Screening of Metabolites Products of Fusarium oxysporum and Determination of Its Antibacterial and Antifungal Activity Using Medicinal Plants Extract

DOI Number: 10.5958/0976-5506.2018.00243.7 Screening of Metabolites Products of Fusarium oxysporum and Determination of Its Antibacterial and Antifungal Activity Using Medicinal Plants Extract Abeer Fauzi Al-Rubaye 1, Imad Hadi Hameed 2, Sabreen A Kamal 1 1 Department of Biology, College of Science for Women, University of Babylon, Hillah city, Iraq, 2 Biomedical Science Department, University of Babylon, College of Nursing, Hillah city, Iraq ABSTRACT The aims of this study were screening of the secondary metabolite products and evaluation antimicrobial activity. Bioactives are chemical compounds often referred to as secondary metabolites. Twenty one bioactive compounds were identified in the methanolic extract of Fusarium oxysporum. The identification of bioactive chemical compounds is based on the peak area, retention time molecular weight and molecular formula. Melissa officinalis was very highly active 6.470±0.25 mm. The results of anti-fungal and anti-bacterial activity produced by Fusarium oxysporum showed that the volatile compounds were highly effective to suppress the growth of Aspergillus fumigatus (5.893±0.20) and Streptococcus pyogenes (6.001±0.19). Based on the significance of employing bioactive compounds in pharmacy to produce drugs for the treatment of many diseases, the purification of compounds produced by Fusarium oxysporum can be useful. Keywords: Antifungal, Antibacterial, Fusarium oxysporum, GC-MS, Secondary metabolites. INTRODUCTION Fusarium oxysporum is a common inhabitant of soil and produces three types of asexual spores; macroconidia, microconidia and chlamydospores. Infection by Fusarium oxysporum f.sp. cubense triggers the self-defense mechanisms of the host plant causing the secretion of a gel. This is followed by the formation of tylose in the vascular vessels which blocks the movement of water and nutrients to the upper parts of the plant 1-5. The tips of the feeder roots are the initial sites of infection which then moves on to the rhizome. The leaves begin to wilt and may buckle at the base of the petiole. As the disease progresses, younger leaves are affected, turn yellow and crumple and the whole canopy begins to consist of dead or dying leaves. F. oxysporum is primarily spread over short distances by irrigation water and contaminated farm equipment 6-14. Corresponding author: Imad Hadi Hameed Biomedical Science Department, University of Babylon, College of Nursing, Hillah city, Iraq; Phone number: 009647716150716; E-mail: imad_dna@yahoo.com The fungus can also be spread over long distances either in infected transplants or in soil. Although the fungus can sometimes infect the fruit and contaminate its seed, the spread of the fungus by way of the seed is very rare. It is also possible that the spores are spread by wind. Fusarium oxysporum is an asexual fungus that produces three types of spores: microconidia, macroconidia, and chlamydospores 14-27. Microconidia are one or two celled, are produced by Fusarium oxysporum under all conditions, and produced the most within the infected plants. The objectives of this study were analysis of the secondary metabolite products and determination of antimicrobial activity. MATERIALS AND METHOD Interpretation of mass spectrum was conducted using the database of National Institute of Standards and Technology (NIST, USA). The database consists of more than 62,000 patterns of known compounds. The spectrum of the extract was matched with the spectrum of the known components stored in the NIST library. Fusarium oxysporum was isolated and maintained in potato dextrose agar slants. Spores were grown in a liquid culture of potato dextrose broth (PDB) and incubated

400 Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3 at 25ºC in a shaker for sixteen days at 150 rpm. The extraction was performed by adding 50 ml methanol to 150 ml liquid culture in an Erlenmeyer flask after the infiltration of the culture 28-35. The mixture was incubated at 4ºC for 10 min and then shook for 10 min at 130 rpm. Metabolites was separated from the liquid culture and evaporated to dryness with a rotary evaporator at 45ºC. The residue was dissolved in 1 ml methanol, filtered through a 0.2 μm syringe filter, and stored at 4ºC for 24 h before being used for GC-MS. Determination of antibacterial and antifungal activity The test bacterial pathogens were swabbed in Muller Hinton agar plates. 90μl of fungal extracts was loaded on the bored wells. The wells were bored in 0.5cm in diameter. The plates were incubated at 37C for 24 hr and examined. After the incubation the diameter of inhibition zones around the discs was measured. Fusarium oxysporum isolate was suspended in potato dextrose broth. They were flood inoculated onto the surface of Potato dextrose agar and then dried. Standard agar well diffusion method was followed. Fivemillimeter diameter wells were cut from the agar using a sterile cork-borer, and 25 μl of the plant samples solutions were delivered into the wells. The plates were incubated for 48 h at room temperature. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test microorganisms. Methanol was used as solvent control. Amphotericin B and fluconazole were used as reference antifungal agent 36-47. The tests were carried out in triplicate. The antifungal activity was evaluated by measuring the inhibition-zone diameter observed after 48 h of incubation. Results of the study were based on analysis of variance (ANOVA) using Statistica Software. A significance level of 0.05 was used for all statistical tests. Table 1. Major phytochemical compounds identified in methanolic extract of Fusarium oxysporum. Serial No. Phytochemical compound RT (min) Molecular Weight 1. 1,2,3,4-Cyclopentanetetrol, (1α,2β,3β,4α)- 3.150 134.057909 2. 2-Furanmethanol 3.259 98.0367794 3. 2,4,6-Cycloheptatrien-1-one, 4-methyl- 3.751 120.0575147 4. Dihydroxyacetone 3.917 90.031694 5. 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one 3.968 144.042258 6. 2,3,5-Trioxabicyclo[2.1.0]pentane, 1,4-bis(phenylm 3.779 254.094295 7. DL-Arabinose 4.134 150.052823 8. Isosorbide Dinitrate 4.878 236.028066 9. 5- Hydroxymethylfurfural 6.738 126.031694 10. 6-Acetyl-β-d-mannose 6.686 222.073953 11. L-Glucose 7.756 180.063388 12. α-d-glucopyranoside, O-α-D-glucopyranosyl-(1.fw 10.113 504.169035 13. N-(2,5-Dicyano-3,4-dihydro-2H-pyrrol-2-yl)-acetamide 10.960 176.069811 14. 8-Hydroxy-2,6-dimethylocta-2,6-dienoic acid,ethyl 11.458 212.141245 15. 2-Acetylamino-3-hydroxy-propionic acid 11.727 147.053158 16. 5H-Cyclohepta-1,4-dioxin, 2,3,4a,6,7,9a-hexahydro 14.308 154.09938 17. 7-Hydroxy-6-methyl-oct-3-enoic acid 14.525 172.109944 18. trans-2-undecenoic acid 14.662 184.14633 19. 2-Heptanol, 6-methyl- 15.979 130.135765 20. Dodecane, 1-fluoro- 16.362 188.194029 21. Ethylene, 1-nitro-2-[3-benzyloxyphenyl]- 16.608 255.089543

Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3 401 Table 2. Zone of inhibition (mm) of test different bioactive compounds and standard antibiotics of medicinal plants to Fusarium oxysporum. Plant Inhibition (mm) Plant Inhibition (mm) Diplotaxis cespitosa 5.870±0.23 Daucus carota 5.853±0.22 Cassia angustifolia 5.330±0.23 Vitex agnus-castus 5.633±0.24 Euphorbia lathyrus 6.011±0.22 Cressa cretica 6.006±0.25 Rosmarinus oficinalis 5.680±0.24 Citrus sinensis 6.070±0.22 Citrullus colocynthis 4.000±0.17 Ruta graveolens 4.080±0.19 Althaea rosea 5.074±0.20 Thymus vulgaris 6.007±0.25 Coriandrum sativum 6.370±0.25 Passiflora caerulea 5.900±0.23 Origanum vulgare 5.811±0.24 Glycine max 5.767±0.22 Urtica dioica 3.925±0.23 Brassica oleracea 3.908±0.22 Foeniculum vulgare 2.989±0.17 Olea europaea 3.000±0.19 Ocimum basilicum 5.002±0.24 Calendula officinalis 5.087±0.23 Achillea millefolia 5.514±0.27 Taraxacum officinale 2.008±0.20 Medicago sativa 2.982±0.18 Borago officinalis 3.544±0.19 Celosia argentea 3.261±0.21 Sambucus nigra 2.015±0.23 Apium graveolens 4.801±0.23 C. morifolium 5.906±0.19 Brassica rapa 5.973±0.22 Equisetum arvense 6.004±0.24 Cichorium endivia 5.610±0.24 Portulaca oleracea 6.070±0.24 Anethum graveolens 6.006±0.23 Malva neglecta 5.227±0.19 Plantago major 5.002±0.23 L. angustifolia 2.006±0.17 Linum usitatissimum 4.075±0.19 Althaea Officinalis 5.005±0.18 A. esculentus 5.551±0.24 Melissa officinalis 6.470±0.25 Malva sylvestris 4.991±0.23 Control 0.000 RESULTS AND DISCUSSION Identification of biochemical compounds Analysis of compounds was carried out in methanolic extract of Fusarium oxysporum, shown in Table 1. Chromatogram GC-MS analysis of the methanol extract of Fusarium oxysporum showed the presence of thirty one major peaks and the components corresponding to the peaks were determined as follows. Clinical pathogens selected for antibacterial activity namely, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Pseudomonas eurogenosa, Escherichia coli, Proteus mirabilis, Streptococcus pyogenes, and Klebsiella pneumonia maximum zone formation against Streptococcus pyogenes (6.001±0.19) mm. Methanolic extraction of Fusarium oxysporum showed notable antifungal activities against M. canis, Penicillium expansum, Aspergillus flavus, Candida albicans, Aspergillus fumigatus, Trichoderma viride, Saccharomyces cerevisiae, and Aspergillus terreus. Aspergillus fumigatus was very highly active against Fusarium oxysporum (5.893±0.20). In agar well diffusion method the selected medicinal plants were effective against Fusarium oxysporum Table 2. Fivemillimeter diameter wells were cut from the agar using a sterile cork-borer, and 25 μl of the samples solutions (Anastatica hierochuntica (Crude), Cassia angustifolia (Crude), Euphorbia lathyrus (Crude), Rosmarinus oficinalis (Crude), Citrullus colocynthis (Crude), Althaea rosea (Crude), Coriandrum sativum (Crude), Origanum vulgare (Crude), Urtica dioica (Crude), Foeniculum vulgare (Crude), and Ocimum basilicum (Crude), Achillea millefolia, Medicago sativa, Celosia argentea, Apium graveolens, Brassica rapa, Cichorium

402 Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3 endivia, Anethum graveolens, Plantago major, Linum usitatissimum, A. esculentus, Malva sylvestris, Vitex agnus-castus, Cressa cretica, Citrus sinensis, Ruta graveolens, Thymus vulgaris, Passiflora caerulea, Glycine max, Brassica oleracea, Olea europaea, Taraxacum officinale, Borago officinalis, Sambucus nigra, C. morifolium, Equisetum arvense, Portulaca oleracea, Portulaca oleracea, Malva neglecta, L. angustifolia, Althaea Officinalis, and Melissa officinalis) were delivered into the wells. Melissa officinalis was very highly antifungal activity (6.470±0.25) mm. CONCLUSION Twenty one bioactive chemical constituents have been identified from methanolic extract of the Fusarium oxysporum by (GC-MS). In vitro antimicrobial determination of products of Fusarium oxysporum forms a primary platform for further phytochemical and pharmacological investigation. Financial Disclosure: There is no financial disclosure. Conflict of Interest: None to declare. Ethical Clearance: All experimental protocols were approved under the Department of Biology, College of Science, Hillah city, Iraq. REFERENCES 1. Ploetz R. Fusarium Wilt of Banana is caused by Several Pathogens Referred to as Fusarium oxysporum f. sp. cubense. Phytopathology. 2006; 96(6): 653-656. 2. Mohammed GJ, Kadhim MJ, Hameed IH. Proteus species: Characterization and herbal antibacterial: A review. International Journal of Pharmacognosy and Phytochemical Research. 2016; 8(11): 1844-1854. 3. Shireen SK, Hameed IH,, Hamza LF. Acorus calamus: Parts used, insecticidal, anti-fungal, antitumour and anti-inflammatory activity: A review. International Journal of Pharmaceutical Quality Assurance. 2017; 8(3): 153-157. 4. Huda JA, Hameed IH, Hamza LF. Anethum graveolens: Physicochemical properties, medicinal uses, antimicrobial effects, antioxidant effect, antiinflammatory and analgesic effects: A review. International Journal of Pharmaceutical Quality Assurance. 2017; 8(3): 88-91. 5. Altaee, N., Kadhim, M.J., Hameed, I.H. Detection of volatile compounds produced by pseudomonas aeruginosa isolated from UTI patients by gas chromatography-mass spectrometry. International Journal of Toxicological and Pharmacological Research. 2016; 8(6): 462-470. 6. Hussein HM, Hameed IH, Ubaid JM. Analysis of the secondary metabolite products of Ammi majus and evaluation anti-insect activity. International journal of pharmacognosy and phytochemical research. 2016; 8(8): 1192-1189. 7. Hussein HM, Ubaid JM, Hameed IH. Inscticidal activity of methanolic seeds extract of Ricinus communis on adult of callosobruchus maculatus (coleopteran:brauchidae) and analysis of its phytochemical composition. International journal of pharmacognosy and phytochemical research. 2016; 8(8): 1385-1397. 8. Ubaid JM, Hussein HM, Hameed IH. Determination of bioactive chemical composition of Callosobruchus maculutus and investigation of its anti-fungal activity. International journal of pharmcognosy and phytochemical research. 2016; 8(8): 1293-1299. 9. Ibraheam IA, Hussein HM, Hameed IH. Cyclamen persicum: Methanolic Extract Using Gas Chromatography-Mass Spectrometry (GC-MS) Technique. International Journal of Pharmaceutical Quality Assurance. 2017; 8(4); 200-213. 10. Ibraheam IA, Hadi MY, Hameed IH. Analysis of Bioactive Compounds of Methanolic Leaves extract of Mentha pulegium Using Gas Chromatography- Mass Spectrometry (GC-MS) Technique. International Journal of Pharmaceutical Quality Assurance. 2017; 8(4); 174-182. 11. Hadi MY, Hameed IH, Ibraheam IA. Ceratonia siliqua: Characterization, Pharmaceutical Products and Analysis of Bioactive Compounds: A Review. 2017; 10(10): 3585-3589. 12. Hadi MY, Hameed IH, Ibraheam IA. Mentha pulegium: Medicinal uses, Anti-Hepatic, Antibacterial, Antioxidant effect and Analysis of Bioactive Natural Compounds: A Review. Research Journal of Pharmacy and Technology. 2017; 10(10): 3580-3584. 13. Kadhim MJ, Sosa AA, Hameed IH. Evaluation of anti-bacterial activity and bioactive chemical analysis

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