Chapter 1Chapter 1 IntroductionIntroduction &&&& Review...

Chapter 1Chapter 1Chapter 1Chapter 1

IntroductionIntroductionIntroductionIntroduction &&&&

Review of literatureReview of literatureReview of literatureReview of literature

Introduction and Review of Literature

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Secondary metabolites are organic compounds that are not directly

involved in the normal growth, development and reproduction of

organisms (Vining, 1992). The functions or importance of these

compounds to the organism is usually of an ecological nature as they

are used to defend against predators, parasites, diseases and for

interspecies competition, to facilitate the reproductive processes

(colouring agents, attractive smells, etc). Prime importance is given to

these compounds as they are produced from restricted group of

organisms. Secondary metabolites may be the likely candidates for

drug or other technological development directly, or as an inspiration

for unnatural products. This will concern secondary metabolites in

plants, bacteria, fungi and many marine organisms (sponges,

tunicates, corals, snails). It is considered that the cell investment in

secondary metabolite production is almost the confirmation of a

function that should give the organisms certain advantage against

other members of the community (Stone and Williams, 1992).

Secondary metabolites are produced from organisms to inhibit other

organism’s competeting for same ecological niche. Secondary

metabolites are produced after active growth of the organism and are

structurally diversified. The distribution of secondary metabolites is

also unique and some metabolites are found in a range of related

microorganisms, while others are only found in one or a few species.


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The nature has immense potential to provide broad spectrum of

structurally diverse secondary metabolites (Maier et al., 1999). The

structural diversity of secondary metabolites reflects a variety of

biological activities like, antimicrobial agents, inhibitors of enzymes

and antitumor, immunosuppressive and antiparasitic agents (Demain,

1999). Secondary metabolites are generally produced following active

growth, and many have an unusual chemical structure. Some

metabolites are toxic to humans and other animals. Yet others can

modify the growth and metabolism of plants (Griffin, 1994). These

metabolites have been subjected to combinatorial chemistry following

growth in selective media. Interestingly, the most important secondary

metabolites seem to be synthesized from one or a combination of the

biosynthetic pathways: polyketides arising from Acetyl Coenzyme A,

mevalonate pathway that also arises from Acetyl Coenzyme A, and

from amino acids. In addition, genes for the synthesis of some

important secondary metabolites are found clustered together, and

expression of the cluster appears to be induced by one or a few

regulators (Cox, 2007).

1.1 MICROORGANISMS AS SOURCE OF SECONDARY

METABOLITES

History of microbial biotechnology reveals that many desirable

materials like foods, beverages, pesticides and antibiotics were

produced using microorganisms. The studies of Louis pasture for the


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production of penicillin in the nineteenth century results in the

exploitation of microorganisms for valuable metabolites.

Microorganisms are considered as miniatures of chemical factories. In

the 1940’s the discovery of penicillin by Louis pasture initiated

researchers for the exploitation of microorganisms for the production

of secondary metabolites, which revolutionized the field of

microbiology (Demain and Fang, 2000). Inspite of this, until 1970 only

two classes of naturally occurring β-lactam antibiotics, penicillins and

cephalosporins, were known. With the advent of new screening and

isolation techniques, a variety of β-lactam-containing molecules (Wells

et al., 1992) and other types of antibiotics have been identified.

With the emergence of new diseases, screening of microorganisms

for the production of new antibiotics has rapidly increased during the

last three decades. The majority of studies were focused on the

exploitation of antibiotics from fungi and actinomycetes, which are

capable of producing secondary metabolites with widely divergent

chemical structures.

Parallel to the screening for new antibiotics, efforts have been

focused in finding low molecular weight secondary metabolites with

other biological activities. Among others, secondary metabolites with

activity as enzyme inhibitors, plant growth stimulators, herbicides,

insecticides, antihelminthics and immunosuppressant’s have been

obtained. Two main strategies have been used during the screening of


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microbes for the isolation secondary metabolites. The first strategy is

to isolate the known secondary metabolites to evaluate the biological

activities not mentioned in the literature and the second one is to

isolate novel secondary metabolites with various biological activities.

Most of the secondary metabolites were used in the clinical practice as

antibiotics. Some metabolites like mithramycin, bleomycin,

daunomycin and adriamycin were used as antitumor compounds

(Kieslich, 1986). Other properties of secondary metabolites are

anabolics, anesthetics, anticoagulants, anti-inflammatories,

immunosuppressants (cyclosporine-A and tacrolimus), hemolytics,

hypocholesterolemics (statin), and vasodilatories (Bentley, 1997).

More than 23,000 bioactive metabolites of which 17,000 antibiotics

were discovered from the microorganisms in the last 50 years (Janos

Berdy, 2005). Microbial secondary metabolites have specific and

complex chemical structures, with fascinating array of diverse and

unique functional groups. Studies on secondary metabolites revealed

the fact that the microbial secondary metabolites have unique

molecular skeleton which is not found in the chemical libraries which

makes the chemist unable to synthesize more than 40% of the

metabolites (Feher and Schmidt, 2003).

Filamentous microorganisms such as fungi and actinomycetes are

the main source of secondary metabolites with antibiotic activity. The

filamentous microorganisms freshly isolated from soil are the best


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source of secondary metabolites. Some isolates of several

Streptomyces species able to produce more than 180 different

secondary metabolites (Demain and Fang, 2000).

In the group of bacteria, genus Bacillus produces more number of

secondary metabolites. Wide varieties of antibiotics such

moenomycins, difficidins, bacillomycins and bacillaenes have been

isolated from different strains of Bacillus (Patel et al., 1995; Zweerink

and Edison, 1987). Apart from Bacillus, Myxobacterium is another

genus which produces more number of antibiotics. Approximately

80% of the isolated Myxobacteria produced secondary metabolites

with antibiotic activity, many of them exhibiting antifungal activity

(Dolak et al., 1983).

1.2 BIOACTIVITIES OF MICROBIAL SECONDARY METABOLITES

Secondary metabolites produced from microorganisms exhibit

various types of biological activities. The secondary metabolites

produced from microorganisms with incredible array of chemical

structures results in the versatile biological activities. The scope of

search for various bioactive microbial products had however

broadened. The exploration and wide utilization of the antitumor

(doxorubicin) and agricultural antibiotics, (antiparasitic avermectin,

feed additive monensin and herbicide glufosinate), the early

discoveries of utilization of microbial metabolites in the

pharmacological fields (cyclosporin, statins), were important new


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features in the exploitation of microorganisms (Maplestone et al.,

1992). In general secondary metabolites produced from

microorganisms are used in agriculture and medicine directly or used

in the chemical/biological derivitization or used in the rational drug

design.

1.3 BIODIVERSITY AND CHEMICAL DIVERSITY OF

MICROORGANISMS

Biodiversity is usually referring to the number of species (or other

taxonomical entities) in a given ecosystem or geographic area.

Biodiversity is defined as concerning those biological species that are

studied specifically for their value as producers of organic compounds,

so-called secondary metabolites. These compounds are isolated,

characterized and studied by chemists and biologists to evaluate their

biological, pharmacological/chemotaxonomic potential, or simply to

explore chemical diversity.

Till date about 6,000 species of prokaryotes especially bacteria are

known and it is estimated that 106 to 109 bacterial strains exist in the

nature (Ward, 2002). About 70,000 fungal species are known and

expecte to be present at the rate of 1.5 × 106 species in the nature

(Hawksworth, 1991). The above calculation of unexploited

microorganisms provides a hope to the researchers for the exploitation

of secondary metabolites from them. The number of metabolites from

filamentous organisms i.e. actinomycetes and fungi are tremendous


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and accounts for 19,000 molecules representing 60% of total bioactive

microbial secondary metabolites (Berdy, 2005). In fungi certain genera

such as Penicillium, Aspergillus, Trichoderma and Fusarium are the

frequent producers of secondary metabolites (Pelaez and Genilloud,

2003). Fungi are highly diversified microorganisms. Various fungi like

terrestrial fungi, endophytes, entomopathogenic, nematode trapping,

coprophilous, marine and freshwater fungi represents the ecological

diversity (Collado, et al., 2001). New species and even major taxa of

fungi and actinomycetes are being discovered every day, opening

windows of opportunities and providing evidence that our knowledge

of these microorganisms is far from exhaustive.

1.4 BIOTECHNOLOGICAL IMPORTANCE OF FUNGI

Fungi are the most important biotechnologically useful organisms

(Kurtzman, 1983). Fungi have been used to elucidate the complex

biochemistry and genetic principles of eukaryotes. They are important

not only for antibiotics, but also used in commercial production of

flavoring foods, production of biochemicals such as organic acids and

enzymes (Blain, 1975; Eveleigh, 1981). The fungal kingdom offers

enormous biodiversity, with around 70,000 known species, and an

estimated 1.5 million species in total. Most of these are filamentous

fungi, which differ from the yeasts not only in their more complex

morphology and development (e.g. asexual and sexual structures), but

also in their greater metabolic complexity. In particular, they are


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known for production of enzymes and secondary metabolites. Of the

12,000 antibiotics known in 1995, about 22% could be produced by

filamentous fungi (Berdy, 1995).

Antibiotics like penicillin, teteracyclin, cephalosporin and

immunosuppressive agent like cyclosporine-A was reported from fungi

in the early days of antibiotic research. The first known secondary

metabolite mycophenolic acid was never used as an antibiotic where

as its ester, 2-morpholinoethylester was commercialized as

immunosuppressive agent. Lovastatin and pravastatin used as

hypocholesterolemic agents are also of fungal origin (Endo, 1985).

Antitumor agent taxol was also reported to be produced from fungus

Taxomyces andreanae (Stierle et al., 1993). Fermented rice with

Monascus purpureus used as a chinese traditional medicine contains

monascorubramine and rubropunctamine (Juzlova et al., 1996). The

highly valuable product astaxanthin was exploited from Phaffia

rhodozyma (Andrewes et al., 1976).

Ascomycetes and fungi imperfecti are the most frequent producers

of secondary metabolites accounting for 6400 compounds. Aspergillus,

Penicillium and Fusarium species produce 950, 900 and 350

compounds respectively from the group representing ascomycetes.

Basidiomycetes or mushrooms produce 2000 active compounds. From


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yeasts, only 140 and from Myxomycetes (slime moulds) species 60

bioactive metabolites have been isolated (Janos Berdy, 2005).

1.4.1 IMPORTANCE OF FUNGI IN AGRICULTURE

Microbial metabolites have advantage over the synthetic pesticides.

Chemical pesticides cause environmental pollution and impose

deleterious effects on human health. The microbial metabolites are

host specific and have broad range of activity. Synthetic pesticides are

also the derivatives of secondary metabolites. These secondary

metabolites are used as antifeedents, anti parasitic, insecticides,

antihelminthic agents, cestocides, acaricides, nematocides, antiworm

compounds, phytotoxins, plant growth regulators, germination

inhibitors, allelochemicals, phytohormones, chlorosis inducers,

algicides, and other biocontrol agents. Avermectin, monensin and

bialaphos are the most useful microbial compounds in this group.

In agriculture attempts are being made to use fungi as biocontrol

agent to reduce the damage of insects and weeds (Adams, 1988) and

pathogenic microorganisms (Burge, 1988; Gellespic, 1988) and as an

inoculants or biofertilizers to improve crop yield (Lynch et al., 1991;

Whips and Lumpsden, 1989). Association of mycorrhizal fungi

provides an intimate link between soil and nutrient absorbing organs

of plants and optimizes the uptake of phosphorous and increases the

yield (Khan, 1972; Harley, 1989).


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Table 1.1: Secondary metabolites produced by microorganisms and their biological activities

Microorganism Metabolite Bioactivity

Acremonium chrysogenum Cephalosporin Antibacterial Streptomyces clavuligerus Cephamycin Antibacterial Streptomyces venezuelae Chlormomphenicol Antibacterial Streptomyces erythraea Erythromycin Antibacterial Streptomyces aureofaciens Tetracyclin Antibacterial Penicillium chrysogenum Penicillin Antibacterial Amycolatopsis mediterranel Rifamycin Antibacterial Streptomyces spectablis Spectinomycin Antibacterial Streptomyces griseus Streptomycin Antibacterial Streptomyces niveus Novobiocin Antibacterial Micromonospora Gentamicin Antibacterial Zygosporium mansonii Zygosporin-A Antibacterial Streptomyces nodosus Amphoteracin Antifungal Aspergillus flavus Aspergillic acid Antifungal Streptomyces aureofaciens Aureofacin Antifungal Streptomyces griseus Candicidin Antifungal Penicillium griseofulvin Griseofulvin Antifungal Streptomyces nourse Nystatin Antifungal S. diastachromogenes Oligomycin Antifungal Streptomyces antibioticus Actinomycin-D Antitumor Streptomyces verticilus Bleomycin Antitumor Streptomyces speuceticus Doxyrubicin Antitumor Streptomyces lavendulae Mitomycin-C Antitumor Taxomyces andreanae Taxol Antitumor Streptomyces clavuligerus Clavulanic acid Plant enzyme inhibitor Gibberella fujikuroi Gibberelin Plant growth regulator

Continued in next page…


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Microorganism Metabolite Bioactivity

Streptomyces cinnamonensis Monensin Growth promoter Streptomyces fradiae Tylosin Growth promoter Streptomyces hygroscopicus Bialaphos Herbicidal Streptomyces avermitilis Avermectin Insecticidal Streptomyces hygroscopicus Milbemycin Insecticidal Phaffia rhodozyma Astraxanthin Pigment Monascus purpureus Monascin Pigment Trichoderma flavofuscum L-DOPA Parkinson's disease Phycomyces blakesleeanus Ferritin Nutrient Ashybya gossypii Riboflavin Nutrient Altrenaria arborescens Alternariols Mycotoxin Aspergillus Parasiticus Aflatoxins Mycotoxin Penicillium verrucosum Ochratoxin-A Mycotoxin Claviceps purpurea Ergotamines Mycotoxin Aspergillus and Penicillium Patulin Mycotoxin Fusarium Sps Fumonisins Mycotoxin Fusarium Sps Zearalenone Mycotoxin Fusarium and trichoderma Trichothecenes Mycotoxin Aspergillus terrus Lovastatin Anticholestrolemics Monascus ruber Monacolin Anticholestrolemics Penicillium citrinum Pravastatin Anticholestrolemics Beauvaria nivea Cycloporines Immunosuppressive Aspergillus and trichoderma Gliotoxin Immunosuppressive Tolypocladium iflatum Cyclosporin-A Immunosuppressive Streptomyces hygroscopicus Rapamycin Immunosuppressive


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1.4.2 USE OF FUNGAL METABOLITES AGAINST PLANT DISEASES

Secondary metabolites play important role in the control of plant

pathogens. These are used to control the phytopathogenic fungi and

bacteria (Burge, 1988). Trichoderma harzianum produces an

antifungal agent, alkyl-pyrone which is active against wide range of

fungi and bacteria (Claydon et al., 1987). Pisolithus arhizus produce

compounds, hydroxy benzyl formic acid and R-(-)-p-hydroxymendelic

acid active against Truncatella hartigii (Kope et al., 1991). Most of the

Trichoderma sps. produce gliotoxin active against root pathogenic

fungus Rhizoctonia solani (Dennis and Webster, 1971; Harman et al.,

1980). Gliotoxin is also isolated from Gliocladium species active

against wood-rotting fungus, Armillarriab mellea (Lumsden et al.,

1991, 1992).

Trichoderma viridae and Peniophora gigantean are the two fungi

successfully exploited and commercialized against plant pathogenic

fungi. Trichoderma viridae has been used to control Armillaria mellea

on trees, Cerotocystis ulmi on elm, Chondrosterium purpureum on fruit

trees and Eucalyptus and Heterobasidium annosum on pine (Ricard

and Highley, 1988), which parasitizes hyphae of the pathogenic fungi

and produces secondary metabolites (Dennis and Webster, 1971).

Peniophora gigantean is used to control infestation caused by

Heterobasidium annosum, root rot of pines (Lynch, 1990).


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Coniothyrium mimitans parasitizes sclerotia of Sclerotium

trifoliarum, Sporidesmium sclerotivorum and Sclerotinia minor (Ayers

and Adams, 1979). Rhizoctonia solina was reported to be parasitized

by Gliocladium virens (Howell, 1987) and it also protects damping off

caused by Pythium ultimum and Rhizoctonia solina (Howell, 1982).

Ampelomyces quisqualis is used to prevent powdery mildews (Chet,

1990). Verticellium chlymadosporium showed strong inhibitory effect

on M. phaseolina, R. solani and F. solani both in vitro and in vivo

(Ehteshamul-Haque., et al., 1994). Talaromyces flavus which

parasitizes hyphae of Rhizoctonia solina (Boosalis, 1956) also inhibits

the growth of other fungi (Dwivedi and Garrette, 1968; Husain and

McKeen, 1963). Similarly Verticellium chlymadosporium possess

antibacterial activity (Marchisio, 1977) and was found to parasitize

oospore of Phytophthora cactorum (Sneh et al., 1977). Now a day’s

Fusarium oxysporum received attention as a biocontrol agent. Been rot

caused by Fusarium solani and infection in red clover decreased by

non pathogenic Fusarium oxysporum (Lechappe et al., 1988).

1.4.3 FUNGAL SECONDARY METABOLITES AGAINST PLANT

PARASITIC NEMATODES

Soil contains various nematophagous fungi which are natural

enemies to nematodes. Large number of nematophagous fungi with

significant nematicidal activity have been discovered so far (Dayal,

2000). Paeciomyces lilacinus infects eggs of Meloidogyne incognita


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(Jatal, 1985 & 1986). Vericillium chamydosporium also showed

significant activity against the cysts of Meloidogyne spp. and

Heterodera spp. (De Leji, 1992; Kerry, 1990). The hatching of

nematode cysta, Globodra pallaada was adversely affected by the fungi

like Ulocladium Botrytes, Drechslera spp., Gliocladium spp., and

Trichurus spp (Gonazales et al., 1984). The fungus Paecilomices

lilacinus was also widely tested for nematode control (Wainwright,

1992). Penicillium anatolicum produces compounds that acts on the

eggs of nematodes (Jatala, 1986).

1.4.4 FUNGAL METABOLITES AS INSECTICIDAL COMPOUNDS

The use of chemical pesticide and other agro chemicals are getting

reduced / being banned globally because of their toxic effects on

human beings and his live stock, residual toxicity, environmental

problems, pest outbreaks and drastic effects on beneficial insects.

Microbial pesticides have advantage over the synthetic pesticides.

Most of the compounds practiced in agriculture are the derivatives of

the natural products.

Various formulations of microbial pesticides are commercially

available. Formulations of Bacillus thuringiensis, Trichoderma sps,

Metarhizium sps, Beauveria sps, Verticillium sps and Baculovirus are

used against agriculture pests, Helicoverpa & Spodoptera and

commercially available in India.


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Table 1.2: List of metabolites isolated from fungi from 1998-2003 and their biological activity*

Metabolite Produced by Activity Year

Trichodenone Trichoderma harzianum Antitumor 1998

Harzialactone Trichoderma harzianum Antitumor 1998

Glisoprenin Gliocladium roseum Antifungal 1998

Curvularin Aspergillus sps Biological 1998

Longibrachin Trichoderma longibrachiatum Antibacterial 1998

Trichorzin Trichoderma harzianum Antibacterial 1998

Pyrenocine Penicillium waksmanii Antitumor 1998

Aranochlor Pseudoarachniotus roseus Antifungal 1998

Terprenin Aspergillus candidus Biological 1998

Haematocin Nectria haematococca Antifungal 2000

Arisugacin Penicillium sps Biological 2000

Curtisian Paxillus curtisii Biological 2000

Melleolide Armillariella mellea Antibacterial 2000

Roseoferin Mycogone rosea Antifungal 2000

Chaetoatrosin-A Chaetomium atrobrunneum Antifungal 2000

Strobilurin Mycena galericulata Antiparasitic 2000

Topopyrone Phoma sps Antitumor 2000

Phellinsin-A Phellinus sps Antifungal 2000

Xanthoepocin Penicillium simplicissimum Antifungal 2000

Neobulgarone Neobulgaria pura Agricultural 2000

Ampullosporin Sepedonium ampullosporum Biological 2001

Continued next page..


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Metabolite Produced by Activity Year

Phoenistatin Acremonium Fusigerum Antitumor 2001

Cephaibol Acremonium Tubakii Antiparasitic 2001

Epicorazine-C Stereum Hirsutum Antibacterial 2001

Spirobenzofuran Acremonium sps Antibacterial 2001

Cladospolide-D Cladosporium sps Antifungal 2001

Lucilactaene Fusarium sps Antitumor 2001

Bisabosqual-D Stachybotrys ruwenzoriensis Antifungal 2001

Altersetin Alternaria sps Antibacterial 2002

Chrysoqueen Chrysosporium queenslandicum Antibacterial 2002

Chrysolandol Chrysosporium queenslandicum Antibacterial 2002

Thielavin Chaetomium carinthiacum Biological 2002

Miyakamide Aspergillus flavus Others 2002

Phenylpyropene Penicillium griseofulvum Biological 2002

Ustilipid Ustilago maydis Biological 2003

Coniosetin Coniochaeta ellipsoidea Antibacterial 2003

Asperaldin Aspergillus niger Biological 2003

Terreulactone Aspergillus terreus Biological 2003

Quinocitrinine-B Penicillium citrinum Antibacterial 2003

Monorden Humicola sps Biological 2003

Malbranicin Malbranchea cinnamomea Antifungal 2003

Acremonidin Acremonium sps Antibacterial 2003

Exophillic acid Exophiala pisciphila Antiviral 2003

*According to “Novel antibiotics data base” The Journal of Antibiotics;

http://www.antibiotics.or.jp/journal/database/database-top.htm


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Entamopathogenic fungi kill the host by inhibiting the growth &

development of insect. Entamopathogenic fungi are widely used

against lepidopteran pests (Balasubramanian, 1994).

Entomopathogenic fungi, Beauveria bassiana and Metarhizium

anisopliae are highly active against various insects and the

formulations are available in the market. Fungal secondary

metabolites with insecticidal activity like Avermectins, destruxins,

ibotenic acid, pantherine, and tricholomic acid are highly active

(Huang and Shapiro, 1971).

Versimide and methyl-α-(methylsuccinimido)acrylate produced

from Aspergillus versicolor are contact insecticides. Acetylene and

furanocoumarins act as inhibitors of acetylcholine esterase, a

neurotransmitter. Tenuazonic acid and diacetoxyscirpenol produced

from Alternaria tenuis and Fusarium lateritium respectively showed

larvicidal activity. Thiolutin, cycloheximide, rubratoxin, patulin,

trichothecin, actinomycin, and scirpene produced by fungi also had

insecticidal activity.

1.5.1 FUNGAL METABOLITES AS ANTIBIOTICS

Antibiotics are the heterogeneous organic molecules of microbial

origin and deleterious to the growth and metabolic activities of other

microorganisms (Thomashow and Weller, 1995). Antibiotic research is

the most important field in the microbial biotechnology to produce

compounds against pathogenic microorganisms. Antibiotics represent


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greatest contribution of drug therapy (Robbers et al., 1996). The

process of new drug discovery is driven largely by the desire to identify

a structurally novel compound that possesses novel and potentially

useful biological activity (Stephen and Horce, 2000).

5.1.1 FUNGAL METABOLITES AS ANTIMICROBIAL AGENTS

Penicillium vericulum produce a vermiculin with antiprotozoal

potential (Fuska et al., 1972). The compound As-186 from Penicillium

asperosporium exihibit acyl-CoA:cholesterol acyltransferase (ACAT)

activity. Lovastatin reported from Aspergilus terrus is used in the

inhibition of cholesterol biosynthesis (Kuroda et al., 1994). Lovastatin

related compounds were isolated from P. brevicompactum and P.

citrinum (Endo et al., 1976). Arohynapenes A and B, anticoccidial

agents (Masuma et al., 1994) were reported from Penicillium sps.

Alfavarin and β-aflatrem, a new antisectan metabolite was isolated

from the sclerotia of Aspergillus flavus (TePaske et al., 1992).

Fusarium solani is known to produce toxin like Neosalaniol, T-2 toxin,

HT-2 toxin diacetoxyseripenol (Ueno and Nishimura, 1973) and

fusarubin reported to have antimicrobial (Arnstein, et al., 1946),

antitumor (Issaq et al., 1977), and phytotoxin activitivities (Kern,

1970). Apart from clinical uses the metabolites produced from

microorganisms are also used as growth stimulant and in the control

of plant diseases (Evans, 1989).


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Fungi produce various types of metabolites used in current

chemotherapy, for example penicillin, cephalosporin and fusidic acid,

which have antibacterial and antifungal activity (Lowe and Elander,

1983). After the discovery of penicillin in 1928 from P. notatum, the

modern era in the research of antibiotics started. A number of

antibiotics have been isolated from microorganisms with a ratio of

70:20:10 from actinomycetes, fungi and eubacterials respectively

(Bredy, 1974). Antibiotics like cephalomycin-C from Streptomyces

lactamdurrans, bacitracin from Bacillus subtilis, and tetracycline from

Streptomyces erythreus and kanamycin from Streptomyces

kanamyceticus (Tyler et al., 1988) produced and used in medicine.

Tolypocladium inflatum and T. geodes as well as strains of

Acremonium, Beauvaria, Fusarium, Paecilomyces and Verticillum Sps

(Dreyfus et al., 1976; Good et al., 1985) were know to produce

Cyclosporine-A. Multipliolides-A and B, 10-membered lactose

compounds were produced from Xylaria mutiplex (Boonphong et al.,

2001).

Most of the β-lactam antibiotics like pencillin, cephalosporin and

their relatives are produced from Penicillium and Cephalosporium

group where as polyene antibiotics are produced from Aspergillus sps

(Egoron, 1985). Griserofulvin is a metabolic product of Penicillium

nigricans, P. urticae and P. raistrickii used as therapeutic agent in the

treatment of dermatomycosis (Brain, 1960).


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The genus Streptomyces of actinomycetes is most potent producers

of antibiotics. They produce antibiotics like tetracyclins,

aminoglycosides, macrolides, ivermectin, rifamycin and others. Other

strains of actinomycetes such as Actinoplanes, Streptosporangium,

Nocardia, Streptoverticillium, and Micromonospora also produce

antibiotics. Bacillus polymyxa produce polymyxin and Bacillus subtilis

produce bacitracin, which belongs to polypeptide group. Zwittermicin

is produced by Bacillus cereus, where as nisin and thryothricin is

produced by Streptococcus lactis. Eubacteriales produce more number

of antibiotics in which Bacillus accounts for 70% and 13% for

Pseudomonas (Hugo and Russell, 1998).

Fusarium solani and Fusarium oxysporum produces several

napthaquinones with antibacterial properties (Baker, 1990). From

Talaromyces flavus, azole fungicide FKI-0076 was isolated. Fusidic

acid and fucidin produced from Acromonium fusidioides have anti

bacterial activity against gram negative bacteria (Godtfredson and

Lorck, 1963). An antibiotic, botrydiplodin produced by Botryosphaeria

rhodina was found active against both Gram-positive and Gram-

negative bacteria (Sen Gupta et al., 1966).

Cochliodinol produced by Chaetomium cochlioides exhibit both anti

bacterial and antifungal activities whereas chaetomin was active

against gram positive bacteria (Brewer et al., 1970). Cryptosporiopsis

guercina produce crytocandin, a lipopeptide antibiotic showed strong


21

inhibitory activity against human pathogenic fungi like Candida

albicans, Trichophyton mentagrophytes, Trichophyton rubrum and

plant pathogenic fungi like Sclerotinia sclerotium and Botrytis cinerea

(Strobel et al., 1999). Epidithiadiketo-piperazine and chaetomin

produced by Chaetomium globosum showed strong antifungal activity

(DiPietro et al., 1992). Glioclabium virens produced compounds,

gliovirin having antibiotic activity towards Pythium ultimum (Howell

and Stipanovic, 1983) and viridian, fungistatic in nature (Jones and

Hancock, 1987).

1.5.2 FUNGAL METABOLITES AS ANTITUMOR AGENTS

Apart from antibiotics compounds such as steroid derivatives,

ergot alkaloids, immnoregulators and antitumor agents were isolated

from fungi. Aspergillus sps., produce several quinine antitumor agents

(Sultan and Ahmad, 1994). Fusarium griseum produce fusidienol

which inhibits farnesyl protein kinase transferase responsible for

cancer (Singh et al., 1994). A potent protein kinase-C inhibitor,

Balanol, is produced by Verticillium balanoides (Kulanthaviel et al.,

1993). Antitumor compounds such as SCH-49210, 53516 were

reported from Naurassia mangifera and Preussia isomera (Chu et al.,

1994; Weber et al., 1990).

Myrothecium sp. produce roridins having cytotoxic activity against

colon tumor cell lines (wagennar and Clardy, 2001). Cytotoxic


22

compound Viscoltricin produced from Fusarium tricinctum inhibits the

growth of several human tumor cell lines (Visconti and Solfrizzo,

1995). Pentostatin, peplomycin, and epirubicin are the commercialized

antitumor agents isolated from fungi (Butler, 2005). Taxomyces

andreanea was the first endophytic fungus that was found to produce

taxol and taxane. Pestalotiopsis microspora produce high amount of

taxol. On the other hand may be useful in producing taxol much more

cheaply. Other fungi like Nodulisporium sylviforme and Taxodium

distichurn also produces taxol (jia yao et al., 1996).

Increased clinical needs, multi drug resistance strains, new

emerging pathogens, neoplastic and viral diseases led to the search for

discovery of new pharmacophores. In the agriculture field to prevent

the fungal pests and pathogens there is a need to discover newer

metabolites with high biological activity. The present work describes

the screening of various indigenously isolated fungal strains for the

isolation of bioactive secondary metabolites. Further the extracts were

bioassayed for antimicrobial against a panel of microorganisms and

larvicidal activity against agriculture pest Spodoptera litura. The

results showed, fungi Aspergillus funiculosus, A. gorakhpurensis and

Curvularia oryzae were found to be active. Ethyl acetate extracts were

further purified by chromatographic techniques and fractions were

isolated. Structures of the isolated fractions were determined by NMR

and mass spectral data. The isolated compounds were bioassayed for


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antibacterial, antifungal and larvicidal activities. The mode of action of

the isolated compounds was established against bacteria and S. litura

larvae.


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AIM OF THE STUDY

The main aim of this study is to isolate bioactive secondary

metabolites from fungi with antibacterial, antifungal and insecticidal

efficacy.

To achieve this goal the following specific objectives were pursued

� Screening of fungal species for their antimicrobial and larvicidal

efficacy

� Large scale cultivation of the lead fungi

� Isolation and chemical characterization of the secondary metabolites

from Aspergillus funiculosus, Aspergillus gorakhpurensis and

Curvularia oryzae

� Bioevaluation of the isolated compounds against bacteria, fungi and

Spodoptera litura larvae

� Determination of mode of action of metabolites against bacteria and

insect larvae (S. litura)

Chapter 1Chapter 1 IntroductionIntroduction &&&& Review...

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