Outline of Research study TOC o “1-3” h z u Abstract PAGEREF _Toc527161355 h 3Introduction PAGEREF _Toc527161356 h 4Research Question PAGEREF _Toc527161357 h 4Hypothesis PAGEREF _Toc527161358 h 4Objectives of Research PAGEREF _Toc527161359 h 5Literature Review PAGEREF _Toc527161360 h 5Material and Methods PAGEREF _Toc527161361 h 6Collection and identification of samples

Outline of Research study
TOC o “1-3” h z u Abstract PAGEREF _Toc527161355 h 3Introduction PAGEREF _Toc527161356 h 4Research Question PAGEREF _Toc527161357 h 4Hypothesis PAGEREF _Toc527161358 h 4Objectives of Research PAGEREF _Toc527161359 h 5Literature Review PAGEREF _Toc527161360 h 5Material and Methods PAGEREF _Toc527161361 h 6Collection and identification of samples: PAGEREF _Toc527161362 h 6Isolation of Endophytic Fungi from collected plants: PAGEREF _Toc527161363 h 6Characterization: PAGEREF _Toc527161364 h 6Identification of Isolated Fungi by DNA extraction, DNA amplification and phylogenetic analysis: PAGEREF _Toc527161365 h 6Extraction of Secondary Metabolites: PAGEREF _Toc527161366 h 7Activity analysis of Secondary Metabolites against pathogens: PAGEREF _Toc527161367 h 7Time Frame PAGEREF _Toc527161368 h 8References PAGEREF _Toc527161369 h 9

Production of bioactive secondary metabolites with subsequent analysis of their stability from isolated endophytic fungal species of Curcuma zedoaria: An antimicrobial assay
AbstractCurcuma zedoaria, determined due to various beneficial properties. It is being used in herbal medicines and also used as a cure for wounds. In ancient times, it is used for the treatment of cancer. Almost every plant has the particular type of symbiotic relationship with diversified microbiota. In this study Endophytes (microorganism inhabiting in the plant), are in either commensalism or mutualism relationship. These organisms are reported to be the producer of secondary metabolites that shows the antagonistic nature against several group of microorganisms, cancer cells. Some metabolites are also found to be insecticidal in nature and thus reflect ability of endophytes to protect plant from damage caused by insects. With accordance to these findings there is a need to produce these compounds on a large scale and also to find the hidden potential to treat infection or even diseases. Due to lack of in vitro gene expression and less production yields, there are some compounds used by the researcher to either increase the production or increase the release of secondary metabolites in surrounding.

IntroductionCurcuma zedoaria commonly referred to as white turmeric known for its medicinal properties. This genus is classified in the family of ‘Zingiberaceae’ (SULISTIYANI et al., 2014). It is native or indigenous to Bangladesh, Srilanka, India, but due to the tremendous benefits from the product of these pants; it is now widely cultivated in China, Japan, Indonesia and in some regions of Pakistan. The extract from this plant contain bioactive compounds, might be originating from plant or the living entities colonizing or inhabiting the tissues of plants as intracellular or intercellular. They are termed as Endophytic microorganisms which can be fungi or bacteria (Muhrani et al., 2016). Most of the studies reported endophytic fungi capability to produce bioactive compounds in the form of secondary metabolites that are of great significance; for example Taxol is the first anticancer drug by endophytic fungi Taxomyces andreanae reported in 2007 (SULISTIYANI et al., 2014; Tan et al., 2018). The activity of producing beneficial compounds is not only specific for Curcuma sp., but there are some studies suggest the presence of endophytes in variety of plants (Garcia et al., 2012; Katoch et al., 2017; Gonález-Menédez et al., 2018; Tan et al., 2018). Moreover, diversified bioactive compounds located in different regions of plants are influenced by the different ecological niches and plant age (SULISTIYANI et al., 2014). Although the compounds produced by these plants are less toxic and effective in various aspects like antagonistic effect on pathogens, but they are produced in very minute quantities. Their production can be increased on a large scale by incorporating resins in fermentation media. This will not only influx the production but also protects the newly synthesized compounds from biotransformation or post degradation (Gonález-Menédez et al., 2018). Mostly, Endophytic fungi belongs to the phylum Ascomycota, Zygomycota classified among these orders are Xylariales, Pleosporales, and the most prominent genera found in previous studies was Fusarium sp., Colletotrichum sp., Alternaria sp., Aspergillus sp. and Penicillium sp.(Garcia et al., 2012; Katoch et al., 2017; Gonález-Menédez et al., 2018 & Tan et al., 2018)
Research QuestionHow activity of secondary metabolites would remain the same after 5 weeks?
HypothesisIf Secondary Metabolites dissolved in Dimethyl sulphoxide (DMSO) and kept in 4°C for 5 weeks then it will give somewhat similar zone of inhibition in every week.

Objectives of ResearchTo detect the bioactive potentialities of extracted secondary metabolites of fungus isolated from different parts of Curcuma zedoaria against human pathogens.

To identify active producers of secondary metabolites on genomic level.

To determine the activity of extracted compounds for 5 weeks.

Literature ReviewWheat leaf rust disease in different plant caused by fungal species; suppressed by the methanol extracted bioactive substances from the endophytic species of plant C. zedoaria (Han et al., 2018).

Alpha glucosidase inhibition by the activity of actinomycetes isolated from Curcuma aeruginosa (SULISTIYANI et al., 2014).

Extracted Compounds of endophytic Penicillium species isolated from C. zedoaria plant showed antibacterial activity on S. aureus (Muharni et al., 2016).

Extracts isolated from endophytes Fusarium sp. and Cladosporium sp. of Dysosma versipellis plant shows antifungal activity against Aspergillus fumigatus and Candida tropicalis respectively and found to be a potent antagonist against S. aureus, E. coli, B. subtilis (Tan et al., 2018).

Anticancer activity in Secondary metabolites from Aspergillus sp. and Fusarium sp. against cancer cell lines HCT-116, PC-3, A549, and MCF-7 (Katoch et al., 2017)
PTox used in the formation of anticancer drugs are produced by Fusarium sp. isolated and cultivated from D. versipellis (Tan et al., 2018).

Inhibition of phytopathogens, Magnegorthe grisea, Colletotrichum acutatum and human pathogens Aspergillus fumigatus, Candida albicans by the crude extract obtained from the endophytic species of those plants isolated from arid region of Andalusia (Gonález-Menédez et al., 2018).

Nothapodytes foetida inhabited Endophytic fungi Aspergillus nidulans, synthesized nanoparticles using its secondary metabolites in the presence of precursor molecules (Vijayanandan et al., 2018)
Material and MethodsCollection and identification of samples:White turmeric (C. zedoaria) whole plant will be collected from Centre for plant conservation, University of Karachi. Botanist will verify the Plant species according to its physical appearance.

Isolation of Endophytic Fungi from collected plants:Leaves flowers roots and stem will be excised or separated from the original plant and with distilled and deionized water soil particles or dust will be removed. Washed off parts will be disinfected sequentially with 95% Ethanol, 25 % Sodium Hypochlorite and again 95% Ethanol. From every tissue, a 5 to 6 mm piece will be cut by the help of sterile scalpel. Each piece will be transferred to Potato dextrose agar plates with streptomycin and oxytetracyclin antibiotics in a concentration of 50 (µg/ml) incorporated in to the media to only favors the growth of Endophytic Fungi (González-Menéndez et al., 2018). Petri plates will be wrapped with Para film or scotch tape and incubated at 25±2 °C for a week. Plates will be checked for the growth daily.

Characterization:Mycelical growth obtained will be characterized according to morphological and cultural characteristics (SULISTIYANI et al., 2014).

Identification of Isolated Fungi by DNA extraction, DNA amplification and phylogenetic analysis:Isolated Aerial mycelia or hyphae will be crushed into fine powder followed by the addition of extraction buffer, vigorous mixing by the help of vortex, incubation at 65°C, and chloroform addition. It will give rise to the formation of aqueous layer containing DNA. The DNA will be precipitated by the help of Ethanol and Sodium acetate. Precipitated DNA will be separated by centrifugation at 8000 rpm. The pellets will be subjected to air dry and dissolved in 20 µl of DNAse free water (Katoch et al., 2017).

DNA fragments will be amplified by using primers of the sequences ITS1-5.8S-ITS2-28S, PCR Mix (QiaGen ™), Thermo cycler (Bio-Rad). Amplified products will be sequenced using Big Dye Terminator cycling sequence kit. Sequences will be compared with GenBank Databases of NCBI by operating BLAST application (González-Menéndez et al., 2017)
Phylogenetic tree will be generated by the alignment of downloaded sequences of nearest neighbors in MEGA4 software. The maximum composite likelihood method will be use and to access the robustness of tree, the bootstrap analysis with tree 1000 replicates will be used (Katoch et al., 2017).

Extraction of Secondary Metabolites:5mm plug of fungal growth obtained from every tissue will be inoculated into freshly prepared Potato Dextrose Broth (PDB) having same antibiotics as in PDA. All the inoculated media will be incubated at 25±2°C for at least 10 to 14 days so the secondary metabolites will be released in liquid media or broth. The media will be filtered out and mycelial biomass will be discarded. Filtered fermentation broth will be mixed with the equal volume of methanol and ethyl acetate and bioactive secondary metabolites will be separated from solvent by separating funnel and rotator vacuum evaporator at 40°C. Bioactive secondary metabolites will be dissolved in DMSO and kept at 4°C for further analytical procedures (González-Menéndez et al., 2014).

Activity analysis of Secondary Metabolites against pathogens:Antibacterial and antifungal activity will be analyzed by Agar Disc Diffusion Method (Tan et al., 2018). Activity will be checked against, McFarland adjusted stock cultures of Bacillus subtilis, Escherichia coli, Salmonella typhiricum, Shigella dysentrae, Klebsiella pneumonia, Staphylococcus aureus and Candida albicans obtained from ATCC. 20 µl of bacterial culture and fungal culture will be spread on Nutrient agar plates containing fluconazole and PDA media plates containing ampicillin respectively. Bioassay will be performed using 100 µg sterile disc impregnated with 20 µl test material (10mg/ml) obtained from endophytic fungi. Streptomycin and amphotericin B commercially available 100µg/ disc will be used as positive controls for bacteria and fungi respectively. The analysis will be performed in triplicates to neglect any false positive results (Katoch et al., 2017).

Above experiments was repeated periodically after every 5 days till 5 weeks in order to detect the stability of extracted biomolecules.

Time FrameResearch activity Duration
Sample collection, Gathering Resources for Research, Grants and permission for Instruments calibration and use 2 months
Research work Isolation 2 months
Characterization 2 months
Bioassay 3 months
Assembling of data using ANOVA software 1 month
Research Findings, outcome, future perspective, conclusions, thesis writing and submission 8 months
Total months 18 months

ReferencesGonzález-Menéndez, V., Crespo, G., de Pedro, N., Diaz, C., Martín, J., Serrano, R., … & Vicente, F. (2018). Fungal endophytes from arid areas of Andalusia: high potential sources for antifungal and antitumoral agents. Scientific reports, 8(1), 9729.

Gonzalez-Menendez, V., Martin, J., Siles, J. A., Gonzalez-Tejero, M. R., Reyes, F., Platas, G., … & Genilloud, O. (2017). Biodiversity and chemotaxonomy of Preussia isolates from the Iberian Peninsula. Mycological Progress, 16(7), 713-728.

González-Menéndez, V., Asensio, F., Moreno, C., de Pedro, N., Monteiro, M. C., de la Cruz, M., … & Tormo, J. R. (2014). Assessing the effects of adsorptive polymeric resin additions on fungal secondary metabolite chemical diversity. Mycology, 5(3), 179-191
Garcia, A., Rhoden, S. A., Bernardi-Wenzel, J., Orlandelli, R. C., Azevedo, J. L., & Pamphile, J. A. (2012). Antimicrobial Activity of Crude Extracts of Endophytic Fungi Isolated from Medicinal Plant Sapindus saponaria L. Journal of applied pharmaceutical science, 2(10), 35.Han, J. W., Shim, S. H., Jang, K. S., Choi, Y. H., Dang, Q. L., Kim, H., & Choi, G. J. (2018). In vivo assessment of plant extracts for control of plant diseases: A sesquiterpene ketolactone isolated from Curcuma zedoaria suppresses wheat leaf rust. Journal of Environmental Science and Health, Part B, 53(2), 135-140.

Katoch, M., Phull, S., Vaid, S., & Singh, S. (2017). Diversity, Phylogeny, anticancer and antimicrobial potential of fungal endophytes associated with Monarda citriodora L. BMC microbiology, 17(1), 44.
Muharni, M., Fitrya, F., Purwaningrum, W., & Nugraha, A. Y. (2016). Secondary Metabolite from Endophytic Fungi Aspergillus Sp. The Leave of Kunyit Putih (Curcuma Zedoaria (Berg) Roscoe. Molekul, 11(1), 125-134.

SULISTIYANI, T. R., Lisdiyanti, P., & Lestari, Y. (2014). Population and diversity of endophytic bacteria associated with medicinal plant Curcuma zedoaria. Microbiology Indonesia, 8(2), 4.Tan, X., Zhou, Y., Zhou, X., Xia, X., Wei, Y., He, L., … & Yu, L. (2018). Diversity and bioactive potential of culturable fungal endophytes of Dysosma versipellis; a rare medicinal plant endemic to China. Scientific reports, 8(1), 5929.

Vijayanandan, A. S., & Balakrishnan, R. M. (2018). Biosynthesis of cobalt oxide nanoparticles using endophytic fungus Aspergillus nidulans. Journal of environmental management, 218, 442-450.