Antibacterial potential of fungal endophytes isolated from Boerhaavia diffusa

Article history: Received on: 14/07/2016 Revised on: 09/08/2016 Accepted on: 22/08/2016 Available online: 29/10/2016 Boerhaavia diffusa L. is widely used for its ethno-medicinal properties. The present study assessed the antibacterial activity of endophytic fungi harbored in this plant. The endophytic fungi isolated belonged to various genera like Aspergillus, Cladosporium, Colletotrichum, Curvularia, Fusarium, Pestalotiopsis and Zygorhynchus and the overall colonization frequency of endophytic fungi was found to be 24%. Among the isolated fungi Aspergillus fumigatus, Cladosporium sphaerospermum, Fusarium sp. and Zygorhynchus sp. showed antibacterial activity in preliminary screening against one or all the test bacteria. Further, after fermentation, the ethyl acetate extracts of the selected endophytic fungi displayed variable antibacterial activity with maximum 54.75 mm and a minimum 27.50 mm zone of inhibition against test bacteria in disc diffusion assay. The lowest minimum inhibitory concentration (MIC) was 0.156 mg ml -1 in the broth dilution assay against a B. subtilis, S. typhi and Staph. aureus, while it was 0.312 mg ml -1 against E. coli which was offered by the ethyl acetate extracts of A. fumigatus.


INTRODUCTION
Endophytic fungi are an interesting group of microorganisms associated with the tissues of plants and are often regarded as plant-defending mutualists (Saikkonen et al., 2004).Plant endophytic fungi are defined as the fungi that spend the whole or part of their life cycle colonizing inside the healthy tissues of the host plant, typically causing no apparent symptoms of disease (Rodriguez et al., 2009) and these fungal endophytes lie under the widely neglected component of plant communities.Endophytic fungi are distributed world-wide and have been isolated from a variety of plant types including mosses, liverworts, ferns and higher plants (Higgins et al., 2007;Rosa et al., 2010;U'Ren et al., 2012).Endophytic fungi have diverse application as they may play role in plant growth (Mucciarelli et al., 2003); plant defense (Gao et al., 2010); affect plant resistance under stressful conditions (both biotic and abiotic stresses) (Marquez et al., 2007;Rodriguez et al., 2008); decompose plant litter (Sun et al., 2011).Apart from these, endophytic fungi are also known to produce many valuable bioactive metabolites including anti-microbial, anti-insect, anticancer, anti-diabetic and immunosuppressant compounds along their great potential applications in agriculture, medicine and food industry (Verma et al., 2009;Zhou et al., 2010;Zhao et al., 2011;Hema et al., 2015).There are various reports on fungal endophytes that produce biologically active compounds including fungicidal and herbicidal properties (Petersen et al., 2004).
The probability of sampling specific medicinal plants that harbor antibiotic producing endophytes might increase significantly if one is guided by the medicinal role of native people (Raviraja et al., 2006).Hence, there is need to isolate new antimicrobial compounds from endophytic fungi that can overcome the difficulties related to the treatment of infections caused by resistant pathogens.Thus, in the present study endophytic fungi associated with Boerhaavia diffusa L.-a medicinal plant with high medicinal properties was screened for their antibacterial potential against test pathogens.

Collection of plant material
Healthy (showing no visual disease symptoms) mature stem of Boerhaavia diffusa L. were collected from Mysore region, Karnataka.For sampling selection, the plant was randomly collected from different sites in the same vicinity for the study.The healthy stems of the plant were collected and brought to the laboratory and processed immediately to reduce the risk of contamination.

Isolation and identification of endophytic fungi
The collected plant samples were washed thoroughly 2-3 times in running tap water to remove the dust and debris present on the surface of explants, followed by repeated washing in distilled water.After proper washing, stem samples were cut into small pieces (1 to 2 cm length) under aseptic conditions using sterile scalpel.Further, they were subjected to surface sterilization by immersing the plant samples in 70% ethyl alcohol for one minute, followed by immersion in 4% sodium hypochlorite solution for three minutes and then rinsed with deionized sterile distilled water three times to remove the sterilants present on the explants and blot dried on sterile blotter sheets.The efficiency of surface sterilization procedure was ascertained for every segment of tissue following the imprint method of Schulz et al. (1993).About 08 to 10 stem segments were placed on Petri plates containing 20 ml of potato dextrose agar (PDA) medium supplemented with antibiotic chloramphenicol to avoid the emergence of endophytic bacteria.Then the Petri plates were sealed using para film and incubated at 25 ± 2 C for 15 days.Endophytic fungal colonies emerging from their host were picked with sterile fine tip needle and sub cultured on to Petri plates containing PDA devoid of antibiotic to obtain pure cultures.The fungi were identified based on their morphological, conidial and cultural characters (Barnett and Hunter, 1998).All the fungal isolates were maintained in test tubes and Petri plates on PDA media.

Test organisms
All the isolated endophytic fungal isolates were screened for antibacterial activity.The indicator bacteria included both Gram-positive (Staphylococcus aureus MTCC 7443 and Bacillus subtilis MTCC 121) and Gram-negative (Escherichia coli MTCC 7410 and Salmonella typhi MTCC 733) bacteria obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India and used throughout the study.All the microbial cultures were adjusted to 0.5 McFarland standards, which is visually comparable to a microbial suspension of approximately 1.5 x 10 8 cfu ml -1 .

Preliminary screening
The preliminary screening of antibacterial activity was carried out done following agar plug method (Devaraju and Satish, 2011).The nutrient agar (NA) medium were poured into Petri plates and inoculated with 100 µl of the bacterial suspension and spread uniformly.The mycelial discs (6 mm) of each endophytic fungal isolate (15 day-old) grown on PDA were obtained from actively growing margins using a sterile cork borer and placed on the surface of the NA medium previously seeded with test organisms.PDA media (6 mm discs) devoid of any fungal colony served as negative control.The plates were sealed using Para film and incubated at 37 C for 24 h.After incubation, antibacterial activity was confirmed by the visualization and measurement of inhibition zones.Each experiment was carried out in triplicates and measured diameters of zone of inhibition surrounding the agar disc were averaged and expressed in milli meter (mm).The mean value of the zone of inhibition was recorded.

Fermentation of endophytic fungi
The endophytic fungal isolates that offered antibacterial activity in primary screening were subjected to fermentation for metabolite production.Each endophytic fungi (5 -10 discs) were picked from actively growing margins and were fermented in ml Erlenmeyer flasks containing 250 ml of potato dextrose broth (PDB) for 4-6 weeks at 25 ± 2 C under static conditions devoid of antibiotic.After incubation, the culture broth was filtered through Whatmann No. 1 filter paper to separate fungal mat and the resulting culture filtrate was extracted with equal volume of ethyl acetate in total three times and evaporated to dryness using flash evaporator.

Secondary screening
The fungal isolates with relatively broader antibacterial spectrum or stronger activities shown in the preliminary assay were selected for secondary assay.The secondary antibacterial screening was done by following disc diffusion method (Elecyinmi, 2007).The resulting crude extract of the fungal isolates were dissolved in ethyl acetate and tested for secondary assay.The test bacteria were seeded onto the surface of NA media and uniformly spread.Each sterile disc (6 mm) were loaded with 50 µl of fungal extract (concentration 1 mg disc -1 ) and 50 µl of ethyl acetate and equidistantly placed on NA plates.Streptomycin discs were also placed as standard.The inoculated plates were incubated at 25 ± 2 C for 24 h and measured for its antibacterial activity by measuring the inhibition zones.

Minimum Inhibitory Concentration (MIC) by Micro broth dilution assay
Minimal Inhibitory Concentration (MIC) was determined by broth micro-dilution technique according to the method of Sarker et al., (2007).The crude ethyl acetate extracts of selected endophytic fungi were diluted to a concentration of 50 mg ml -1 (stock solution).The 96 well plates were prepared by dispensing 100 μl of broth and 100 μl of the extract to the first well.A twofold serial dilution was made in the row up to 12 well and final concentrations from well one to 12 were 2.5-0.001mg ml -1 .A μl inoculum suspension of each bacterial strain was added to each well.The wells containing nutrient broth with inoculum and solvent served as negative control.The plates were incubated at 37 ºC for 24 h and the absorbance was measured at 620 nm using micro plate reader.The lowest concentration that inhibited visible growth of the test bacteria was recorded as the MIC based on the readings.The MIC was also detected by adding 10 μl well -1 of TTC (2, 3, 5-triphenyl tetrazolium chloride) (Sigma) dissolved in water (TTC 2 mg ml -1 ) and incubated under appropriate conditions for 30 min (Qaiyami, 2007).Viable organism reduced the dye to pink color.The lowest concentration at which the colour change occurred was taken as the MIC value.All MIC tests were repeated in triplicates.

Statistical Analysis
Data from three replicates were analyzed for each experiment and analysis of variance (ANOVA) using SPSS Inc. 16.0.Significant effects of treatments were determined by F values (P ≤ 0.05).Treatment means were separated by Tukey's Honestly Significant Differences (HSD) test.

Isolation and identification of endophytic fungi
A total of 12 endophytic fungi were isolated from healthy stem segments of B. diffusa.The overall colonization frequency of endophytic fungi was found to be 24%.The isolated endophytic fungi were classified into seven different taxa of Zygorynchus sp., Pestalotiopsis sp., Fusarium sp., Aspergillus sp., Cladosporium sp., Colletotrichum sp., and Curvularia sp.Among the isolated fungi, Cladosporium sphaerospermum was the dominant fungus showing 25% of colonization frequency, followed by Fusarium oxysporum (16.6%) and all the other isolated fungi had a colonization frequency of 8.3% (Table 1).

Table 1 :
List of endophytic fungi isolated from of B. diffusa.

Table 2 :
Antibacterial activity of isolated endophytic fungi against test bacteria by agar plug method (Zone of inhibition in mm).

Table 3 :
Antibacterial activity of ethyl acetate extracts of selected endophytic fungi against test bacteria by disc diffusion assay (Zone of inhibition in mm).

Table 4 :
MIC of ethyl acetate extracts of selected endophytic fungi against test bacteria (mg ml -1 ).