Bangladesh Journal of Pharmacology Volume: 15; Number 4; Year 2020 Cite this article as: Jalil MTM, Ibrahim D. Antimicrobial activity of volatile compounds produced by endo- phytic fungi associated with Ocimum sanctum. Bangladesh J Pharmacol. 2020; 15: 100-12. Antimicrobial activity of volatile compounds produced by endophytic fungi associated with Ocimum sanctum Sir, Endophytic fungi are known as biosynthesizers inside their host plants (Owen and Hundley, 2004). Many endophytes capable of secreting bioactive compounds that may be used against pathogenic microorganisms (Daoud et al., 2013). The endophytic fungi Chaetomium cupreum (isolated from Mussaenda luteola; Shylaja et al., 2018), Cryptosporiopsis ericae (isolated from Coptis chinensis; Zhang et al., 2018), Phoma sp. (isolated from Fucus serratus; Hussain et al., 2014), Nigrospora sphaerica URM-6060 (isolated from Indigofera suffruticosa; Santos et al., 2015), Trichoderma virens and Aspergillus terreus (isolated from Cyperaceae sp.; Ratnaweera et al., 2018). The essential oil of Ocimum sanctum showed significant antimicrobial agent (Sharma et al., (2014). The current study focused on the evaluation of antimicrobial activity of bioactive compounds produced by endo- phytic fungi isolated from the medicinal plant, Ocimum sanctum. To test the antimicrobial production, plate to plate method was carried out (Stinson et al., 2003). The ino- culum suspension of the test microorganism was spread on the agar media respectively. Then, the 14- days-old endophytic isolate cultures were physically attached to the agar plates seeded with test micro- organisms. The two plates were sealed using two layers of Parafilm® and kept at 4ºC for 7 days to allow the complete fumigation process of the volatile compounds. Then, the plates were transferred and incubated at 30ºC for 48 to 96 days for test fungi and at 37ºC for 24 hours for bacteria and yeasts. For fungi, the diameter of the test fungal culture was measured as an indication of the growth rate, whereas the colony-forming units were counted for yeast and bacterial cultures. Untreated test microorganisms were used as a control. Besides, the mycelia of the test fungi that showed the positive result were transferred to fresh potato dextrose agar media whereas the single colony of the test bacteria and yeast A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2020; 15: 110-112 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v15i4.48496 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor Table I Antimicrobial activity of volatile compounds produced by endophytic fungi against pathogenic microorganisms Test microbes %Inhibition IBRL OS-27 IBRL OS-64 IBRL OS-94 IBRL OS-98 Bacteria (Gram positive) MRSA ATCC 33591 94.3 ± 1.3 82.5 ± 2.0 87.3 ± 1.2 - S. aureus 94.2 ± 1.2 84.3 ± 3.1 89.4 ± 0.9 - S. mutans 88.3 ± 2.7 - 95.2 ± 1.1 - Bacteria (Gram negative) K. pneumoniae ATCC 13883 - - - 58.5 ± 2.0 S. typhimurium - - - 80.4 ± 0.8 Y. enterocolitica - 70.9 ± 3.1 66.3 ± 2.2 - Yeast C. albicans IBRL 26.2 ± 2.1 - - - C. utilis IBRL - - 64.4 ± 4.1 44.2 ± 3.5 Fungi T. rubrum IBRL SA1 - - 37.6 ±2.6 - M. fulvum IBRL SD3 - - 45.7 ± 3.6 - were streaked onto fresh nutrient agar and Sabouraud dextrose agar, respectively to maintain their viability. Table I shows only four (IBRL OS-27, IBRL OS-64, IBRL OS-94, and IBRL OS-98) out of 148 endophytic fungal isolates (volatile in nature) from O. sanctum exhibited significant antimicrobial activity against several test microorganisms. The highest inhibition percentage was observed on Gram positive bacteria for all the endophytic fungal isolate with the inhibition value ranged from 82 to 95%, except for IBRL OS-98. As for Gram negative bacteria, only isolate IBRL OS-98 was able to inhibit both Klebsiella pneumoniae ATCC 13883 and Salmonella typhimurium with the inhibition of 58.5 and 80.4%, respectively. However, isolate IBRL OS-64 and IBRL OS-94 were only able to inhibit Yersinia enterocolitica with the inhibition value of 70.9 and 66.3%, respectively. For yeast, isolates IBRL OS-94 and IBRL OS-98 were able to inhibit Candida utilis IBRL compared to other isolates with a moderate inhibition of 64.4 and 44.2%, respectively. Besides that, the isolate IBRL OS-94 was observed to have the capability to inhibit Trichophyton rubrum IBRL SA1 and Microsporum fulvum IBRL SD3 with an inhibition value of 37.6 and 45.7%, respectively. The results indicated that most of the fungal isolates exhibited better inhibitory activity towards Gram positive bacteria. It is noteworthy to observe that the Gram negative bacteria, Salmonella typhimurium was not susceptible to both of the fungal isolates, IBRL OS-27 and IBRL OS-98. The finding was in contrast to the previous data of disc diffusion assay (data not shown) whereby both isolates showed a good inhibitory effect on Gram negative bacteria. This may be explained by the different constituent of volatile compounds with a different mode of action and most probably the compounds were targeted regardless of the cell wall of bacteria. Preliminary identification showed IBRL OS-27 might be categorized in the genus of Trichoderma spp. This type of species is a plant pathogen such as T. harzianum that could be characterized as a green mold that grows aggressively with white mycelium, which rapidly infected the host and might cause a soft decay (Sokovic and Van-Griensven, 2006). Fungal isolate IBRL OS-94 was morphologically characterized and identified as Muscodor sp. The isolate showed a vast volatile antimicrobial activity since it can inhibit bacteria, yeast, and fungi. It is noteworthy that the fungal isolate was observed to produce volatile bioactive compounds that able to kill Microsporum fulvum IBRL SD3 since it did not survive in the viability test. Figure 1 illustrates a significant growth reduction of M. fulvum IBRL SD3 (in diameter) fumigated with the fungal isolate IBRL OS-94 relative to growth control. Based on observation, the mycelial production of the test fungus (Figure 1A) was less dense compared to control (Figure 1B). Muscodor spp. is one of a well-known fungus that able to produce volatile antimicrobial compounds. The antimicrobial activity of volatile organic compounds produced by Muscodor crispans against Pythium insidiosum was reported (Krajaejun et al., 2012) They revealed that volatile organic compounds from M. crispans (B23) able to inhibit and thus killed P. insidiosum in vitro. Likewise, B23 also was observed to reduce the growth of all P. insidiosum isolates tested even in low concentrations and the inhibition rate was dose-dependent. According to Strobel (2006), ketones, lipids, esters, acids, and alcohols are major components of antimicrobial activity of volatile organic compound found in Muscodor. The authors are grateful to Universiti Sains Malaysia for awarding the RUI research grant scheme (AC: 1001/ PBIOLOGI/811326) to support this study. Mohd Taufiq Mat Jalil and Darah Ibrahim Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia. Corresponding author: email: taufiqjalil28@gmail.com References Daoud H, Sadrati N, Zerroug A, Dahamna S, Bouharati S. Screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated from wheat (Tricum durum). J Plant Protect Res. 2013; 53: 128-36. Hussain H, Kock I, Al-Harrasi A, Al-Rawahi A, Abbas G, Green IR, Shah A, Badshah A, Saleem M, Draeger S, Schulz B, Khrohn K. Antimicrobial chemical constituents from endophytic fungus Phoma sp. Asian Pac J Trop Med. 2014; 699-702. Krajaejun T, Lowhnoo T, Yingyong W, Rujirawat T, Fucharoen S, Strobel GA. In vitro antimicrobial activity of volatile organic compounds from Muscodor crispans against the pathogenic oomycete Pythium insidiosum. SE Asian J Trop Med Public Health. 2012; 43: 1474-83. Owen NL, Hundley N. 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