Evaluation of Antibacterial activity of Endophytic Fungi Isolated from Medicinal plants of Mid-Himalaya

 

Amita Kumari*, Sonika Mehta, Suman Lata, Ankita Singh, Deepika

School of Biological and Environmental Sciences, Faculty of Basic Sciences, Shoolini University, Solan (HP), India

 

ABSTRACT:

The present study is based on the isolation and identification of endophytic fungi from common medicinal plants of mid-Himalaya and the screening of selective isolated endophytic fungi for their antimicrobial activity against pathogenic bacteria (Escherichia coli and Staphylococcus aureus). Five species of endophytic fungi were identified from the selected plants. Among them ethanolic extract of three endophytic fungi (Alternaria sp., Fusarium sp. and Aspergillus sp.) were selected for the screening of antimicrobial activity. Both the tested bacterial pathogens were found susceptible to the fungal extracts. Results showed significant increase in the inhibition zone diameter with increase in the concentration of fungal extract (100-400 µg/ml). Against E. coli, Alternaria sp. and Fusarium sp. were found to be more effective than Aspergillus sp., whereas against S. aureus, Alternaria sp. and Fusarium sp. showed almost similar activity. The Aspergillus sp. was observed with significant activity but lesser than the other two fungal extracts. The study concludes the potential antimicrobial activity of the endophytic fungi against E. coli and S. aureus which could be due to bioactive secondary metabolites. Further study required the isolation of novel bioactive compounds for use in medicines. 

 

 

 

INTRODUCTION:

Medicinal plants are known for their medicinal values since the ancient times. The medicinal properties in plants are due to some chemical substances, called secondary metabolites that when enter in the human body, produce a definite function. Researchers have now started to isolate these chemical substances from the entophytic fungi residing in the tissues of medicinal plants because, both medicinal plants and their endophytes are important source of discovery of natural products. Endophytic fungi are basically the group of fungi that colonize living internal tissues of plants without causing any immediate or harm effect^[1] except when the host is under stress conditions.

 

 

 

The benefit of symbiotic relationship for the endophyte is that the host plant is able to supply the necessary nutrients and compounds required for the endophyte to complete its life cycle and unlike the host plant, many endophytes are able to survive under quite extreme and inhospitable condition. They play an essential role to provide protection to their host against attack by other pathogen and environmental condition. They can co-evolve with plant host and possess species-specific interaction.

 

Approximately, one million species of entophytic fungi residing in plants^[2]. Over the last decade, numerous studies have been done on fungal endophytes of medicinal plants^[3-10]. In addition, endophytes have also been investigated in search for new secondary metabolites^[11-15]. 

 

A verity of relationship exists in fungal endophytes and their host plant ranging from mutualistic or symbiotic to antagonistic or slightly pathogenic^[16]. Because of their contribution to the host plant, endophyte may produce a large amount of substances having potential use to modern medicine, agriculture and industries as anti-cancer compounds^[3,17] and antimicrobial drugs etc. Therefore, present study is based on the isolation and identification of endophytic fungi from some common medicinal plants and the screening of selective endophytic fungi for their antimicrobial activity. The investigations of the antimicrobial activity of natural products have opened new ways for drug development in the control of antibiotic resistant pathogens. Therefore, initial objectives of this work are to isolate and identify the endophytic fungi from some common medicinal plants viz., Silibium marianum (Asteraceae), Mentha officinalis (Lamiaceae), Cinnamomum camphora (Lauraceae), Aegle marmelos (Rutaceae) Citrus limon (Rutaceae), Withania somnifera (Solanaceae) and Vitex negundo (Lanimaceae).

 

METHODOLOGY:

Site of sample collection:                                               

Medicinal plant materials were collected from the mid-Himalaya (from the vicinity of Shoolini University, Dist. Solan, Himachal Pradesh) in the month of February, 2017. Leaves of selected medicinal plants except W. somnifera and C. camphora were collected during February-March, 2017. For C. camphora leaf and stem were collected whereas for W. somnifera only stem was selected for endophytic fungi isolation. Leaf/ stem samples from selected plants were randomly cut off with an ethanol-disinfected sickle and placed separately in sterile polythene bags and stored in an icebox. Chilled samples (4.5º) were further used to isolate endophytic fungi within 48 h of collection.

 

Isolation of endophytic fungi:

Collected samples (leaves and stem) of selected medicinal plants were thoroughly washed with running tap water and then air dried. The samples were again washed with autoclaved distilled water for 2-3 times. After that surface sterilization was done by submerging collecting leaves and stem in 0.1mg of mercuric chloride solution for 5 minutes. Again plant samples were washed with autoclaved distilled water 2-3 times. After sterilization each leaf were randomly cut into small equal discs and stem was cut into small pieces. The discs and small pieces of leaves and stem were treated with 70% ethanol for a few seconds and then dried with blotting paper. Then dried discs and stem pieces were placed on agar media supplemented with kanamycin (100 mg/l) to suppress the bacterial growth. All plates were placed in BOD at 28º until the fungal growth appeared. The plates were observed once a day for the growth of endophytic fungi.

 

Pure culturing:

The plant discs were observed with in few days for the growth of endophytic fungi. Hyphal tips growing out the plated discs were immediately transferred into potato dextrose agar for making pure culturing. The fungal isolates were identified based on their morphological characters.

 

Identification of endophytic fungi:   

For the identification of endophytic fungi, small thin hyphae of unknown endophytic fungi were isolated through forceps from fungal petriplate (pure culture) and kept on glass slide. The thin hyphae of fungus treated with methylene blue stain. The fungal hyphae or mycelium structure then observed under the microscope in 10X and then 40X. Morphological features of fungus were recorded and photograph were taken using camera lucida. Further identification of fungi was done on the basis of morphological structure using standard identification manuals and previously reported literature ^[18,19].

 

Mass cultivation of endophytic fungi:

Mass cultivation of selected endophytic fungi was done by following the method of previous study on fungal endophytes^[20]. Fungal endophytes were mass cultivated on PDA broth by placing agar blocks of actively growing pure culture (3mm in diameter) in 250 ml Erlenmeyer flask containing 100 ml of the medium. The flasks were incubated at room temperature for 3 weeks with periodical shaking at 150 rpm. After the incubation period, the cultures were taken out and filtered through sterile cheesecloth to remove the mycelial mats. Mycelia mats were dried and used for antimicrobial activity.

 

Evaluation of antimicrobial activity of endophytic fungi:

Extraction of metabolites from endophytic fungi:

Ethanol solvent systems was used for metabolites extraction. Three species of fungi isolated from M. officinalis, W. somnifera and S. merianum were extracted with ethanol. The solvent was taken in a separating funnel and shaken vigorously with dried mycelia mats.  The solution was then allowed to stand, the cell mass got separated and the solvent so obtained, was collected. Solvent was evaporated on hot water bath to yield the crude extract^[21].              

 

Selection of test organisms:

Two most common human pathogens (Escherichia coli, Staphylococcus aureus) were used to evaluate the antimicrobial activity of endophytic crude extracts. Both test pathogens were obtained from Department of Microbiology, Shoolini University, Solan. The selected microbial cultures were maintained using sub culturing techniques. Nutrient agar slants were used for bacterial culture maintenance. Bacterial cultures after 24 hours’ incubation period at 37º, were kept in refrigerator.

 

Evaluation of antimicrobial activity of endophytic fungi:

Antimicrobial activity against selected gram negative and positive bacteria was evaluated by following the method of Wang^[22]. The crude extract was dissolved in dimethyl sulphoxide (DMSO) for antimicrobial bioassay. 1 ml of DMSO was taken and 20 mg weight of crude was dissolved for stock preparation. 5-20 µl of fungal extract containing 100-400 µg/ml was taken from stock solution on sterilized whatman filter paper discs. Total five discs were placed on nutrient broth media on a petri plate. One disc has Cephalosporin of 20 µl (5mg/ml) was used as positive control.

 

The magnitude of antimicrobial activity was assessed by the diameter of inhibition zones relative to those of positive and negative controls. The zone of inhibition was measured and compared with the control. Three replicates were maintained in each case. The plates were incubated at 37° for 24 h. After incubation the diameter of the clear zone was measured, the averages were calculated.

 

Data Analysis:

Data analysis was done using PRISM software.

 

RESULTS:

Isolation and identification of endophytic fungi:

Endophytic fungi was isolated from the medicinal plant leaves (sterilized). It took 7-15 days of time period for the growth of fungi from the discs of sterilized leaves. For checking whether it is endophytic or epiphytic fungi the procedure was repeated 3 times. Results of pure culturing are shown in Fig. 1. For the identification of endophytic fungi the first attempt involved comparing their features as exhibited in culture with those of known species of fungi. By measuring morphological character six fungi were identified (Fig. 2 & Table 1). Alternaria sp. was isolated from S. marianum, C. limon and V. negundo. Aspergillus sp. was isolated and identified from M. offiicinalis and W. somnifera. The other fungi Rhizopus sp. and Colletotrichum sp. were isolated from M. officinalis and C. camphora, respectively. Fusarium sp. was isolated from A. marmelos and W. somnifera. The characteristics features of all the isolated fungi are shown in Table 1.

 

 

Figure 1: Pure culture of endophytic fungi isolated from leaves of  M. officinalis (a); S. marianum (b); A. marmelos (c); C. camphora (leaf) (d); V. negundo (e); C. limon (f) and stem of W. somnifera (g); C. camphora (h).

 

 

Figure 2: Endophytic fungi (a) Alternaria sp. from S. merianum (b) Aspergilus sp. from M. officinalis (c) Fusarium from A. marmelos (d) Rhizopus sp. from M. officinalis (e) Alternaria sp. from C. limon (f) Colletotrichum sp. from C. camphora (g) Alternaria sp. from V. negundo (h) Fusarium sp. from W. somnifera (i) Aspergillus sp. from W. somnifera.

 

 

Table 1: Characteristics/ identifying features of isolated fungi from different medicinal pants

Fungus identified	Plants	Family	Characteristic/ Identifying features
Alternaria sp.	Silibium marianum Citrus limon Vitex negundo	Asteraceae Rutaceae Lamiaceae	i.     Presence of pale and brown to olive brown colored conidia and conidiophores ii.     Individual conidiophores arise directly from substrate forming bushy heads consisting of 4-8 large catenate conidia chains iii.     Secondary conidiophores are generally short and one celled
Aspergillus sp.	Mentha officinalis Withania somnifera	Lamiaceae Solanaceae	i.     Presence of black and brown colony ii.     Individual fungus looks like dandelions with metulae that supports a dark coloured head shot, through with black lines called phialides, which radiate out from the centre.
Rhizopus sp.	Mentha officinalis	Lamiaceae	i.     Presence of stolon ii.     Hyphae are branched and coenocytic iii.     Sporangium (large apophysate columella) present at top a long stalk
Colletotrichum sp.	Cinnamomum camphora	Lauraceae	i.     Mycelium threads are thick and filamentous hyphae are septate and branched ii.     Presence of spores
Fusarium sp.	Aegle marmelos Withania somnifera	Rutaceae Solanaceae	i.     Long thin filamentous threads ii.     Hyphae  are septate and asporulated

 

 

Antimicrobial activity of endophytic fungi:

Different concentration of fungal extracts were used for antimicrobial activity assay. Both the tested bacterial pathogens were found susceptible to the fungal extracts (Table 2 and Fig. 3). There was significant increase in the inhibition zone diameter with increase in the concentration of fungal extract (100-400 µg/ml). Against E. coli, Alternaria sp. and Fusarium sp. were found to be more effective than Aspergillus sp., whereas against S. aureus, Alternaria sp. and Fusarium sp. showed almost similar activity. The Aspergillus sp. was observed with significant activity but lesser than the other two fungal extracts.

 

 

Figure 3: Antimicrobial activity of (A) Alternaria sp. (B) Aspergillus sp. (C) Fusarium sp. against Escherichia coli and Staphylococcus aureus at different concentration (a-100µg/ml; b-200 µg /ml; c-300µg/ml; d- 400µg/ml).

 

Table 2: Antimicrobial activity of ethanolic extract of Fusarium sp. isolated from C. camphora and against pathogenic bacteria.

Endophytic fungi	Inhibition zones diameter of ethanolic extract of fungi in mm against E. coli at different conc.
	100µg/ml	200µg/ml	300µg/ml	400µg/ml
Alterneria sp.	10.00±0.00	11.66± 0.33	12.00± 0.00	13.33± 0.34
Aspergillus sp.	0.54±1.23	2.53± 0.67	4.23± 0.83	6.67± 3.333
Fusarium sp.	10.00±0.00	11.66± 0.33	12.00± 0.00	13.33± 0.34
Cephalosporin (5mg/ml)	18.667±0.3333
Endophytic fungi	Inhibition zones of ethanolic extract of fungi in mm against S. aureus at different conc.
	100µg/ml	200µg/ml	300µg/ml	400µg/ml
Alterneria sp.	10.66±0.33	11.33±0.33	12.00± 0.57	13.33±  0.33
Aspergillus sp.	10.66±0.33	10.33±0.33	11.00± 0.57	11.66± 0.66
Fusarium sp.	10.00±0.00	11.00±0.57	11.66± 0.66	13.00± 0.57
Cephalosporin (5mg/ml)	20.00±0.577

 

DISCUSSION:

Endophytic fungi has ubiquitous nature and found to be associated with the inner tissues of the plants. These fungi positively interact with its environment/plant and benefits its host in various ways. As endophytic fungi have many important properties and one of them is to protect the plant species against harmful pathogens by producing potential metabolites like alkaloids, terpenoids, quinones, lignins, phenols etc.^[4,23]. Hence, to fulfill the needs of agriculture and pharmaceutical industries a large scale production of these bioactive compounds must be necessary.

 

From the last few decades, endophytic fungi have been investigated in search of potential antibacterial activity. For example, endophytic fungi (Phomopsis, Alternaria, Colletotrichum and Nigrospora) isolated from the leaf tissues of Tectona grandis and Samanea saman have shown antimicrobial potential against pathogenic bacteria^[24]. Similarly, endophytic fungi isolated from five Garcinia plants have also verified the antimicrobial potential of metabolites of 70 fungal isolates against Staphylococcus aureus, Candida albicans, Cryptococcus neoformans and Microsporum gypseu bacteria^[25]. According to authors some endophytic fungal genera like Aspergillus, Botryosphaeria, Eutypella, Fusarium, Guignardia, Penicillium, Phomopsis and Xylaria have greater antibacterial activity than others.

 

In the present study, five fungal species (Alternaria sp., Aspergillus sp., Rhizopus sp., Colletotrichum sp. and Fusarium sp.) were isolated from the studied medicinal plant species (Table 1). Among all extracted species, three (Alternaria sp., Aspergillus sp. and Fusarium sp.) were selected for identification of antimicrobial activity against E. coli (gram negative bacteria) and S. aureus (gram positive bacteria). The ethanolic extract of selected fungal species have shown successful activity against the pathogenic bacterial species (Fig. 3 & Table 2). The inhibition diameter increased with increasing concentration of fungal extract (100-400µg/ml) and results showed that Alternaria sp. and Fusarium sp. were more effective than Aspergillus sp. against E. coli whereas against S. aureus  similar antimicrobial activity was observed (Table 2).

 

Present study observed a wide range of host adaptability of Fusarium, Alternaria and Aspergillus sp. in medicinal plants with potential antimicrobial activity against human pathogenic bacteria. Similar to present study, total 1160 endophytic fungi have been isolated by Huang et al. from 29 traditional medicinal plants and observed Alternaria sp. and Colletotrichum as dominant fungal endophytes^[26]. On the other hand, Marcellano et al. also isolated seventeen endophytic fungi from the bark of Cinnamomum mercadoi and among them Colletotrichum, Aspergillus, Fusarium were the major endophytes^[27]. They also observed the role of Aspergillus and Colletotrichum fungi in antimicrobial activities against E. coli and S. aureus and Fusarium against S. aureus, B. cereus and E. aerogenes.  Zhang et al. also observed the antimicrobial activity of Fusarium sp. against gram positive (S. aureus) and gram negative (E. coli) bacteria and suggested its role in antimicrobial potential^[28].

 

Eight endophytic fungi (Aspergillus fumigatus, A. niger, A. repens, A. alternata, Alternaria sp., Phoma hedericola, Fusarium solani and F. oxysporum) were isolated from Mentha viridis and shown their antibacterial potential against E. coli bacteria^[29]. Most of research on endophytic fungi belong to Aspergillus, Fusarium and Alternaria genus and showed good antibacterial activity^[30].

 

The study concluded that endophytic fungi (Fuserium sp., Aspergillus sp. and Alterneria sp.) isolated from the studied medicinal plants have potential antibacterial activity against E. coli and S. aureus which may be due to bioactive secondary metabolites. The present study serve as preliminary work and further study is required for the isolation and identification of responsible chemical compounds having potent antimicrobial activity from endophytic fungi.

 

ACKNOWLEDGEMENT:

Authors are thankful to Department of Microbiology, Shoolini University, Solan for providing microorganisms for antimicrobial activity.

 

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Accepted on 12.05.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(8): 3624-3628.