1

CHAPTER 1
INTRODUCTION

Actinomycetes

are

the

most

widely

distributed

group

of

microorganisms in nature which primarily inhabit the soil (Oskay et al 2004.).

They are widespread in nature and can be separated into two sub-groups,
oxidative forms, mostly found in soil and fermentative forms, living in natural
cavities of humans and animals (Lechevalier and Pine 1977). Actinomycetes
are

Gram-positive bacteria belonging to

the

order

Actinomycetales

(Lechevalier and Lechevalier 1981), characterized by the formation of substrate

andaerial mycelium on solid media, presence of spores and a high guanine and
cytosine (GC) content of the DNA (60-70 %).The name Actinomycetes derived
from the Greek word aktis (a ray beam) and mykes (fungus) was observed
from their initial morphology.
1.1

TAXONOMY OF ACTINOMYCETES
Actinomycetes belongs to the
Order

: Actinomycetales

Superkingdom

: Bacteria

Phylum

: Firmicutes

Class

: Actinobacteria

Subclass

: Actinobacteridae

Based on the Bergeys manual actinomycetes are divided into the

following eight diverse families: Actinomycetaceae, Mycobacteriaceae,
Actinoplanaceae,

Frankiaceae,

Dermatophilaceae,

Nocardiaceae,

Streptomycetaceae, Micromonosporaceae (Holt 1975) and also comprises

about 63 genera (Nisbet and Fox 1991).
Actinomycetes constitute the most important group of bacteria, in
which Streptomyces having the high potential in producing secondary
metabolites e.g. antibiotics (Mellouli et al 2003). Streptomycetes are soil
bacteria having DNA with a high G+C content similar to that of other
actinomycetes genera (Anderson and Wellington 2001). In addition, it was well
known to have the capacity to synthesize bioactive secondary metabolites
which include vitamins, enzymes, herbicides and pesticides. Almost 80% of the
worlds antibiotics are mostly from the genera of Streptomyces and
Micromonospora (Pandey et al 2004). As they are the most widely distributed
microorganisms in nature and constitute a significant component of the
microbial populations in most soils (Barakate et al 2002). Terkina et al (2002)
reported that Streptomyces also plays a majorrole in the carbon cycle due to
their ability to grow at low concentrations of carbon and todegrade recalcitrant
organic compounds.
1.2

CHEMOTAXONOMY
Here, chemotaxonomy refers to grouping organisms based on their

cell wall constituents and membranes (Zaitlin and Watson 2006). The
composition of cell wallsvaries greatly among different groups of
actinomycetes. Most actinomycetes possess cell wall type I and IV with
peptidoglycans containing L-diaminopimelic acid (DAP) and glycine (type-I),
meso-DAP and glycine (type-II), meso-DAP (type-III) or meso-DAP,
arabinose and galactose (type-IV) (Goodfellow 1989, Lechevalier 1970). The

genus Streptomyces is best known for its cell wall type-I. They are able to
catabolize sugars, alcohols, amino acids, organic acids and some aromatic
compounds. This is accomplished by producing extra cellular hydrolytic
enzymes that allow the catabolism of organic molecules (Madigan et al 2000).
Table 1.1 Major constituents of the four cell wall types of actinomycetes
(Lechevalier and Lechevalier 1970)
Cell wall
type

DAP
Meso

LL

Glycine

Arabinose

Galactose

III

IV

Chemotaxonomy also involves the analysis of other macromolecules

such as the lipids (lipopolysaccharides and fatty acids including mycolic acids),
polysaccharides and related polymers and proteins (Ward and Goodfellow
2004). The genus Streptomyces was classified in the family Streptomyceae on
the basis of morphology and subsequently by the constituents of the cell wall
(Williams and Wilkins 1994). The development of numerical taxonomy
systems, which utilized phenotypic traits, helped to resolve the intergeneric
relationship within the family Streptomycetaceae (Anderson and Wellington
2001).
1.3

SIGNIFICANCE OF ACTINOMYCETES
Actinomycetes produce secondary metabolites with diverse chemical

structures and biological activities of industrial and pharmaceutical interest.

The secondary metabolites of actinomycetes are produced during the life cycle.
Many of the industrially important antibiotics and secondary metabolites

produced by Streptomyces species have been well studied; therefore by

isolating actinomycetes from an environment such as Eastern Ghats, the
potential of discovering novel secondary metabolites of industrial significance
is increased. Actinomycetes play an important role in the rhizosphere region
where they influence plant growth and protect plant roots against invasion by
pathogenic fungi (Goodfellow and Williams 1983). Some genera have also
been shown to produce herbicidal and insecticidal compounds (Crawford et al
1993). Members of the actinomycetes genus like Frankia can fix nitrogen from
atmospheric air to plant rhizosphere regions. They have a broad host range and
can form root nodule symbioses with more than 200 species of flowering plants
(Mincer et al 2002).
Mostly secondary metabolites from natural source found to exhibit
high potential antagonistic activity. Actinomycetes are one such organism,
because of their ability to produce important secondary metabolites. Of these,
the genus Streptomyces is the largest antibiotic-producing genus, accounting
for approximately 80% of the actinomycetes derived natural products reported
(Jensen et al 2005). Although thousands of antibiotics have been described,
these are thought to represent only a small fraction of therepertoire of bioactive
compounds that members of the genus Streptomyces are able to produce
(Watve et al 2001).
The genes encoding many antifungal characteristics are currently
being used by agribusiness to create genetically modified plants that have
increased fungal resistance in the field. Genomic fingerprinting assays using
randomly amplified polymorphic DNA (RAPD) proved in differentiating and
tracking specific genetic elements within a complex genome (Zaki et al 2003).
Majority of these microorganisms in the soil that are potential drug sources
remain uncultivable and therefore inaccessible for novel antibiotic discovery.
However, there are numerous reports (Whitman et al 1998) based on 16S
rRNA gene sequence data which suggest that more than 99% of

microorganisms in natural habitats are uncultured or unculturable. Clearly this

unseen majority encompasses an enormous genetic diversity for exploitable
biology (Bull et al 2000, Bull 2004).
Actinomycetes play a major role in recycling of organic matter
production of novel pharmaceuticals, nutritional materials, cosmetics, enzymes,
antitumour agents, enzyme inhibitors, immune-modifiers and vitamins.
Streptomyces are especially prolific and can produce a great many antibiotics
(around 80% of the total antibiotic production) and active secondary
metabolites (Williams 1978).
Many of them are known to have the capacity to synthesize bioactive
secondary metabolites which include enzymes, herbicides, pesticides and
antibiotics. The filamentous actinomycetes are well known as prolific
producers of biologically active secondary metabolites of economic
significance to the chemical and agricultural industries. There is a continued
need to bioprospect alternative sources of natural products and one strategy is
to harness the chemical diversity from actinomycetes genera previously underrepresented in natural product screening collections (Donadio et al 2002).
1.4

EASTERN GHATS
Eastern Ghats are a discontinuous range of mountains along Indias

eastern coast. Eastern Ghats run from West Bengal state in the north, through
Orissa and Andhra Pradesh to Tamil Nadu in the south. The southern-most of
the Eastern Ghats are the low Sirumalai and Karanthamalai hills of southern
Tamil Nadu. North of the Kaveri river is higher Kollimalai, Pachaimalai,
Shevaroy (Servaroyan), Chitteri, Palamalai, and Mettur hills in northern Tamil
Nadu state.
One of the biggest characteristics of Eastern Ghats lie in its being
extremely fertile. In fact, the Ghat is said to be the watershed of many rivers as

the Ghat gets higher average waterfall. Due to higher rainfall, the fertile land
results into better crops. Often referred as Estuaries Of India, Eastern Ghats
gift its inhabitant the popular profession of fisheries as its coastal area is full of
fishing opportunity. Like Western Ghats, thus, Eastern Ghats also carry a heap
of ecological importance.
The diversified ecological niches and environmental situation
provide habitat for rich fauna. Eastern ghats is home to largest number of
Asiatic elephants in the world. Other large animals such as Nilgiri
Tahr, Leopards, Gaurs, Sambar, and tigers abound the landscape. Apart from
this, these ghats are known for the wide variety of birds species. Eastern ghats
also holds the rich floral system. It is region where you may find large number
of medicinal plants.
Eastern Ghats has a rich array of tropical forests including of moist
deciduous, evergreen and semi-evergreen forests. About 2600 angiosperms,
gymnosperms, pteridophytes and 160 cultivated plants are known, which also
includes over 530 tree species, 1800 medicinal and 450 endemic plants
(Jonathan 2006). The biodiversity richness in the region of the southern Eastern
Ghats has 143 lianas and 272 tree species which are reported (Muthumperumal
et al 2010 and Pragasan et al 2010). Eastern Ghats has several unique taxa such
as Shorearox burghii G. Don, red sanders, Dyer and some wild varieties of
rice. Eastern Ghats contributes significantly to both species richness and
endemicity of the Indian region. However, the forests of Eastern Ghats are
relatively under-studied and have received less attention for conservation
compared to the relatively better-known Western Ghats. It has been left with
insufficient data for several groups of organisms.
1.4.1

Yercaud (Shevaroy hills)

Yercaud (Shevaroy hills) is one of the world's biodiversity hotspots

located in Eastern Ghats of Tamil Nadu, which is having various types of

vegetation including evergreen, tropical deciduous, scrub, subtropical

temperate forests and grasslands. The diversity of higher plant flora and fauna
has been studied in great detail by Kadavul and Parthasarathy (1999). Although
there has been substantial research in terms of medicinal and ecosystem values,
the lower plants such as lichens, bryophytes, pteridophytes, algae and fungi
have not received desired attention.
Yercaud hills or otherwise called as Shevaroy Hills is situated at
1500 meters (4,920 feet) above sea level (MSL) high in the state of Tamil
Nadu, it is quiet little hill station on the Eastern Ghats. The total area of these
hills is about 383 sq.kms with latitude of 11.4 to 11.5oN. Yercaud's temperature
never rises above 30C or falls below 13 C. The relative humidity ranges from
58 to 75%. The average rainfall is about 45 cm to 60 cm from both the
Southwest and Northeast monsoons. It receives an annual rainfall of 1572 mm.
The soil is lateritic with a depth of 0.52 to 1.55m. The soil pH varies from 4.75
to 6.25.
The aromatic coffee and tea plantations, orange groves, jack fruit,
guava, nutmeg, cardamom, cinnamon and black pepper are being cultivated in
these hills. The Yercaud hills have many faunal and floral similarities to the
Western Ghats. A total of 199 species belonging to 175 genera covering 85
families are recorded as wild or naturalized plant resources from the National
Orchidarium and Experimental Garden, Yercaud. 46 species are additions to
the medicinal plants of Shevaroy hills. 6 species are 'endemic', rare or 'scare' in
Shevaroy hills of Salem district, which need special conservation (Pandey
2004).
1.4.2

Kolli Hills
Kolli hills are a small mountain range located in central Tamil

Nadu in Namakkal district of India. Kolli hills are also called as Sathuragiri or
Square hill. The mountains are about 1000 to 1300 m in height and cover an

area of approximately 280 km. The Kolli hills have 70 hair pin bends to reach
the top of the hills. The Kolli hills are part of the Eastern Ghats, which is a
mountain range that runs mostly parallel to the east coast of southern India.
These hills are located at latitude: 1012 - 117 N, longitude: 76-7717 E at
altitudes ranging from 1000 to 1300m above MSL. Rice and minor millets are
the locally produced staple food. Important fruit crops are the jackfruit and hill
banana, well known for its flavour and heady scent. Other crops are coffee,
pineapple, black pepper and other spices. More recently tapioca has entered as
an industrial crop rapidly replacing the small millets grown over centuries and
threatening its genetic diversity.
Objective of the present study

Study the diversity of actinomycetes in the rhizosphere of

medicinal plants collected from Yercaud and Kolli hills (Eastern
Ghats) of Tamil Nadu, southern India.

Isolation,

purification

and

characterization

of

potential

actinomycetes through various in vitro bioassays.

Identification of the most promising strains of actinomycetes

which shown the potential of antimicrobial and anticancerous
activities through molecular techniques (16S rRNA, RFLP and
Dendrogram analysis) at species level.

Study the biocontrol mechanisms exerted by the selected strains

against the pathogen under in vitro to validate the PCR results.

Biosynthesis of silver, gold and copper nanoparticles from the

selected strains of Streptomyces spp. and subjected to evaluate
the efficacy based on antimicrobial and anticancerous activities.