I= STUDIES ON ANTIBIOTICS FROM SOIL FUNGI
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Title of Thesis
STUDIES ON ANTIBIOTICS FROM SOIL FUNGI

Author(s)
Shamim Akhter Qureshi
Institute/University/Department Details
University of Karachi, Pakistan
Session
2003
Subject
Biochemistry
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
antibiotics, soil fungi, penicillin, rhizoplane, fungi, culture filtrates, rhizosphere, chaetomium flavum omvik

Abstract
Since the discovery of penicillin, a number of antibiotics have been discovered mostly from soil inhabiting microorganisms. Plant roots surface (rhizoplane) and soil around the roots (rhizosphere) are the zones of intensified microbial activity due to secretion of root exudates (which contains sugars, amino acid vitamins etc.) resulting in competition among the microbes for nutrition. The competitive fungal species utilize their toxic metabolites or antibiotics for their establishment in these regions, hence providing unexhausted source of antibiotic. In the present study, 680 root samples with adhering soil of 65 plant species belonging to 59 genera and 19 families viz., Amaranthaceae (Amaranthus virides L., Aerva javanica (Burm. f.) Merrill, Digera muricata (L.) Mart.); Asclepediaceae (Calotropis procera (Willd.) R. Br.); Boraginaceae (Heliotropium europeam L.); Cannaceae (Canna indica L.); Caricaceae (Carica papaya L.); Chenopodiaceae (Beta vulgaris L., Chenopodium album L., Spinacea oleracea L.); Compositae (Conyza bonariensis (L.) Cronq., Helianthus annuus L., Lactuca sativa L., Launea nudicaulis Hook. F.); Convolvulaceae (Convolvulus arvensis L.); Crucifcrae (Brassica juncea (L.) Czern & Coss, B. oleracea L. var. capitata L., B. rapa L., Rhaphaus sativus L.); Cucurbitacea (Citrllllus lanatus (Thumb.) Matsum. & Nakai, Cucumis sativus L., Cucurbita moschata L., Lagenaria siceraria (Mol.) Stand., Luffa aegyptiaca Mill., Momordica charantia L.); Cyperaceae (Cyperus rotundus L.); Euphorbiaceae (Euphorbia hirta L.); Fabaceae (Alhaji maurorum Medik, Arachis hypogaea L., Cicer arietinum L., Cyamopsis tetragonoloba (L.) Taub., Glycine max (L.) Merr., Lens culinaris MediK., Leucaena leucocephala Lam. De Wit., Medicago sativa L., Melilotus alba Medik, Phaseolus vulgaris L., Pisum sativum L., Sesbania sesban (L.) Merr., Trifolium alexandrinum (L.) Spergue ex Turrill, Trigonella foenum-graecum L., Vigna mungo (L.) Hepper, v. radiate (L.) Wilczek.); Gramineae (Avena sativa L., Cenchrno setigerus Vahl., Cynodon dactylon (L.) Pers., Oryzae sativa L., Pennisetum americanum (L.) Leeve, Sataria verticilata (L.), Sorghum bicolor L., Triticum aestivum L., Zea mays L.); Malvaceae (Abelmoschus esculentus (L.) Moench., Abutilon indicum L. Sweet; Gossypium arboreum L.); Pedaliaceae (sesamum indicum L.); Piperaceae (Piper betel L.); Solanaceae (Capsicum annuum L., C. annuum var. shimla, Lycopersicon esculentum Mill., Solanum melongena L., S. nigrum L., S. surrenttense Burro. f.); Umbelliferae (Coriandrum sativum L., Daucus carota L., spp. sativus ) were collected from Karachi University Campus, Memon Goth, Darsano-Chano, Kathor, Shah Faisal colony, Thatta from Sindh Province and Hub from Baluchistan Province.

Fungi isolated from rhizoplane were identified as Cunninghamella echinulata (Thaxt.) Thaxt., (10 hosts), Helicocephalum sp. (1 host), Rhizopus stolonifer (Ehrenb ex. Link) Lind. (12 hosts) from Zygomycetes of Phylum Zygomycota; Aspergillus candidus Link ex Link (1 host), A. flavus Link ex Gray (25 hosts), A.fumigatus Fres. (5 hosts), A. nidulans (Eidam) Winter (13 hosts), A. niger Van Tieghem (32 hosts), A. terreus Thorn (19 hosts), Paecilomyces lilacinus (Thorn.) Samson (3 hosts), Chaetomium flavum Omvik (1 host), C. globosum Kunz ex Staud. (14 hosts), C. indicum Corda (6 hosts) from Ascomyctes of Phylum Ascomycota; Alternaria alternata (Fr.) Ktissler (35 hosts), Cladosporium sp., (5 hosts), Curvularia clavata Jain (4 hosts), C. lunata (Wakker) Boedjin (19 hosts), Drechslera australiensis (Bugni) Subram. & Jain ex M.B. Ellis (40 hosts), D. halodes (Drechsler) Subram. & Jain (8 hosts), D. hawaiiensis (Bugni) Subram. & Jain ex M.B. Ellis (3 hosts), Humicola grisea Traaen (1 host), Macrophomina phaseolina (Tassi) Goid. (44 hosts), Memnoniella echinata (Riv.) Galloway (1 host), Myrothecium roridum Tode ex Steudel (1 host), Nigrospora oryzae (Berk. & Br.) Petch (2 hosts), Phialophora fastigiata (Lagerb. & Melin) Conant (1 host), Rhizoctonia solani Kuhn (40 hosts) from Deuteromycetes of Phylum Ascomycota; Fusarium culmurum (W.G. Sm.) Sacc., (2 hosts), F. moniliforme Sheld (6 hosts), F. oxysporum Schlecht emend. Snyd. & Hans (19 hosts), F..semitectum Berk & Rav., (3 hosts), F. solani (Mart.) Appel & Wollenw. emend. Snyd. & Hans (61 hosts) and Trichoderma viride Pers ex. Gray (4 hosts), Verticillium chlamydosporium Goddard (1 host) from Pyrenomycetes of Phylum Ascomycota; Unidentified fungi viz., Ascomycetes (1 host), Basidomycetes (1 host), Sclerotial fungus (4 hosts), Sterile mycelium including black (3 hosts), yellow (2 hosts) and white (9 hosts).

Fungi isolated from rhizosphere were identified as Cunninghamella echinulata (Thaxt.) Thaxt., (6 hosts), Rhizopus stolonifer (Ehrenb ex. Link) Lind. (12 hosts), from Zygomycetes of Phylum Zygomycota; Aspergillus candidus Link ex Link (6 hosts), A. flavus Link ex Gray (57 hosts), A.fumigatus Fres. (7 hosts), A. glaucus Link (2 hosts), A. nidulans (Eidam) Winter (22 hosts), A. niger Van Tieghem (61 hosts), A. sulphureus (Fres.) Thorn & Church. (11 hosts), A. terreus Thorn (48 hosts), A. versicolor (Vuill) Tiraboschi (1 host), Chaetomium flavum Omvik (4 hosts), C. globosum Kunze ex Staud. (11 hosts), C. indicum Corda (8 hosts), Paecilomyces lilacinus (Thorn.) Samson (10 hosts), Penicillium aspermum (Shear) n. Comb (1 host), P. citrinum Thorn (1 host), P. chrysogenum Thorn (3 host), P.funiculosum Thorn (3 hosts), P.javanicum Van Beijma (1 host), P. luteum Zukal (7 hosts), P.purpurenscens (Sopp) n. Comb. (1 host), P.purpurogenum Stoll (5 hosts), P. raistrickii Smith (2 host), P. regulosum Thorn (6 hosts), from Ascomyctes of Phylum Ascomycota; Cephalosporium sp., (8 hosts), Fusarium culmurum (W.G. Sm.) Sacc., (2 hosts), F. moniliforme Sheld (8 hosts), F. oxysporum Schlecht emend. Snyd. & Hans (12 hosts), F. proliferatum (Matsushima) Nirenberg (1 host), F. semitectum Berk & Rav., (7 hosts), F. solani

(Mart.) Appel & Wollenw. emend. Snyd. & Hans (26 hosts), Trichoderma harzianum Rifai (4 hosts), T. koningii Oudem (1 host), T. viride Pers ex. Gray (7 hosts) from Pyrenomycetes of Phylum Ascomycota; Alternaria alternata (Fr.) Keissler (37 hosts), Articulospora sp., (1 host), Cladosporium sp., (9 hosts), Curvularia clavata Jain (3 hosts), C. lunata (Wakker) Boedjin (3 hosts), Drechslera australiensis (Bugni) Subram. & Jain (32 hosts), D. hawaiiensis (Bugni) Subram. & Jain ex M.B. Ellis (lhost), Gliomastix murorum Grey (l host), Macrophomina phaseolina (Tassi) Goid., (22 hosts), Memnoniella echinata (Riv.) Galloway (l host), Monodictys puterdinis (Wallr.) Hughes (2 host), Myrothecium cinctum (Corda) Sacco (3 hosts), M. roridum Tode ex Steudel (5 hosts), Nigrospora oryzae (Berk. & Br.) Petch (2 hosts), Philophora fastigiata (Lagerb & Melin) Conant. (1 host), Rhizoctonia solani Kuhn (5 hosts), Scopulariopsis brumptii Salvanet-Duval (2 hosts), Stachybotrys atra Corda (2 hosts), S. parvispora Hughes (l host) from Deuteromycetes of Phylum Ascomycota; Unidentified fungi viz., Ascomycetes (l host), Basidomycetes (2 hosts), Sclerotial fungus (8 hosts), Sterile mycelium including black (3 hosts), Yellow (4 hosts), White (19 hosts) and Yeast (6 hosts). Paecilomyces lilacinus from Citrul / us lanatus, Luffa aegyptiaca and Cyamopsis tetragonoloba; Memnoniel / a echinata from Sorghum bicolor and Stachybotrys parvispora from Coriandrum sativum and Zea mays have been reported first time from Pakistan.

In the present study, fifty seven fungal isolates were tested in vitro for their antifungal activity against plant pathogens viz., Macrophomina phaseolina, Rhizoctonia solani, Fusarium oxysporum, F.solani and human pathogens viz., Aspergillus fumigatus and Candida albicans . Fusarium proliferatum, Paecilomyces lilacinus (S-1 & S-3) and Trichoderma viride (S-I) inhibited all the six test pathogens. Aspergillus candidus, Chaetomium jlavum, P.lilacinus (SA), Stachybotrys atra (S-2) and S.parvispora were found active against five pathogens except C.albicans. Whereas P.lilacinus (S-2) was active against five pathogens except Afumigatus. Aspergillus jlavus (S-2), Memnoniella echinata, Penicillium raistrickii, T.pseudokoningii and Verticillium chlamydosporium showed activity against four test pathogens. These fungal isolates were also tested in vitro against 5 bacterial species viz., Bacillus subtilis (Ehrenberg) Cohn, Staphylococcus aureus Rosenbach (Gram-positive) and Escherichia coli (Migula) Castellani and Chalmers, Pseudomonas aeruginosa (Schraeter) Migula and Salmonella typhimurium (Loeffler) Castellani and Chalmers (Gram-negative). Aspergillus jlavus (S-2), Curvularia clavata, Fusarium oxysporum, F.solani (S-2), Penicillium citrinum, P.raistrickii, Stachybotrys atra (S-1 & S-2) and V.chlamydosporium were found to inhibit all the five test bacteria and produced inhibition zones against them. Whereas Aspergillus candidus, Cephalosporium sp., Drechslera australiensis (S-I), Memnoniella echinata, Myrothecium roridum, P.lilacinus (S-I, S-2, & S-3) and Stachybotrys parvispora inhibited four test bacteria except Saureus. Aspergillus flavus (S-3), A.niger (S-2), A.sulphureus(S-I), D.hawaiiensis,

F.solani (S-l), Macrophomina phaseolina, and T. viride (S-2) also inhibited four test bacteria except P.aeruginosa. Antibiotic production of some fungi were tested in vitro against test fungal pathogens using cellophane paper method; all the test fungi including Aspergillus candidus, Ajlavus, Afumigatus, Chaetomium flavum, C.globosum (S-l), C.indicum, Fusarium solani, F.oxysporum, Gliomastix murorum, Memnoniella echinata, Myrothecium cinctum, M.roridum, Paecilomyces lilacinus (S-l, S-2, S-3, & S-4), Penicillium raistrickii, Stachybotrys atra (S-l & S-2), Sparvispora, Talaromyces flavus and Trichoderma spp., were found to produce fungistatic metabolites against M.phaseolina, R.solani, F .solani, F.oxysporum. Metabolites of A.candidus and Sparvispora showed fungicidal activity against M.phaseolina. Culture filtrates of twenty five isolates of fungi tested showed better antibacterial activity than antifungal activity. Lyophilization of culture filtrates was found to enhance antibacterial activity of culture filtrates.

Plant parasitic nematodes constitute one of the most important and destructive pest groups of economic crops causing billion dollars losses each year. In the' present study, forty-eight culture filtrates of fungal isolates were tested for nematicidal activity against Meloidogyne javanica, root knot nematodes. Significant nematicidal activity was observed by culture filtrates of Aspergillus sulphureus, Paecilomyces lilacinus (S-l), Scopulariopsis brumptii.. Stachybotrys atra (S-2), Trichoderma harzianum, and Verticillium chlamydosporium by producing more than 50 % mortality of nematodes after 48 hours of exposure.' Promising strains of fungi could be exploited for the isolation of nematicidal compounds.

The search for cancer chemotherapeutic natural products has been rapidly accelerating in recent years. The cytotoxicity of plant or fungal materials is considered as the presence of antitumour compounds. In the present study, fifty-five culture filtrates of fungal isolates were tested against Artemia salina (brine shrimp) for determining their cytotoxic activity. Highest percent death of Artemia salina was observed by five fungi viz., Aspergillus niger (S-l) (Lc50 43.68 ug/ml), Penicillium citrin urn (Lc50 78.18 ug/ml), P. purpurescens (Lc 50 169.7 ug/ml), P.rugulosum Lc50 604.0 ug/ml ) and Penicillium sp. Ā«Lcso 161.7 ug/ml) after 24 hours. Fungal metabolites also offer a potential source of anti-tumor compounds.

Ethyl-acetate fractions of culture filtrates of Ajlavus (S-2), Cjlavum, C.globosum (S-l & S-2), C.indicum, M.echinata, P.lilacinus (S-l & S-2), P.raistrickii, S.atra (S-l), Sparvispora, Talaromyces flavus, Trichoderma harzianum, and T.koningii were found more active against test bacteria than n-hexane fractions, indicating the presence of intermediary polar nature of antimicrobial compound(s) produced by these fungi. It is interesting to note that n-hexane fraction of Some fungi were also produced inhibition zones against some bacteria where inhibitory activity was not observed by ethyl-acetate tractions of these fungi. Similarly, few antibacterial activities were also observed by water soluble tractions of culture filtrates viz., Trichoderma koningii and T viride (S-l & S-2) against B,.subtilis, Aspergillus candidus, Trichoderma harzianum, T viride (S-2) and Verticillium chlamydosporium against S.aureus, and by A.candidus, F.oxysporum, Tkoningii and T viride (S-2) against E.coli.

Physical and chemical mutagenesis used to modify the gene of microorganism to improve biosynthesis of the active product. During the present study, mutagenesis with UV or acridine orange has induced or enhanced antimicrobial activity in some fungi although some strains has lost or reduced their activity.UV mutatIon induced antibacterial activity in Chaetomium flavum against B.subtilis and S.typhimurium and enhanced against S.aureus. Alteration of genetic make up by mutation offers a simple and convenient method for obtaining superior strains of antibiotic producing fungi. In the present study, mycelium of twenty-six fungal isolates were extracted with methanol and fractionated into n-hexane, chloroform and methanol soluble portions. Less antimicrobial activity was found in solvent tractions of mycelium as compared to culture filtrates. However methanol and chloroform soluble tractions of mycelium of A.candidus, C. globosum (S-2), M.echinata, and species of Fusarium, Stachybotrys and Trichoderma were found inhibitory against some test bacteria. Methanol soluble tractions of M.echinata and Sparvispora were also produced significant inhibition zones against M.phaseolina. Fungi like Chaetomium flavum, Memnoniella echinata and Stachybotrys parvispora which showed significant antimicrobial activity and have no or a few reports on their chemical investigation with antimicrobial activity were further investigated. Saturated and unsaturated fatty acid esters were isolated from column chromatography of ethyl-acetate soluble traction of culture filtrate of C.flavum and identified on the basis of spectral data of GC-MS. Through GC-MS spectrum, another waxy traction obtained from the same column of Chaetomium flavum showed the presence of saturated and unsaturated hydrocarbons. These fatty acid esters and hydrocarbons were found to be inhibitory against B.subtilis, Saureus, Styphimurium and P.aeruginosa and are reported first time from this source. Saturated and unsaturated hydrocarbons were also isolated from column of ethyl-acetate soluble traction of culture filtrate of M.echinata and identified through GC-MS ,technique. These hydrocarbons showed antibacterial activity against B.subtilis, E.coli, , S.aureus, S.typhimurium and r.aeruginosa. Similarly, five saturated aliphatic hydrocarbons of chain length varying trom C22-C29 and an unsaturated 17 carbons containing hydrocarbon were isolated from column of ethyl-acetate soluble fraction of S. parvispora.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 1 Introduction 1
1280.37 KB
  1.1 Importance Of Fungi 1
  1.2 Importance Of Antibiotics 5
2 2 Materials And Methods 14
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  Collection Of Samples 14
  2.2 Isolation Of Fungi From Rhizosphere 14
  2.3 Isolation Of Fungi From Rhizosplane 14
  2.4 In Vitro Antifungal Activity Of Soil Fungi By Duel Culture Plate Method 15
  2.5 In Vitro Antibacterial Activity Of Soil Fungi By Agar Disc Diffusion Techniques 15
  2.6 In Vitro Antibiotic Production By Soil Fungi 15
  2.7 Preparation Of Culture Filtrates 16
  2.8 Preparation Of Lyophilized Culture Filrates 17
  2.9 Fractionation Of Culture Filtrates 17
  In Vitro Antimicrobial Activity Of Culture Filtrates And Their Fractions 17
 
  2.11 In Vitro Nematicidal Activity Of Culture Filrates Of Soil Fungi 19
  2.12 In Vitro Cytotoxic Activity Of Culture Filrates Of Soil On Artemia Salina (Brine Shrimp ) 20
  2.13 Extraction And Fractionation Of Mycelium Of Soil Fungi 21
  2.14 Mutation Of Some Soil Fungi 21
  2.15 Chromatography 22
  Spectroscopy Of Oily Fractions Eluted From Column 23
 
3 3 Experimental Results 24
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  24 Isolation And Identification Of Fungi From Rhizosphere And Rhizoplane Of Wild And Cultivated Plants
  3.2 In Vitro Antifungal Activity Of Soil Fungi 81
  3.3 In Vitro Antibacterial Activity Of Soil Fungi 96
  3.4 In Vitro Antibiotic Production By Soil Fungi 106
  In Vitro Antimicrobial Of Culture Filtrates Of Soil Fungi 113
  3.6 In Vitro Nematicidal Activity Of Culture Filtrates Of Soil Fungi 141
  3.7 In Vitro Cytotoxic Activity Of Soil Fungi On Artemia Salina (Brine Shrimp ) 147
  3.8 In Vitro Antimicrobial Activity Of Fractions Culture Filtrates 156
  In Vitro Antimicrobial Activity Of Mycelium Of Soil Fungi 183
 
  3.10 Use Of Physical And Chemical Mutagens To Enhance The Antimicrobial Activity Of Soil Fungi 208
  3.11 Chaetomium Flavum Omvik 220
  3.12 Memnoniella Echinata ( Riv ) Galloway 256
  3.13 Stachybotrys Parvispara Hughes 270
4 4 Discussion 288
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5 5 Conclusion 300
376.12 KB
6 6 References 304
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7 7 Acknowledgements 345
111.88 KB
8 8 Publications 346
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