Exploitation of Soil and Rhizosphere Inhabiting Fungal Species for Biologically Active Secondary Metabolites

Abstract

In quest for new bioactive compounds the present study deals with isolation, identification and bioassay screenings of fungi and purification and structural elucidation of pure compounds isolated from fungal strains under study. For production of fungal bioactive secondary metabolites fungal strains were isolated from soil and rhizosphere of Mentha piperita using Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA) media. After preliminary antimicrobial testing, two fungal strains were selected for metabolites production, their bioassays and subsequent structural characterization. The rhizospheric strain was identified as Aspergillus specie while soil isolate was identified to be Penicillium specie. For secondary metabolites production both fungal strains were cultured in Czapec Yeast Broth (CYB) medium at 28 o C for 14 days at 150 rpm. Crude secondary metabolites produced by fungal strains were then extracted and tested for their respective bioassays. In our study fungal metabolites were tested for five different activities i.e. antibacterial, antifungal, cytotoxic, phytotoxic and herbicidal activities. In antibacterial bioassay crude extracts of Aspergillus and Penicillium species were tested against eight pathogenic bacterial strains i.e. Xanthomonas oryzae, Klebsiella pneumonia, Bacillus subtilus, Proteus vulgaris, Staphylococcus aureus, Escherichia coli, Salmonella typhi and Shigella flexeneri. A dose concentration from 1 to 500 µL mL-1 of the crude extracts was tested against pathogenic bacterial strains and zone of inhibition was recorded in millimeters (mm). A dose concentration of 500 µL mL-1 of ethyl acetate extract of Aspergillus sp. inhibited growth of all test bacterial strains but the highest zones of inhibition were recorded against B. subtilus (47.5 mm) and S. flexeneri (45 mm) while lowest zone of inhibition was recorded against X. oryzae (9 mm). At 500 µg/mL of ethyl acetate extract of Penicillium sp growth of P. xxii vulgaris, E. coli, S. aureus, B. subtilus and S. flexeneri was inhibited by forming zones of 34, 28, 24, 23 and 21 mm respectively. Lowest zone of inhibition was recorded against S. typhi (9.5 mm) while considerable inhibition was noted against K. pneumoniae (16 mm) and X. oryzae (13 mm) at 500 µL mL-1 of Penicillium sp. extract. Antifungal activities were tested against six different pathogenic fungal strains i.e. Fusarium solani, Aspergillus flavus, Microsporium canis, Candida albicans, Candida glabrata, and Trichophyton longifusus. Different dose concentrations i.e. from 1 to 1000 µL mL-1 of both fungal strains were prepared and tested against pathogenic fungal species. 1000 µL mL-1 of Aspergillus extract inhibited the growth of M. canis, C. glabrata and A. flavus by 60.5%, 52% and 44.5% respectively. C. albicans and F. solani showed 35 and 38.5% growth inhibition while lowest inhibition was recorded against T. longifusus (27.5%). A dose of 1000 µL mL-1 inhibited the growth of all test pathogenic fungal species, however, comparatively T. longifusus, M. canis and C. glabrata showed highest inhibition i.e. 70%, 63.5% and 52% respectively. In cytotoxicity assay three dose concentrations i.e. 10, 100 and 1000 µL mL-1 of crude extract of both fungal strains were used to test the toxicity of fungal metabolites against A. salina. At µL mL-1 highest mortality percentage for Aspergillus metabolites was recorded as 98.33 while at this concentration highest mortality percentage for Penicillium metabolites was 63.33 after 24 hrs. Crude metabolites of both fungal strains were tested against Lemna minor for phytotoxicity. At a dose of 1000 µL mL-1 , 85% mortality was recorded for Aspergillus extract while 56.66% mortality was recorded for Penicillium extract against the Lemna minor. Extracts of both fungal strains showed strong herbicidal activities against seeds of Silybum marianum L. by completely inhibiting seed germination. xxiii In light of above mentioned activities isolation of compounds was performed using column chromatography and high performance liquid chromatography (HPLC) techniques. Structure elucidation of isolated compounds was performed using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques. From the organic extracts a total of 5 compounds were isolated among which 3 were known compounds while 2 were new compounds. The two new compounds were 3-allyl-6-(hydroxy (phenyl) methyl) piperazine-2, 5-dione and (2R, 4S) -2, 4-dimethyl-4-((E)-2-((3S, 4S)-2, 4, 5-trihydroxy-3-methoxy-4-phenyl-1, 2, 3, 4- tetrahydroquinolin-6-yl) vinyl) cyclohexanone while the three known compounds were (E)-3- ((3S,4R)-8-hydroxy-3,4-dimethyl-1-oxoisochroman-7-yl) acrylic acid, 2-(1, 4-dihydroxybutan-2- yl)-1,3-dihydroxy-6, 8-dimethoxyanthracene-9, 10 (4aH, 9aH)- dione and 5-hydroxy-2- (hydroxymethyl)- 4H-pyran-4-one. This thesis presents a research work on isolation and structure elucidation of secondary metabolites from soil and rhizosphere inhabiting fungi that may have potent biological molecules.