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Antibacterial Ability of Endophytic Bacteria Isolated from Kemenyan (Styrax benzoin L.)

Kemenyan is one of plant commodities in North Sumatera that has been used as medicinal plants to treat infection and illness. The aim of this study was to isolate endophytic bacteria from kemenyan (Styrax benzoin) and evaluate their antibacterial properties against pathogenic bacteria. Sixteen isolates were found from leaf, bark and root samples of kemenyan. Two isolates, RS01 and RS13 showed considerable antibacterial activities against Staphyloccocus aureus, Escherichia coli and Streptococcus mutans.


Introduction
Kemenyan is one of native commodity plants in North Sumatera and Malaysia that produce benzoin gum with high economical and medical value. In North Sumatera, two species of Kemenyan can be found namely kemenyan Toba/ Styrax sumatrana J.J.SM and kemenyan durame/ Styrax benzoin Dryand. [1]. Both species have been used in traditional remedies as topical medicine to treat various illness. Extensive studies have reported some of its biological properties. Benzoin gum produced from the stem, contained flavonoid and tannins possessing antibacterial properties [2,3].
Ethanolic extract from its leaves also possessed antibacterial properties against skin pathogens, Propionibacterium acne and Staphylococcus epidermidis [4]. Recent information of species distribution have showed that species is currently facing serious exploitation. The use of kemenyan is quite simple in many traditional practices. Continuous harvesting upon certain plant parts as traditional medicine may hinder plants to grow sustainably and may lead to species decline in the future. One of alternative ways in utilizing kemenyan as biological remedies without harvesting most of its parts, is by exploring and exploiting microbial endophytes living within. Microbial endophytes are community of microorganisms that inhabit internal environment of a healthy plant through various entry sites [5].
Endophytes are known to produce extracellular enzymes and metabolites to cope within host environment [6,7]. Endophytes are also known to synthesize similar compounds with their hosts that become key feature to utilize kemenyan in a more sustainable way [8]. Information regarding antibacterial properties from endophytic bacteria residing within kemenyan is still less reported. In this study, we reported that vegetative plant parts of kemenyan are known to harbor microbial endophytes, especially from bacteria group and antibacterial properties are also evaluated.

Isolation of endophytic bacteria from kemenyan
Endophytic bacteria were isolated from leaf, bark and root of kemenyan, sampled from Parbuju village, North Tapanuli Regency, North Sumatera Province. Samples from healthy plant were collected randomly. Samples were immediately rinsed with water and stored in cold temperature prior laboratory test. In laboratory, plant parts were again rinsed with water and then cut into 1-3 cm pieces. Sample pieces of stems and roots were surface sterilized by soaking into solutions: 70% ethanol for 1 min, 5.25% NaOCl for 5 min, 70% ethanol for 30 secs. Sample pieces of leaves were surface sterilized using 96% ethanol and 5.25% NaOCl in the same manner. Sample pieces were rinsed with sterile distilled water prior plating on top of Nutrient Agar (NA) medium supplemented with Nystatin (0.01% b/v). Plates were incubated in ambient temperature for 7 days. Growing bacterial colonies from sample pieces were recovered and stored in stock medium.

Morphological and biochemical characterization of endophytic bacteria
Bacterial isolates were morphologically characterized by observing their colony shape, color, elevation and margin using standard observation. Gram staining was performed to observe cellular shape, arrangement and gram reactions. Biochemical tests was performed to observe physiological features such as: starch hydrolisis, gelatin hydrolisis, citrate utilization, sugar utilization, hydrogen sulfide production, motility and catalase test.

Antagonistic assay of endophytic bacteria
Antagonistic assay was performed against three pathogenic bacteria: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Streptococcus mutans ATCC 251715 using disk diffusion assay [9]. Discs impregnated with 10 µL endophytic bacteria (OD 600 ≈10 8 CFU/mL) were placed on top of pathogenic bacteria lawns grown on NA medium. Assay was done in duplicate. Clear zones around discs showed antibacterial activity and diameter of zones were measured.

Characterization of Endophytic Bacteria from Kemenyan
Sixteen bacterial isolates were found in this study: 5 isolates from leaf, 6 isolates from bark and 5 isolates from root. Each plant parts used as isolation source showed no significant differences in terms of number of bacterial isolates which may indicate that plant parts of kemenyan are evenly harbored by endophytic bacteria without any obvious limiting factor exposed by different parts. From morphological and biochemical characters, it may be concluded that all isolates were different eachothers (Table 1).
Reports on finding endophytic bacteria within members of Styracaceae are still limited. Eight bacterial isolates are reported to harbor vegetative parts of S. officinalis from Iran [10]. Evidence showed that endophytic colonization occurred in leaf part of S. camporum from Brazil, indicating the assumption of metabolite co-production from endophytic bacteria in secretory glands of plant [11]. Interesting finding that most bacteria colonizing kemenyan were gram positive bacteria. Secondary metabolites from kemenyan which is mostly benzoin may affect the presence of certain group or even species of bacteria.   Most morphological characteristics of isolated bacteria were dominated by irregular form, undulate and entire margin, and cream-colored colonies. Most isolated endophytic bacteria (14 isolates) were categorized as gram positive in which 3 isolates of roots, 6 isolates of barks, 5 isolates of leaves while 2 isolates were gram negative, isolated from roots. The dominance of gram positive group found in this study is assumed to show the adaptive ability of the endophytic bacteria to withstand towards secondary metabolite produced from S.benzoin.
Sumatran benzoin tree is known to contain secondary metabolites, i.e. benzoic acid, cinnamic acid, p-coumaryl cinnamate, p-coumaryl benzoic and isovanillin [12][13][14]. The adaptive properties of gram positive endophytic bacteria varies greatly within host cells (plant tissues/organs), starting from the synthesize of pigments, spores and secondary metabolites. In addition, gram positive bacteria may secrete various compositions of secondary metabolites. In other study, 16 isolates of gram positive endophytic bacteria were successfully isolated from roots, stems and leaves of five medicinal plants namely Vinca rosea L., Curcuma longa L., Eucalyptus globules Dehnh, Pongamia glabra Vent, and Musa Paradiasiaca L. The study showed that isolated bacteria produced secondary metabolites such as enzymes, antibacterial, antifungal and antioxidative compounds [15].

Antagonistic Assay of Endophytic Bacteria from Kemenyan
Most bacterial isolates showed antagonisms against tested pathogens: Staphylococcus aureus, Escherichia coli and Streptococcus mutans with varying abilities ( Table 2). Most bacterial isolates were able to inhibit three pathogenic bacteria as shown from inhibition zones measuring from 7.00 to 15.00 mm. Two selected isolates namely RS01 and RS13 were chosen as potential isolates and were subjected to the next step. Both isolates showed the best inhibition against S.aureus with diameter of inhibition zones reaching 15.00 and 13.00 mm for RS01 and RS13, respectively. Endophytic bacteria isolated from Raru (Cotylebium melanoxylon) and tea (Camellia sinensis) exhibit stronger inhibition towards S. aureus than other pathogenic bacteria tested [16,17]. The gram positive bacteria, Staphylococcus aureus is more sensitive against secondary metabolites produced by the endophytic bacteria. The isolate, RS13 displayed different antagonistic activities to the three pathogenic bacteria. Similar metabolites are assumed to be synthesized by endophytic bacteria residing within benzoin tree [18]. Antibacterial activity of benzoin tree compounds is already reported [19]. The metabolites are manifested as adaptive properties of endophytic bacteria to reside within The isolate, RS01 showed prominent antagonistic activities against S. aureus ATCC 25923 and E. coli ATCC 25922. The isolated endophytic bacterial strain was originated from root samples. Preliminary assumptions are that endophytic bacteria colonizing the roots of benzoin tree tissue synthesized higher variety of antibacterial secondary metabolites than the barks and leaves. Endophytic bacteria of benzoin tree may also provide protection against environmental stress and pathogenic bacteria to thrive within root tissues. The interaction between endophytic bacteria and plant rhizosphere is known to be initiated through specific chemical compounds secreted by both symbionts [20].

Conclusion
Sixteen isolates were found from leaf, bark and root samples of Kemenyan. Two isolates, RS01 and RS13 showed considerable antibacterial properties against Staphyloccocus aureus, Escherichia coli and Streptococcus mutans.