitable after studying their properties
hence they are selected as the experimental plant and collected from the nursery of VIT
University, Vellore, Tamil Nadu which is maintained in highly suitable environment.
3.2. Sampling area and collection of plant material
Different part like leaf, root and stem of the selected medicinal plant is taken for extraction
of endophyte. In order to get proper results every part should be properly sterilize.
3.3. Processing of sample for isolation of endophytes
3.3.1. Surface sterilization
Samples taken from the selected plants were cleaned under running tap water for 5 min and
then air-dried. Before surface sterilization, the cleaned leaves, stems and root parts were
cut into pieces 5 cm long. Different fragments of parts of plant were surface sterilized by
immersion in 70% ethanol for 1 min followed by washing with 5% sodium hypochlorite
solution for 5 min and sterile distilled water for 1 min two times. The surface-sterilized
leaves, roots and stems were cut into small pieces using a sterile blade and placed on sterile
water agar plates for incubation at 30 °C.
3.3.2. Isolation of endophytes
Endophytes are extracted from the explants by culturing them into the appropriate media.
Endophytic bacteria In order to culture endophytic bacteria from the explant a well
enriched media is prepared. Nutrient agar media is prepared and sterilized part of the plants
are gently placed on the media. Petridish were kept in incubator maintained at room
temperature. For further purification and extraction sub culturing from the mother culture
is done in order to get the isolated colonies of the bacteria for further activities test.
Endophytic fungi Isolation of endophytic fungi, leaves, roots and stems samples were
inoculated on potato dextrose agar and cultivated in Petri dishes for 4-7days, at 27 ͦ C. A
proper growth can be observed in the petriplate after 4-5 days. After getting the mother

29
culture sub culturing is done on appropriate media to obtain sufficient growth and pure
culture for the fungi.
Preservation of culture
After the development of good strains of microorganisms by following the appropriate
protocol, it is very important to preserve it carefully, so it can be used in future if needed.
If the cultures have not been preserved properly it can result into decline into characteristic
features after certain period. Consequently amount of product produced also gets decreased.
Therefore, different methods have been developed for maintaining the microorganisms in
viable conditions over a long period of time. These methods vary according to strains.
Preservation by Drying in Vacuum: it is one of the easiest technique in which cultures are
dried over CaCl2 in a vacuum and then stored in a refrigerator. At such conditions, the
organisms will survive for longer period than the air dried cultures.
Storage in Saline Suspension: salt is very important for bacteria as it is used in many
metabolic activities. But when it is present at high concentration, salts act as bacteriostatic
which inhibits bacterial growth temporarily. Hence, bacterial culture can be preserved in
1% salt concentration in screw-capped tubes at room temperature.
Agar Slant Culture Covered with Mineral Oil: After incubation the cultures are incubated
at a specific temperature to facilitate the growth of organism. Then the cultures are then
covered with mineral oil to a depth of 1 cm above the slant. They are sub-cultured after the
storage of about 1 year.
Preservation in refrigerator: It is one of the most used way to store petriplate media culture.
After the incubation period the petriplate are covered with new paraffin strips to avoid
moisture inside the plates and carefully stored in the refrigerator. Commented [m11]: which technique did you use

3.3.3. Characterization of endophytes

Gram staining tests The Gram stain is a very important preliminary step in the initial
characterization and classification of bacteria. It is also a key procedure in the identification
of bacteria based on staining characteristics, enabling the bacteria to be examined using a
light microscope. The bacteria present in an unstained smear are invisible when viewed
using a light microscope. Once stained, the morphology and arrangement of the bacteria
may be observed as well. Gram staining is carried out by making smear of the test organism
on sterilize glass slide. Smear is air-dried and heat fixed.
Add crystal violet stain over the fixed culture (1min). Add the iodine solution on the smear,
enough to cover the fixed culture (1min), add a few drops of decolorizer so the solution

30
trickles down the slide (5sec). Counterstain with basic fuchsin solution for 40 to 60 seconds.
Wash off the solution with water. Observe under light microscope at 10x, 40x and for clear
appearance 100x.
Lactophenol cotton blue (LPCB) staining Lactophenol Cotton Blue Stain is
recommended for mounting and staining yeast, fungi and molds. Fungal organisms are
very difficult to visualize without the aid of special staining procedures hence LPCB is used
for fungal staining. First step of this staining procedure is making a proper smear of sample
organism on a clean sterilize glass slide. Then add 2 drops of the lactophenol cotton blue.
Gently press a cover slip to make a thin mount. Also gently warming will aid in clearing
the mount and scan under low power with reduced lighting. Switch to high power to check
for the presence of suspected fungal elements
Biochemical test The IMViC tests are useful for differentiating the Enterobacteriaceae,
especially when used alongside the urease test. When used alone, the IMViC tests are
particularly useful for differentiating Escherichia coli, Enterobacter
aerogenes, Enterobacter cloacae, and Klebsiella pneumonia (although colonial
morphology and the presence of capsules can also be used to differentiate Klebsiella).
Indole test The Isolates were inoculated in point on peptone water broth. Incubated
at 37 c for 48-96 h. After incubation we added 0.5 ml of kovac’s reagent and shake
it gently. Pink/Red color in the alcohol layer showed positive reaction. By using
filter paper in saturated solution of oxalic acid we tried to do this test by hanging it
in culture tube with the help of cotton plug for 24 hrs. Positive test was indicated if
the paper turns red/pink.
Methyl red test Mixed acid fermentation - Many gram-negative intestinal bacteria
can be differentiated based on the products produced when they ferment the glucose
in MR-VP medium. Escherichia, Salmonella, and Proteus ferment glucose to
produce lactic, acetic, succinic, and formic acids and CO2, H2, and ethanol. The
large amounts of acids produced lowers the pH of the medium - Methyl red (a pH
indicator) will turn red when added to the medium if the organism was a mixed acid
fermenter. Many of these organisms also produce gas. After incubation the broth
must be turbid. A clear broth indicates that your organism did not grow and cannot
be tested. Remove 1 ml of broth and place into a sterile tube before performing the
methyl red test if you are going to use the same broth for the VP test. Add 3-4 drops

31
 of methyl red to the original broth. DO NOT SHAKE THE TUBE. A positive result
has a distinct red layer at the top of the broth. A negative result has a yellow layer.
Voges-Proskauer test Inoculate a loopful of bacteria into MRVP broth. Incubate 3
to 5 days. Negative test is indicated by lack of color change after the addition of
Barritt’s A and Barritt’s B reagents. A positive Voges-Proskauer test is indicated
by the development of red-brown color after the addition of Barritt’s A and Barritt’s
B reagents. Organisms that are negative in the methyl red test may be producing 2,
3 butanediol and ethanol instead of acids. These non-acid products do not lower the
pH as much as acids do. Enterobacter, Serratia and some species of Bacillus
produce these substances. There is no satisfactory test for determining production
of 2, 3 butanediol. A precursor of 2,3 butanediol called acetoin can be detected with
Barritt's reagent.
Citrate test Streak a loopful of bacteria onto a citrate agar slant, do not stab the butt.
Incubate 24 to 48 hours, longer for Bacillus species. Incubate with a loose cap.
Simmon's citrate agar tests for the ability of an organism to use citrate as its sole
source of carbon. This media contains a pH indicator called bromthymol blue. The
agar media changes from green to blue at an alkaline pH. After incubation a positive
reaction is indicated by a slant with a Prussian blue color. A negative slant will have
no growth of bacteria and will remain green.
Identification of endophytic bacterial strains The selected endophytic bacterial strains
were identified by morphological, biochemical and physiologically characterization and by
16S rRNA gene sequencing. In morphological characterization, macroscopic and
microscopic features of the selected isolates were studied, additionally an array of
biochemical and physiological tests including catalase test, oxidase test, starch utilization
test, nitrate reduction test, motility test, H2S production test and sugar utilization test were
performed following the methods described by Gerhardt
16S rRNA gene sequencing High-molecular weight DNA of the 10 selected strains
was isolated by using a genomic DNA isolation kit. The PCR amplification of 16S
rRNA gene of the selected strains was done by using the forward and reverse
primers including 27f 5/-AGAGTTTGATCCT GGCTCAG-3/ and 1522r 5/-
AAGGAGGTGATCC ARCCGCA-3/. Amplification was performed in an
automated thermo cycler with the amplification profile as: 94°C for 5 min followed
by 36 cycles of 94°C for 20 sec, 50°C for 20 sec, 72°C for 2 min each and followed
by a final extension step of 5 min at 72°C. The amplified PCR product was extracted
32
 by using gel extraction kit. The pure extracted DNA was sequenced, the sequence
data obtained was refined and was BLAST analyzed at NCBI website:
[Link] to determine the similarity of the sequence with
already reported gene sequences in the GenBank.
Enzyme Assay for isolated endophytes
Proteolytic activity Gelatin hydrolysis was observed using nutrient agar with 1% bacterial
gelatin (gl-1) and acidic HgCl2 Solution. Nutrient broth 8.0, glucose 1.0, and agar 18.0, pH
is used as a media. 15.0 ml of skimmed milk is added after autoclaving. The media is
inoculated with the sample microorganism and after the growth period 2.0 ml of HCl 0.1
mol l-1 was added to the plates and the presence of clear halos around the colonies were
observed.
Amylolytic activity Nutrient media agar with 1% starch at PH 6 is used for this test.
Inoculate with microorganism and after the incubation period treat the cultural plates with
the iodine solution and allow to observe clear zone around the colonies.
Lipase activity The isolated organisms are grown on media in which separately sterilize
tween 20 is added to the media just before the inoculation. Lipase activity is indicated by
the clear zone formed around the colonies.
Esterase activity For esterase activity media was used as suggested by Sierra (1957).
Previously sterilized Tween 80 was added separately after autoclaving in concentration of
1% (v/v).Inoculation of organisms was done and allowed for incubation to observed clear
Zones around the colonies.
Cellulolytic activity Organisms isolated were grown on the selective medium with 0.1%
sodium carboxymethyl cellulose formation of zone around the colonies form is result in the
determination of the fungal strain .The surface of the media with the developed fungi
colonies was mixed with 0.1% Congo red and incubated for 15 minutes at room
temperature. The dye was then removed and NaCl 1 M solution was added, followed by
incubation for a further 10 minutes at room temperature. We then measured the ratio of the
diameter of the clear zone to the diameter of the colony.
3.3.4. Phytochemical analysis
Chemical test were carried out on the aqueous solution and the powdered or crude sample.
For this chemical tests crudes is obtained using the separating funnel technique. The crude
for test is extracted by using separating funnel using three different solvents which are ethyl
acetate, hexane and benzene for bacteria. In this separation process first paste is formed

33
from the mat obtained from the broth culture of fungi. The paste is made up to volume 20ml
using water. Twice the amount of ethyl acetate is mixed with the prepared sample into the
separating funnel. Mix it for 10 min and allow it to stand still for 30 min, three different
layers are formed. The immiscible middle layer is separated and again mix with twice the
amount of water and separated again for further purification. In the same manner
compounds are extracted from the fungal supernatant, bacterial pellet and supernatant as
well. After obtaining the sample it is kept in the hot air oven to get it dry and obtain it in
powder form. Weigh the sample and use it for the phytochemical analysis.
Test for tannins Take 20ml of water and add 0.5 g of the dried powdered samples and
boiled in a test tube and then filtered. Add few drops of 0.1% ferric chloride and observed
for brownish green or a blue-black coloration.
Test for phlobatannins Deposition of a red precipitate when an aqueous extract of each
plant sample was boiled with 1% aqueous hydrochloric acid was taken as evidence for the
presence of phlobatinins.
Test for saponin Take 2 g of the powdered sample and boiled in 20 ml of distilled water
in a water bath and filtered. Mix 10ml of the Filtrate with 5 ml of distilled water and shake
it vigorously for a stable persistent froth. The froth formed is mixed with 3 drops of olive
oil and shake it vigorously, then observed for the formation of emulsion.
Test for steroids Add 0.5 g ethanolic extract of each sample to 2ml of acetic anhydrite,
add few drops of H2SO4. The colour changed from violet to blue or green in some samples
indicating the presence of steroids.
Test for terpenoids Five ml of each extract was mixed in 2 ml of chloroform, and
concentrated H2S04 (3 ml) was carefully added to form a layer. A reddish brown
colouration of the inter face was formed to show positive results for the presence of
terpenoids.
Test for flavonoids Three methods were used to determine the presence of flavonoids in
the plant sample. 5 ml of dilute ammonia solution were added to a portion of the aqueous
filtrate of each plant extract followed by addition of concentrated H2S04. A yellow
colouration observed in each extract indicated the presence of flavonoids. The yellow
colouration disappeared on standing. Few drops of 1% aluminium solution were added to
a portion of each filtrate. A yellow colouration was observed indicating the presence of
flavonoids. A portion of the powdered plant sample was in each case heated with 10 ml of
ethyl acetate over a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate

34
was shaken with 1 ml of dilute ammonia solution. A yellow colouration was observed
indicating a positive test for flavonoids.
Test for Alkaloids
Dragendorff’s test 1 ml of extract, add 1 ml of dragendroff’s reagent (potassium
bismuth iodide solution). An orange- red precipitate indicate the presence of alkaloids.
Mayers test 1 ml of extract, add 1ml of Mayers reagent (potassium mercuric iodide
solution). Whitish or cream colored precipitate indicate presence of alkaloids.
Hager’s test 1 ml of extract, add 3 ml of Hager’sreagent (saturated aqueous solution of
picric acid). Yellow colored precipitate indicates the presence of alkaloids.
Wagner’s test 1 ml of extract, add 2 ml of Wagner’s reagent (iodine in potassium
iodide). Reddish brown colored precipitate indicates presence of alkaloids.
3.3.5. Biological Applications
Extraction of phytochemicals and Determination of Antimicrobial activity The test Commented [I12]: Write about extraction of crude compounds

organisms i.e. biofilm forming bacterial strains were obtained from the culture
collection of the department of Microbiology and Molecular Genetics (MMG),VIT
University Vellore , India. The endophytic bacterial isolates were cultivated as shaking
cultures in LB-broth for 48 hour at 37°C. The culture broth of each of the isolate was
centrifuged at 3000 rpm to get cell free supernatants. The test plates of biofilm forming
bacteria were prepared by pouring 14 mL of LB-agar as base layer; after it has solidified,
it was overlayed with 4 ml of the inoculated seed layer. The agar wells (5 mm diameter)
were made by using sterile cork borer and 60 µl of the supernatant was loaded in each well.
The test plates were left at room temperature for 2 hours to diffuse the supernatant and were
incubated at 37°C for 18-24 hours. After 18-24 hours the diameter of the zones of inhibition
was measured in mm.

35
 CHAPTER 4
RESULT AND DISCUSSION
4.1. Isolation and characterization of endophytic organism
Six different endophytic fungi was isolated from the stem of Asparagus plant and one
bacteria from the leaf explant of Lemon grass. The six different fungi can be differentiated
by merely looking with naked eyes on the basis of color. The mother culture of both fungi
and bacteria was preserved for further use for biological and chemical tests.

A)

B) C)

Figure 3. Endophytic fungal culture isolated from Asparagus racemous: A)

Pure culture of endophytic bacteria isolated from lemon grass, B) Mother
culture of the surface sterilized plant placed on PDA and C) Pure culture of the
fungal colony (green)
One strain of fungi was selected as experimental species and further experiment was
performed. First Lactophenol Cotton Blue (LPCB) Staining was done for the sample fungi
and it was identified as Penicillium sp. Similarly the Gram staining was performed for the
identification of bacterial strain and it was identified as Gram negative cocci shaped bacteria.

36
Figure 4. Cocci shaped Gram negative Figure 5. Penicillium sp. Identified by
bacteria Lactophenol Cotton Blue (LPCB)

4.2. Biochemical characterization of endophytic bacteria

Indole test was used to identify the ability of bacteria to produce tryptophanase enzyme
which converts tryptophan amino acid into indole and two more products. The bacteria was
found to give positive test for indole test which is confirmed by appearance of pink colored
ring after adding Kovac’s reagent.
For Citrate utilization test bacteria was grown on simmon’s citrate agar media. The
growth and change in media color from green to blue confirm the positive result for citrate
utilization test

.
Figure 6. Citrate utilization test showing positive result
4.3. Molecular characterization of endophytes
DNA was isolated and subjected to PCR analysis using universal primers and the DNA
obtained was found to have ------ bp. The aligned sequence of the amplified--------- bp 16S
rDNA fragment from the isolate was submitted to Genbank. The sequence of the isolate
was found 99% similar with Streptomyces ... and the strain was coined as Streptomyces

37
 sp VITS111 A phylogenetic relationship was establishment through the alignment and
cladistic analysis of homologous nucleotide sequences from bacterial species (Fig) Commented [m13]: For sequencing call divya mam both here
in results and materials and methods

4.4. Enzyme Assay for endophytic microorganisms

4.5. Phytochemical screening of endophytes
The result of qualitative phytochemicals analysis inferred that the fungal ethyl acetate
extract contain alkaloid, phenol and tannin. But saponin, flavonoid, steroid and cardiac
glycosides was absent in fungal extract.
Table 2. Preliminary qualitative phytochemicals screening result

Endophytic Cardiac
Solvent Alkaloids Flavonoids Phenols Tannins Steroids Saponins
extract glycosides
Fungal
+ - + - - - -
crude
Bacterial
+ - - + - - -
crude Ethyl
Fungal actetate
+ - - - - - -
supernatant
Bacterial
- - - - - - -
supernatant
Fungal
- - - - - - -
crude
Bacterial
- - - - - - -
crude
Benzene
Fungal
- - - - - - -
supernatant
Bacterial
- - - - - - -
supernatant

38
4.6. Anti-microbial activity of crude secondary metabolite extracts
from endophytes
The fungal and bacterial extracts were screened for antimicrobial activity against the
following bacteria: Escherichia coli, Staphylococcus aureus and bacillus. The culture is
spread over Nutrient media agar and well of 60 µl diameter are made in which the test crude
sample extracted from both fungi and bacteria are poured. The plates are incubated for 48
hrs. At 36 degree Celsius. The fungal crude acetate and fungal crude benzene sample shows
zone of inhibition with Staphylococcus aureus. Whereas the bacterial supernatant acetate
and with benzene shows zone of inhibition with bacillus species.

Figure 7. Crude extracts of fungi from Figure 8. Crude extracts of bacterial

solvent Ethyl acetate and benzene from solvent Ethyl actetate and benzene

39
 CHAPTER V
DISCUSSION
Endophytic microorganisms, a group of organisms relatively unstudied, represent an important
genetic resource for biotechnology, and has been target of ecological and taxonomic studies.
Despite the environmental and biotechnological importance of fungi, most of these organisms
remain uncharacterized and there is no considerable knowledge about evolutionary relationship
in many taxa. In addition, microbial characterization becomes an important aspect of
biotechnological application, in order to avoid problems associated with the use and release of
potential plant and/or animal pathogens in the environment. Development of molecular
techniques has been opening new perspectives for the taxonomic characterization and
relationship inference of complex group of organisms. Since in microbial-plant relationship
endophytes contribute with substances that possess various types of bioactivity, researchers
had been conducting screening for bioactive metabolites and endophytic fungus antimicrobial
activity. Endophytes are the chemical synthesizers within plants. Many of them are capable of
synthesizing bioactive compounds that can be used by plants for defense against pathogens and
some of these compounds have been proved for useful drug discovery Up till date, most of the
natural products from endophytes are antibiotics, anticancer agents, biological control agents’
antivirals, antidiabetic agents and other bioactive compounds by their different functional roles
(Guo et al., 2008). The endophytic fungi secrete different metabolite which are useful for the
host plant. The natural products were subjected to an antimicrobial activity, which differed
from one bacteria extract to another against the different test organism. Most of the bacterial
extracts exhibited activity against the five test organisms. The difference in the in vitro activity
among the various crude extracts could be due to the production of either a broad spectrum
antimicrobial compound, or several compounds with different activities (Foldes et al., 2000).
In a study conducted by Omura (1992), he concluded that the differences in levels of
antagonism are dependent on concentration of the active substance(s). Comparing the activity of
the positive controls and the fractions against S. aureus, [Link] and [Link], the positive controls had
more inhibition activity. This could be due to the fact that the standard drugs may be more concentrated,
hence show more activity. The zones of inhibition of the isolates fractions may be similar to the standard
drugs, but if they have different modes of activity, this would still make them promising (Fatope, 1995).
However some of the test isolates had fairly high activity as well. It is possible that there were novel

40
compounds from the test isolates and this could be promising agents to replace drugs which resistance
has developed. However further chemical analysis is required to determine this that is, if there are novel
compounds. From the results, the different natural products fractions, are a potential in the
provision of the basis for the synthesis of novel therapeutics, that would aid in the fighting of
life threatening diseases once resistance builds up and since gram negative bacteria are among
the recalcitrant pathogens found in hospitals that have acquired resistance to several antibiotics
in the past, the search for the secondary metabolites that could enable the synthesis of drugs
should be encouraged. Commented [m14]: Discussion first part is okay explain your
results more specially phytochemical tests and the significance of
your results in discussion and future prospective

41
 CONCLUSION
Endophytic Fungi and bacteria are one of the main sources of secondary metabolites and it’s a
budding field in Biotechnology. Pharmaceutically important products can be identified and
isolated in large amounts from the endophytes which are found in the different part of many
useful medicinal plants. The endophytic fungi isolated from Asparagus plant is one among such
nominee which can be further exploited for the production of antimicrobial compounds. The
endophytes was identified using different techniques , one of them which is mostly used is 16S
rDNA sequencing technique , using which endophytes obtained from asparagus are identified
as Penicillium sp. and other one related to Streptomyces sp. Different metabolic compounds
secreted by these endophytes can be identified and checked for anti-microbial activity, has
shown that these metabolite reduce the growth of bacillus and Staphylococcus aureus. From
this type of inhibition compounds new pharmaceutical antibiotics and useful drugs can be
discovered and can be used to improve medical condition of a patient by reducing or inhibiting
the growth of harmful bacteria and fungi.

Commented [m15]: For introduction, literature review and

materials and methods please put references and end arrange them
in alphabetical order

42
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