International Journal of Advanced Life Sciences (IJALS)

Bagyalakshmi et al., IJALS, Vol.2. Feb April : 2012

ISSN 2277 758X RESEARCH ARTICLE

A novel endophytic fungus Pestalotiopsis sp. inhabiting Pinus caneriensis with antibacterial and antifungal potential
Bagyalakshmi* Thalavaipandian A*, Ramesh V*, Arivudainambi U.S.E** and Rajendran A* * Department of Botany, VHNSN College, Virudhunagar, Tamilnadu, India, * Department of Chemistry, VHNSN College, Virudhunagar, Tamilnadu, India. Email : arvhnsnbotany@yahoo.co.in

Abstract
An endophytic fungus Pestalotiopsis sp. was isolated from the leaves of Pinus caneriensis and screened for its antimicrobial and antifungal potentials. The fungus was identified based on the morphology of fungal culture and the characteristics of the spores. The crude extracts of hexane, ethyl acetate, dichloromethane and methanol of endophytic fungal isolate were screened for antimicrobial activity. Among the four extractions, the extract of dichloromethane showed potent antimicrobial activity by inhibiting the growth of all tested gram positive (Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis) and gram negative (Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Pseudomonas aeruginosa and Salmonella typhi) bacterial pathogens. The dichloromethane extract exhibited maximum inhibition zone of 19 mm 16 mm and 20 mm against Bacillus subtilis, Escherichia coli and Candida albicans respectively. The minimal inhibitory concentration (MIC) of dichloromethane extract against the bacterial and fungal pathogens ranged from 80 to 160 g/ml. This study reveals that the endophytic fungus serves as a potential source for the production of effective bioactive compounds. Keywords: Endophytic fungus, Pestalotiopsis sp., Bacillus subtilis, Staphylococcus aureus and antimicrobial activity.

Corresponding Author Dr. A. Rajendran Associate Professor & Head in Botany, VHNSN College, Virudhunagar-626 001, Tamilnadu, India. arvhnsnbotany@yahoo.co.in

Introduction
World health problems caused by drug-resistant bacteria and fungi are increasing. An intensive search for newer and effective antimicrobial agent is needed. At present, endophytes are viewed as outstanding source of novel bioactive natural products because many of them occupying literally millions of unique biological niches (higher plants) growing in so many unusual environments (Strobel et al., 2004). Fungal endophytes are microorganisms that colonize living internal tissues of plants without causing any immediate, overt negative effects (Bacon and White, 2000). They have proven to

be promising sources of new and biologically active natural products which are of interest for specific medicinal or agrochemical applications (Strobel, 2002). In 1993, a novel paclitaxel-producing fungus, Taxomyces andreanae, from the yew Taxus brevifolia and a number of endophytic fungi were isolated from the medicinal plants (Strobel et al., 1996). The fungal species of the genus Pestalotiopsis have been demonstrated to be rich source of bioactive secondary metabolites with diverse structural features. In the present study, the fungal isolates were isolated from Pinus caneriensis for production of metabolites having potent antimicrobial activity that might facilitate detection of antimicrobial compounds.

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International Journal of Advanced Life Sciences (IJALS)

Bagyalakshmi et al., Materials and Method Isolation of endophytic fungi
The endophytic fungal strains were isolated from the Pinus caneriensis according to the procedure described by Schulz et al. (2000). The leaves of Pinus caneriensis were washed with water and surface sterilized by immersing in 70% aqueous ethanol (3 min), followed by 5 % aqueous sodium hypochlorite and finally with 70% aqueous ethanol (1 min). After these procedures, the leaves were rinsed in sterilized water and incubated in Petri dishes to ensure the elimination of all epiphytic microorganisms. Small pieces of the leaves were excised and placed in Petri dishes containing potato dextrose agar (PDA) medium supplemented with ampicillin (200 g/mL) and streptomycin (200 g/mL) to inhibit the bacterial growth. Individual hyphal tips of the emerging fungi were removed and transferred to fresh PDA (Potato Dextrose Agar) slants separately and were kept at 4 C after being cultured at 28 1 C for 7 days.

ISSN 2277 758X RESEARCH ARTICLE

IJALS, Vol.2. Feb April : 2012

different solid media like PDA, PDYEA, MEA, CAM, and CZA. The mycelial disck (5 mm) were inoculated at the centre of the each Petri plate containing the respective medium and incubated for 5 days at 30C. At every 24 h interval the diameter of mycelial growth was measured.

Effect of pH on the growth of Pestalotiopsis sp.

The growth of fungal isolate was estimated in different pH under static condition. The fungal isolate was grown in PDYE broth with the initial pH adjusted to 5.0, 5.5, 6.0, 6.5, 7.0 and incubated for 21 days at 30oC. The growth was determined by estimating the dry weight of the fungal mycelium.

Estimation of dry weight of Pestalotiopsis sp. PDYE Broth medium

100 ml of potato dextrose broth was taken in 250 ml Erlenmeyer flask and sterilized at 121C for 20 min at 15 psi. The mycelial disc of the fungal isolate was incubated under static condition at 30C. The dry weight of 21 days old mycelia culture was determined. The sample was kept in hot air oven for 2 h at 70C. After 2 h the dry weight of the fungal mycelium was measured by using the following formula.
Mycelial dry weight = weight of dried filter paper with dried mycelium Weight of filter paper

Microscopic Analysis Identification of the endophyte

The endophytic fungal strain was identified by the morphological methods. The morphological examination was performed by observing the fungal colony, the mechanism of spore production and the characteristics of the spores. All experiments and observations were repeated at least twice. The strain was identified as Pestalotiopsis sp. The fungus was grown on PDA at 30C for 7 - 9 days, and the conidia of the isolate were examined under a microscope. A slide culture technique was also used to observe the morphology of the isolated fungius

Fermentation
The endophytic fungus was grown on potato dextrose agar (PDYEA) at 30C for 5 days and inoculated into 500 ml Erlenmeyer flasks containing 300 ml potato dextrose broth (PDYEB) at 30C for 4 weeks. After incubation period, the fungal cultures were harvested and filtered through 2 layers of cheese cloth. The filtrate was extracted three times with an equal volume of Hexane, ethyl acetate, dichloromethane and Methanol. The solvent was evaporated to dryness under reduced pressure to obtain a crude extract.

Radial growth of fungal isolate on different solid media

Radial growth of the fungal isolate was studied on

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International Journal of Advanced Life Sciences (IJALS)

Bagyalakshmi et al., IJALS, Vol.2. Feb April : 2012

ISSN 2277 758X RESEARCH ARTICLE

Endophytic fungal isolates was grown on PDA plates at 30oC for 7-14 days depending on growth rate. Six pieces of the grown culture were cut from the plate and inoculated into a 1000 ml Erlenmeyer flask containing 200 ml of PD broth. After incubation at 30oC for 21 days under stationary condition, the fungal culture was filtered to remove mycelium. The filtered broth was then extracted with 200 ml of hexane, ethyl acetate, dichloromehane and methanol. The organic phase was evaporated to dryness under reduced pressure using a rotary evaporator and stored at 4C for further use.

loaded onto the well. The fungal culture was incubated at 30oC for 48-72 h and the zone of inhibition was measured around the well. Nystatin disc (100 g) was used as positive control.

Determination of minimum inhibition concentration (MIC)

Minimum inhibitory concentration was determined by micro broth dilution assay technique (Lorian, 1996). Overnight culture of each test organisms was seeded on petri plates containing Muller Hinton Agar medium and the partially purified fraction C was placed into the wells at concentration ranged from 80 - 160 g/ml. The plates were incubated at 30oC for 48 - 72 h. Minimum inhibitory concentration (MICs) was determined after 24 h for the bacteria and 48 h for fungi. MIC was determined as the least concentration of the partially purified fraction that inhibited the growth of the test microorganisms.

Antimicrobial activity Test Organisms

Altogether Eleven (eight bacterial and three fungi) strains such as Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Aspergillus sp., Candida albicans and Saccharomyces cervisiae were used to evaluate the antimicrobial activity.

Results and discussion

A total of 11 endophytic fungi were isolated from the healthy needles of Pinus caneriensis. Mycelium is branched, septate; hyaline, pale brown or grayish white. Conidiomata is acervular, separate or confluent, composed of hyaline to dark brown, thin or thick walled texture angularis; dehiscence irregular. Setae are sparse. Conidiogenous cells are enteroblastic to phialidic, hyaline. Conidia are fusiform, apices obtuse 9 24 X 3-4.5 m (Fig -2). The texture of the mycelium of the isolates is spongy, cottony and flocculate. The colour of the colony is bright white at young stage and light pale orange at mature stage. Conidigenous cells are lining the base of the conidia. The conidia are septate, 5 celled, cylindrical to fusiform, 24.0 9 m and versi coloured. The upper two median cells of conidia are darker than the lower

Antibacterial activity
The agar plate diffusion assay was used to evaluate the antimicrobial activity against test microorganisms. A 100 l of bacterial liquid culture, in an exponential growth phase, was spread onto the surface of Muller Hinton agar plate. Immediately, 100 l of partially purified fraction of the fungal isolate was loaded onto the well. The culture was incubated at 30oC for 18 h and the zone of inhibition was measured. Standard discs of Tetracycline (10 g) and Chloramphenicol (10 g) was used as positive control for antibacterial activity.

Antifungal activity
A 100 l of fungal culture/spore was spread out onto the surface of potato dextrose agar medium. Immediately, 100 l of partially purified fraction was

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International Journal of Advanced Life Sciences (IJALS)

Bagyalakshmi et al., IJALS, Vol.2. Feb April : 2012

ISSN 2277 758X RESEARCH ARTICLE

median cell, bearing appendages. The upper two cells are dark brown and the lower cells are pale brown. Based on the morphological characteristics of fungal colony and conidia, the fungal isolate was identified as Pestalotiopsis sp. The radial growth of the fungal isolate was studied on different solid media like PDA, PDYA, MEA, CZA and GPY. Among the various media, maximal growth was observed on PDYEA medium (Fig -1). The fungus was grown in different pH (5.0, 5.5, 6.0, 6.5 & 7.0) of PDYE Broth medium for 21 days at 30oC and the dry weight of mycelial was studied. Maximum dry weight of the mycelium was obtained at pH 6.0 when compared with the growth on other pHs of the medium (Fig - 4). Antibacterial resistance especially among gram-negative bacteria is an important issue that has created a number of problems in treatment of infectious diseases and necessitates the search for alternative drugs or natural anti-bacterial. The antibacterial activity of endophytic fungi isolated from the leaves of Pinus caneriensis was screened by agar disc diffusion method against pathogenic bacteria and fungal pathogens. The maximum activity against bacteria and fungal pathogens was recorded. Among them, the endophytic fungus which showed strong antibacterial activity against the growth of pathogens was used for further studies. The antibacterial activity of endophytic fungal extract against gram positive and negative organisms was shown in Table - 1. Among the four different extraction dichloromethane extract showed an effective antibacterial activity against all the tested organisms with the highest zone of inhibition on Bacillus subtilis (19 mm), E.coli (16 mm) and Candida albicans (20 mm). Whereas other solvent extract had moderate antibacterial activity against gram positive and negative organisms. Similarly,

Hellwig et al. (2002) reported that the altersetin, a natural product isolated from Alternaria sp., showed potent antibacterial activity against several pathogenic Grampositive bacteria.

Fig-1: Colony morphology of Pestalotiopsis sp.

Fig-2: Conidiospore of Pestalotiopsis sp.

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International Journal of Advanced Life Sciences (IJALS)

Bagyalakshmi et al., IJALS, Vol.2. Feb April : 2012

ISSN 2277 758X RESEARCH ARTICLE

Table 1. Antimicrobial activity of crude extract of endophytic fungus Pestalotiopsis sp.

Zone of inhibition in mm Test micro organisms used Bacillus subtilis Staphylococcus aureus Streptococcus faecalism Escherichia coli Klebsiella pneumonia Proteus mirabilis Pseudomonas aeruginosa Salmonella typhi. Candida albicans Aspergillus sp. Saccharomyces cervisiae Hexane extract 9 8 9 7 7 8 7 7 7 8 9 Ethyl acetate extract 14 12 14 12 10 10 10 12 15 13 14 Dichloromethane extract 19 15 16 16 12 13 12 14 20 15 17 Methanol extract 12 11 12 10 10 11 11 11 10 11 12

Table 2. MIC value of crude endophytic fungal culture extract

Test microorganisms used Bacillus subtilis Staphylococcus aureus Staphylococcus faecalis Escherichia coli Klebsiella pneumonia Proteus mirabilis Pseudomonas aeruginosa Salmonella typhi Candida albicans Aspergillus sp. Saccharomyces cervisiae Minimum inhibitory concentration (g/ml) 80 100 120 100 140 160 120 140 90 140 120

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International Journal of Advanced Life Sciences (IJALS)

Bagyalakshmi et al., IJALS, Vol.2. Feb April : 2012

ISSN 2277 758X RESEARCH ARTICLE

Bacillus subtilis

E.coli

Candida albicans

1-Hexane, 2- methanol, 3-Ethyl acetate, 4- Dichloromethane, C ControL.

Fig-3: Antimicrobial activity of crude endophytic fungal extract against bacterial and fungal pathogens

640 620 D ry w eig h t o f m y celiu m in m g

10 CZA 9 MEA 8 CAM PDA PDYA 6

G r o w th in d ia m e te r (m m )
4.5 5 5.5 6 pH 6.5 7 7.5

600 580 560 540 520 500

3 Incubation in days

Fig-4. Effect of pH on growth of Pestalotiopsis sp. in PDB

Fig 5. PDA- Potato dextrose agar, PDYEAPotato dextrose yeast agar, MEA- Malt extract agar, CAM- Corn meal agar, CZA- Czapek dox agar.

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International Journal of Advanced Life Sciences (IJALS)

Bagyalakshmi et al., IJALS, Vol.2. Feb April : 2012

ISSN 2277 758X RESEARCH ARTICLE

The result of this work indicates that the differences in the zones of inhibition may be directly related to the susceptibility of each test organisms to the fungal extracts. The factors responsible for this high susceptibility of the bacteria to the extracts are not exactly known but may be attributed to the presence of secondary fungal metabolites which is soluble in dichloromethane. The antimicrobial potential of crude dichloromethane extract of endophytic fungal culture extract was investigated using the minimal inhibitory concentration (MIC) assay (Table - 2). The dichloromethane extract showed antimicrobial activity with a MIC of 80 g/ml against Staphylococcus aureus 100 g/ml against Escherichia coli and 90 g/ml against Candida albicans. Similarly, Lu et al. (2000) reported the MIC value of purified bioactive compounds of Colletotrichum sp. against the bacterial pathogens. In the present study, the endophytic fungus Pestalotiopsis sp. isolated from the Pinus caneriensis exhibitied potent antimicrobial and antifungal activity in dichloromethane extract against eleven different pathogenic bacteria and fungal organisms. Endophytic fungi are a poorly investigated group of microorganisms that represent an abundant source of bioactive and chemically novel compounds with potential for exploitation in medical, agricultural, and industrial areas.

Hellwig, V., Grothe, T., Mayer - Bartschmid, A., Endermann, R., Geschke, F.U., Henkel, T. and Stadler, M. 2002. Altersetin, a new antibiotic from culture of endophytic Alternaria spp. Taxonomy, fermentation, isolation, structure elucidation and biological activities. J. Antibiotics., 55: 881- 892. Lorian, V. 1996. Antibiotics in Laboratory Medicine. 4th Edn., Williams and Wilkins, Baltimore, London. Lu, H., Zou, W.X., Meng, J.C., Hu, J. and Tan, R.X. 2000. New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemisia annua. Plant Sci., 151: 67 73. Schulz, B., Boyle, C., Draeger, S., Rommert, A.K. and Krohn, K. 2002. Endophytic fungi a source of novel biologically active secondary metabolites. Mycol. Res., 106 : 996 1004. Strobe1, G.A. 2002. Usefu1 products from rainforest microorganisms. Part 2. Unique bioactive mo1ecu1es from endophytes. Agro. Food. Industry., Hi Tech., 13 : 12-17. Strobel, G., Daisy, B. and Castillo, U. 2004. Natural products from endophytic microorganisms. J. Nat. Prod., 67 : 257 - 268. Strobel, G.A., Hess, W.M., Ford, E., Sidhu, R.S. and Yang, X. 1996. Taxol from fungal endophytes and issue of biodiversity. J. Ind. Microbiol., 17: 417 423.

Reference:
Bacon, C.W. and White, J.F. 2000. Physiological adaptations in the evolution of endophytism in Clavici pitaceae. In: Microbial Endophytes. Marcel Dekker, New York. pp. 237 - 262.

Corresponding Author : Dr.A.Rajendran, Associate Professor and Head in Botany, VHNSN College, Virudhunagar - 626 001, Tamilnadu, India. Email - arvhnsnbotany@yahoo.co.in

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