Fungal Secondary

Metabolites
 The Fungi
 Fungi are one of the largest group of
organisms , world wide distributed and
play a vital role in ecosystems and as one of
the most important tool in biotechnology
 Fungi & Biotechnology
 BIOTECHNOLOGY means “ Use of Fungi to
produce, develop, improve, remove or sustain of
a particular thing which can be useful for man
and nature “
 The most important use of fungi is the
production of Secondary Metabolites
 Such as Antimicrobial Bioactive Substances
that can be beneficial for Medical therapy
The economic significance of Fungi
 Fermentation technology
 Enzymes production technology
Production of acids and chemicals
 Cultivation of fungi for protein
 Food processing by fungi (Bread, cheese)
 Fungi can be used in many applications in
(industry, agriculture, medicine , and
environment)
 Production of Bioactive compounds (Antibiotics)
Secondary metabolites
Organic compounds , with low molecular weight
,which are not essential for fungal growth but
their natural production have certain
significances.
Why secondary metabolites are produced?
 They are chemical compounds produced by many
fungi growing on substrates. They play a role in
competition ,antagonism and self-defense
mechanisms against other living organisms to
allow the fungus to occupy the niche and utilize
the food
Production of SECONDARY METABOLITES
Compounds produced during the stationary and
decline phases of the fungal growth
 They are vary in structures and biosynthetic
pathways . So they differ among the fungal
species and isolates
Fungal metabolites structures
 Fungal secondary metabolites can be obtained by
growing fungi in cultures under certain
conditions (Penicillin is best example)
gs
Types of Fungal secondary metabolites
1. Strobilurin (antifungal)
2. Gibberellins (growth Hormons)
3. Herbicides (control weeds)
4. Mycotoxins (poisneous)
5. Insecticides ( control insects)
6. Enzymes (proteins)
7. Pigments (dyes)
8. Antibiotics (drugs)
9. Pharmacological drugs
 Antibiotics
 Antibiotics: Are chemical substances produced by
fungi which has the capacity to inhibit the growth
of / and or even kill other microorganisms . The
action of an antibiotic is a selective in nature .
History of ANTIBIOTICS discovery
 (1928) Alexander Flimmings discovered the
Penicillin
 (1935) Prontosil, the first sulfa drug was
discovered by Gerhard Domagk
 (1943) Andrew Moyer, industrial production of
Penicillin
 (1943) Selman Waksman discovered the
Streptomycin from soil Bacteria
 (1955) Tetracycline was patented by Lioyd Conover
 (1957) Nystatin was patented
 (1981) Smith Kline Beckham patented Amoxillin
 Since then many antibiotics have been found and
still continued to discover more…….
 Antibiotic can be divided according their
bioactivity into:
 Antifungal antibiotics

 Antibacterial antibiotics

 Antiviral antibiotics

 Antitumoral antibiotics
Mechanism of antibiotic action
 The aim of present study
 1. Screening of local fungi isolates for antimicrobial bioactive
compounds production.

 2. Isolation and purification of the bioactive compounds using

specific techniques

 3. Examining the optimal conditions (Temp, pH, media , C, N

source and some other factors) for a mass production of these
compounds in batch cultures.
 4. Characterization of the purified fungal extracts by using
HNMR and GC Mass.
 5. Testing the inhibition activity of these compounds against a
selected pathogenic microorganisms (Bacteria, Candida,
Dermatophytes and other fungi).
 Most recent publications on bioactive metabolites by fungi
 Last decade several published papers on this aspect: For Examples

 Anke et al. (2004, 2006) from Germany

 Lindquist et al. (2005, 2006) from Germany
 Abad et al. (2007) from Spain
 Stamets P. (2007) from USA
 Johnathan et al. (2010) South Africa
 Muhsin et al (2011) from Iraq

 Pohanka ( 2006 ) PhD. Thesis (Sweeden)

 Roberts (2004). PhD. Thesis (Australia)
 Khalaf KT (2008) PhD. Thesis (Iraq)
 Materials and methods
 1. Sample collection and fungal isolation:
Sample are collecting from different sources (soil , water , air ,
rhizospher , rhizoplane ) for fungal isolation
 Isolation, purification and identification of fungi on solid
media
 Screening of the inhibition bioactivity of each fungal species
against the selected pathogenic microorganisms including:
A. Bacteria B. Candida C. Dermatophytes D. Other fungi
by using fungal discs on solid media.
 2. Fungal cultures Extraction:
The selected fungal isolates will be grown in specific liquid
culture
 (using batch large bottles) under shaking condition for two week
incubation period at 25 C
3. Antimicrobial bioactivity assay

Filter paper discs (0.6 mm) after being

sterilized by autoclave are socked in each
fungal crude extract solution for 5 min.,
Filter paper discs with extracts are placed on
the surface of agar medium in Petri-dishes
streaked with 0.2 ml of bacterial suspension
of Escherichia coli and Staphylococcus
aureus, Candida albicans, Microsporum
gypseum, Fusarium oxysporum.
Plates are incubated at 37 ◦C for 24 hr, an
appearance of inhibition zones around the
filter paper disc indicating the bioactivity of
crude metabolites of the tested fungal isolates
(Casals, 1979).
 4. Extraction of the fungal filtrates
 Five discs (0.5 mm diam.) are cut from the axenic
fungal culture of each isolate by using a cork borer and
amended into PD liquid medium in 500 ml flasks (with
triplicates) and incubation at 25 ◦C for 2 weeks on a
rotary shaker.
 Fungal cultures are filtered on Whatman No 1 filter
paper and the pH adjusted at 3 for each fungal filtrate.

 Filtrate is extracted in ethyl acetate (1:1 vol) by using

separating funnel. The organic layer is collected by
dehydration of water by using Na2SO4. The filtrate is
filtered again and place in Petri dishes then leave to be
dried at room temperature.
 100 ug of the dried extract is dissolved in 1 ml ethanol
as stock extract solution to be used for further
experiments.
5. Minimal inhibitory concentration test 
The MIC values will be determined by the standard 
serial dilution assay (McGinnis, 198o).
Different concentrations of the crude fungal extract 
are used. Filter paper discs (0.5 mm) are soaked
in each extract concentration and will be tested
against each selected microorganisms on solid
medium. The appearance of inhibition zone
around the disc indicates an inhibitory action.
6.Cytotoxicity test 
Cytotoxicity of the fungal crud extracts was examined 
by using human RBC
following a previously described method ( Xian- guo 
and Ursula, 1994).
7. Chemical analysis of fungal crude extracts 
Fungal culture extracts will be chemically analyzed for 
alkaloids, phenols, amino acids, flavenoides and
Tannins according to following the described method
8. Purification of fungal extracts
Crud extract will purified by using TLC and HPLC
technique.
9.Identification and characterization of the
purified extract
By using GC –Mass and HNMR technique
10.Optimal Production of Bioactive metabolites
Experimentation will be conducted to examine the
optimal production of the bioactive compounds in
batch liquid cultures using:
1. Effect of Temperature
2. Effect of pH
3. Effect of Media
4. Effect of carbon and nitrogen sources (4 sources)
5. Effect light and dark period
6. Effect microelements (eg Zn, Fe)

11. Comparison of the bioactivity of the studied

compounds with some commercial antibiotics.
12. If the allows, some other works will be included
Selected fungi for this study
 We try to investigate some fungi
belong to Deuteromycetes in particular
 Rhizoctonia and Trichoderma
For the following reasons:
1. No research so far dealt with these
fungi elsewhere
2. These fungi are highly competitive
against other soil and plant fungi
3. Easy to grow in culture and handle
with