Fungal Secondary Metabolites الأيوض الفطريه الثانويه طالب الماجستير مازن سليم سلمان

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 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)

Types of Fungal secondary metabolites 1. Strobilurin (antifungal) 2 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 gs

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.

8. Purification of fungal extracts 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: Effect of Temperature Effect of pH Effect of Media Effect of carbon and nitrogen sources (4 sources) Effect light and dark period 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: No research so far dealt with these fungi elsewhere These fungi are highly competitive against other soil and plant fungi Easy to grow in culture and handle with