Assessment of Antimicrobial Activity of Endophytic ...

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UGB Journal of Plant Biology and Biotechnology Research Article

Assessment of Antimicrobial Activity of Endophytic Fungi Isolated from Justicia adhatoda L. Soma Barman, Ranjan Ghosh, Nazma Mandal, Surendra K. Gond, Narayan C. Mandal* Mycology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati, Santiniketan-731235, West Bengal, INDIA *Corresponding author: Dr. Narayan C. Mandal, Professor, Mycology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati, Santiniketan731235, West Bengal, INDIA Email: [email protected] Received: August 26, 2016; Accepted: August 27, 2016; Published: August 30, 2016

Abstract Development of resistance in human pathogens against conventional antibiotic necessitates searching of new sources for antibacterial compounds. Eight endophytic fungal strains were isolated from leaves of one important medicinal plant Justicia adhatoda L. collected from Birbhum district of West Bengal. Based on morphological features as well as by 28S rDNA sequence homology two potent endophytic isolates VBAC09 and VBAV19 were identified as Glomerella cingulata and Alternaria alternata respectively. Fungal metabolites from the mycelial homogenate and the culture broth were extracted with ethyl acetate and chloroform. The antimicrobial activities of different fractions were evaluated by disc diffusion method against four Gram positive and four Gram negative bacteria as well as one human pathogenic yeast viz. Candia albicans. Ethyl acetate extract of A. alternata VBAV19 showed very good antimicrobial activities against all of the pathogenic organisms tested. Thin layer chromatographic analyis of ethyl acetate extract revealed the production of two different compounds (Rf values 0.28 and 0.88) by A. alternata VBAV19 with antibacterial potentials.

Keywords: Justicia adhatoda L.; endophytic fungi; Antimicrobial activity

INTRODUCTION Endophytes are the microbes which colonize living, internal tissues of plants without causing any harm to their host (Bacon and White, 2000). Endophytic organisms are present in all vascular plants. The term endophyte was irst proposed by de Bary, 1866. Endophytes refer to bacteria, fungi, actinomycetes and algae which spend their whole life or period of life cycle in the symplast or apoplast region. The most frequently present endophytes are fungi (Stanick et al., 2008) and currently evidenced that all reported endophytes are fungi or bacteria (including actinomycetes). Justicia adhatoda L. (Vasaka) is a primary herb of the ayurvedic system used in treatment of coughs, bronchitis, asthma and symptoms of common cold (Karthikeyan et al., 2009). Inspite of this usefulness of the extracts, they have antimicrobial activity. Vasaka is indigenous to India, where it is found in subHimalayan tract upto an altitude of 1000 m and in Maharashtra especially, in konkan region. Besides India, it is found in Myanmer, Srilanka and Malaya. The plant is not cultivated on commercial scale; it is obtained from garden plants or wild sources. It can be easily propagated by stem cuttings and by seed germination. The plant is obtained in all seasons of the year. It reaches to a height of 2-3 metres. It is also observed that UGB J Plant Biol Biotech - Volume 1 Issue 1 - 2016 ISSN: XXXX - XXXX | www.ugbplantjournal.org © All rights are reserved. Department of Botany, UGB

the plant favourably grows in loamy soil. The leaves have 10-30 cm length and width of 4-10 cm. They are petiolate and exstipulate. The shape is lanceolate. Taste of leaves is bitter and odour is characteristics. It is reported that some medicinal properties of plants due to presence of endophytic fungi living inside the plants (Azevedo et al., 2002). Medicinal plants are known to harbour endophytic fungi that are believed to be associated with the production of phermaceutical products (Strobel, 2002; Zhang et al., 2006). The types of endophytes within the same plant varies with different habitats, different parts of the plant as well as in different seasonal conditions (Fang et al. 2013; Suradkar et al, 2014). In the present endeavor we have tried to isolate and characterize endophytic fungi and to know their antimicrobial potential from Justicia adhatoda collected from two different locations of Birbhum district of West Bengal.

MATERIALS AND METHODS Collection of plant materials Healthy and mature leaves of Justicia adhatoda were randomly collected from the branches at the height of 2.5 m above the ground from the lateritic soil of Santiniketan (site 1), and Basapara (site 2) West Bengal, India. The plant materials were

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ASSESSMENT OF ANTIMICROBIAL ACTIVITY OF ENDOPHYTIC FUNGI

brought to the laboratory in sterilized polythene bags. Fresh plant materials were selected for the isolation of endophytic fungi within two hours of collection. Isolation and puri ication of the endophytes All the samples which were collected were washed thoroughly in running tap water for 10 mins to remove the debris and inally washed with double distilled water to minimize the microbes from the sample surface. The leaves were surface sterilized with 4% sodium hypochlorideand inally washed for three times with sterilized distilled water and cut into 3-4 mm in diameter and 0.5-1 cm in length without midrib. The pieces were placed carefully in petridishes on Malt Extract agar (ME) medium supplemented with streptomycin (150 µg/ml) and incubated for 2-3 days at 28+2°C in a BOD incubator. Tissues were observed after 2-3 days for fungal growth. After emergence of fungal mycelia they were puri ied by streaking and stored in ME slants for further studies. Morphological characterization of the isolated endophytes The endophytic fungi were morphologically characterized according to their microscopic structures and identi ied by their colony morphology, type of hyphae, kinds of reproductive structures by light microscope. To study morphological characteristics of the isolates, well separated mycelia specimens from pure culture of the isolates were taken on a clean glass slide and stained with cotton blue. Excess stain was removed and mounted with a cover glass using lactophenol. The specimens were observed under high power (40×) light microscope (Olympus CH2Oi). Microorganisms used for antimicrobial assay Four Gram positive bacterial strains viz. Bacillus subtilis MTCC121, Listeria monocytogenes MTCC657, Staphylococcus aureus MTCC96 and Staphylococcus epidermidis MTCC2639 as well as four Gram negative bacterial strains viz. Pseudomonas aeruginosa MTCC741, Escherichia coli MTCC1667, Salmonella typhimurium MTCC98 and Pantoea ananetis MTCC2307 were used in antimicrobial assay. All the strains were obtained from Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India and were maintained in nutrient agar medium (HiMedia, Mumbai, India) in slants at 4°C. One human pathogenic yeast Candida albicans MTCC183 was also used in antimicrobial assay was maintained in malt extract (ME) slant at 4°C.

Antimicrobial screening of isolated endophytes To select the endophytes with antimicrobial potentials all of the isolated strains were grown in 300 ml ME and Czapec dox (CD) broth at 28°C for 21 days. After 21 days the broth containing fungal growth were iltered with muslin cloths and centrifuged at 6,000 rpm for 10 min. Antimicrobial activities of cell free supernatants were checked by agar well diffusion method (Ferná ndez-Garayzá bal et al. 1992). 50 µl CFS was added to the wells of agar plates. Zones of inhibition were observed and diameters were measured. Antimicrobial potentials of isolated endophytes were studied against a number of Gram positive and Gram negative pathogenic bacteria. Molecular identi ication of the endophytic fungi Identi ication of two potent representative isolated endophytes VBAC09 and VBAV19 were carried out using www.ugbplantjournal.org

D1/D2 region of large subunit (LSU) of 28S rDNA based molecular technique. Forward and reverse DNA sequencing reactions were carried out with DF and DR primers using BDT v3.1 cycle sequencing kit on ABI 3730xl genetic analyzer. Consensus sequence of D1/D2 region of 28S rDNA gene was generated from forward and reverse sequence data using aligner software. The BLAST analysis was carried out using D1/D2 region of LSU gene sequence with the nrdatabase of NCBI GenBank. Based on maximum identity score irst ten sequences were selected and the phylogenetic tree was constructed using MEGA 4 (Tamura et al., 2007). Organic extraction of culture iltrates and fungal masses of endophytic fungi The fungal masses were crushed by mortar and pastel and extracted at irst by ethyl acetate and then by chloroform using separating funnel. All the extracts were then evaporated to dryness in vacuum rotary evaporator and weighed. The extracts were then dissolved in ethyl acetate and chloroform as they were separated and stored into refrigerator. Study of antimicrobial activity The inhibitory effects of the extracts were tested by discdiffusion method (Bauer et al., 1966). Finally the collected crude extracts were diluted to 0.05 mg/µl for assay. Sterile paper disc (5 mm diameter) was impregnated with the diluted extract and kept under laminar hood for 10 mins. to dryness. The air-dried paper discs were then used to test the activity against the pathogenic bacteria and the fungi. After 24 hrs of incubation zones of inhibition were observed and diameters were measured. Thin layer chromatographic analysis The ethyl acetate fraction of the CFS of the endophytic fungus VBAV19 was analyzed by thin layer chromatography (TLC). The dried fraction was re-dissolved in ethyl acetate at a concentration of 20 mg/ml and 10µl of suspension was spotted on TLC plates (MERCK Silica gel F254). Ethyl acetate and chloroform in the ratio 1꞉1 was used as running solvent. Retention factor (Rf) values for all the bands were calculated under ultra violet light. The bands were then cut and placed on lawn of bacteria on NA plates to check the antibacterial activities. Zones of inhibition were observed after 24 h of incubation. Statistical analysis Each data represented are averages of at least three replicates. Means and standard deviations were calculated using Microsoft Excel 2007 programme.

RESULTS Endophytic fungi were isolated from disease free, healthy leaves of Justicia adhatoda plants (Figure 1). A total of 36 fungal strains were isolated (Figure 2) from 50 leaves segments of which 12 are isolated from site 1 and 24 strains were isolated from site 2. During morphological characterization under light microscope 9 specimens from site 1 were identi ied as Glomerella spp., and other 3 were unidenti ied due to absence of reproductive structures. 21 specimens from site 2 were identi ied as Alternaria spp (Figure 3) and others were unidenti ied. UGB J Plant Biol Biotech 1 (1) 2016 - 5


Figure 1. Plant habit of Justicia adhatoda L.

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Figure 4. Phylogenetic tree of VBAC09 showing its relative position. Related fungal species are denoted by their NCBI accession numbers

Figure 5. Phylogenetic tree of VBAV19 showing its relative position. Related fungal species are denoted by their NCBI accession numberss Figure 2. Emergence of endophytic fungi from leaf segments of Justicia adhatoda L. collected from different locations

Figure 3. Endophytes under light microscope; A. Glomerella sp. and B. Alternaria sp.

Among the 36 different isolates cell free supernatants of two fungal strains viz. VBAC09 from site 1 and VBAV19 from site 2 showed prominent zones of inhibition (Table 1) against all of the Gram positive as well as Gram negative bacterial strains (Figure 6). Among these two strains CFS of VBAV19 was more effective than VBAC09 to produce zones of inhibition against Gram positive and Gram negative bacteria tested (Table 1). Table 1. Zones of inhibition produced by CFS of endophytic fungal strains against pathogenic organisms

Based on the 28S rDNA sequence homology and phylogenetic analysis the two potent endophytes VBAC09 from site 1 was identi ied as Glomerella cingulata and the isolated endophyte VBAV19 from site 2 was identi ied as Alternaria alternata. Consensus sequence of VBAC09 was 571 bp and that of VBAV19 was 594 bp was generated from D1/D2 region of 28S rDNA gene using aligner software. On the basis of antimicrobial potential of the endophytes extracellular CFS as well as intracellular fungal masses of the isolated fungal strains VBAC09 and VBAV19 were extracted with ethyl acetate and chloroform. Ethyl acetate fraction was more effective than chloroform for both of the fungi. Ethyl acetate fraction of VBAV19 was found to be more effective than its chloroform extract (Table 2). The crude ethyl acetate extract of Alternaria alternata VBAV19 showed signi icant antibacterial activity against all eight pathogenic bacteria (Table 2). In L. monocytogenes and Bacillus subtilis intercellular activity was signi icant. The ethyl acetate fraction of extracellular CFS of Alternaria alternata VBAV19 was able to produce zones of inhibition against all of the tested Gram positive and Gram negative bacteria viz. B. subtilis, L. monocytogenes and S. aureus, S. epidermidis, P. ananetis, P. aeruginosa, E. coli, S. typhimurium at a concentration of 0.05 mg/ml (Figure 6). It was also observed that the extract showed maximum zones of inhibition against L. monocytogenes which is a food borne spoilage pathogen (Table 2). Thin layer chromatographic analysis of ethyl acetate fraction of CFS of VBAV19 revealed ive distinct bands (Fig. 7) with Rf values of 0.08 ± 0.032, 0.28 ± 0.024, 0.55 ± 0.019, 0.77 ± 0.027 and 0.88 ± 0.029 under UV light. When the antimicrobial properties of individual bands were studied, band 2 (Rf= 0.28 ± 0.024) and band 5 (Rf= 0.88± 0.029) were able to produce prominent zones of inhibition against all of the Gram positive and Gram negative bacteria.

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Table 2. Antimicrobial activity of different extracts of endophytic fungi isolated from J. adhatoda L.

A- Staphylococcus aureus MTCC96 , B - S. epidermidis MTCC2639, C- Listeria monocytogenes MTCC657, D – Bacillus subtilis MTCC121, E - Pseudomonas aeruginosa MTCC741, F- E. Coli, G - Salmonella typhimurium MTCC98, H - Pantoea ananetis MTCC2307, I - Candida albicans MTCC183

Figure 6. Zones of inhibition produced by different solvent extract of CFS of Glomerella sp. VBAC09 and Alternaria sp VBAV19 against pathogenic bacteria; A. Bacillus subtilis MTCC121, B. Listeria monocytogenes MTCC657, C. Pantoea ananetis MTCC2307, D. Pseudomonas aeruginosa MTCC741

Figure 7. Partial Puri ication of the ethyl acetate crude extract of Alternaria alternata VBAV19 by TLC

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DISCUSSION

CONCLUSION

Plants have long provided mankind with a source of medicinal agents, with natural products once serving as source of all drugs (Balandrin, 1993). Recently, biological controls or the uses of microorganisms or their secretions to prevent diseases offer an attractive alternative or supplement to disease management without the negative impact of chemical control (Petrini, 1991). In most cases these medicinal plants are useful as because of the endophytes residing inside the host plants. Endophytic fungi are the colonizer of healthy plant tissues that get nutrition and shelter from host, and in response produce many functional metabolites. These metabolites have antifeedant activity and provide resistance against various biotic and abiotic stresses, and which may enhance the host itness (Gond et al., 2010). Many reports are there that endophytes in medicinal plants have antimicrobial, antioxidant as well as anticancerous property (Kharwar et al., 2008, Kharwar et al., 2011). Currently, there is demand for a search for new antimicrobial agents because of the development of pathogen resistance to available drugs. There are lots of synthetic drugs and day by day people are generating new synthetic drugs, But these may have adverse effect on the environment. In the present study 36 endophytic fungi were isolated from leaves of J. adhatoda from two different locations of Birbhum district of India. Most of the isolates from site 1 and site 2 were belonging to the genus Glomerella and Alternaria respectively. Suradkar et al. (2014) reported presence of only Pithomyces sp. and Arthrinium sp. as endophytes in Adhatoda vasica where as Shukla and Mishra (2012) reported presence of various species of Aspergillus, Fusarium, Penicillium, Rhizopus, Rhizoctonia, Phoma as endophytes. But till date, there was no report regarding the colonization of Glomerella cingulata and Alternaria alternata as endophytes in leaves of J. adhatoda. Most of the bioactive metabolites from endophytic fungi reported were more effective against Gram-positive bacteria than Gram-negative bacteria and pathogenic fungi (Chareprasert et al., 2006). Both of the isolated endophytes showed inhibitory activity against both Gram-positive and Gram-negative bacteria. Wide spectrum antimicrobial compounds active against Gram-positive and Gram-negative bacteria from endophytic fungi were also reported by several workers (Raviraja et al., 2006 and Gond et al., 2010). Extracellular activity was more than intracellular found in both of the endophytes. Extracellular CFS extract of VBAV19 showed maximum antibacterial activity against Staphylococcus aureus, S. epidermidis, Listeria monocytogenes and in Pseudomonas aeruginosa In L. monocytogenes and Bacillus subtilis intracellular activity was signi icant. Extracellular CFS extract of Glomerella cingulata showed activity against some tested bacteria but did not show any inhibitory effect against the fungus Candida albicans. The intracellular activity was very less compared to VBAV19. As the crude extract showing very encouraging and positive results therefore the puri ied active principle(s) of this metabolite can provide a good alternative natural source for antimicrobial compound(s).

Endophytic fungi Glomerella cingulata VBAC09 and Alternaria alternata VBAV19 isolated from J. adhatoda showed very good antibacterial activities against Gram positive as well as Gram negative pathogenic bacterial strains. It can produce at least two different antibacterial compounds as evidenced from TLC analysis. In addition it also showed anti-candidal activities. Therefore these two endophytes could be very good source of bioactive compounds for development of new drugs.

UGB J Plant Biol Biotech - Volume 1 Issue 1 - 2016 ISSN: XXXX - XXXX | www.ugbplantjournal.org © All rights are reserved. Department of Botany, UGB

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