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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Sandhu et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 5, 678-691.

Research Article

ISSN 2278 – 4357

ISOLATION AND ANTIBACTERIAL PROPERTY OF ENDOPHYTIC FUNGI ISOLATED FROM INDIAN MEDICINAL PLANT CALOTROPIS PROCERA (LINN.) R.BR. Ravindra Prasad Aharwal, Suneel Kumar and Sardul S. Sandhu* Fungal Biotechnology and Invertebrate Pathology Laboratory, Department of Biological Sciences, Rani Durgawati University Jabalpur- 482001, (M.P.) India.

Article Received on 22 February 2014, Revised on 25 March 2014, Accepted on 21 April 2014

ABSTRACT Endophytes are those organisms that colonize the living internal tissues of their hosts without causing any visible symptoms. Several fungal endophytes have been isolated from a variety of plant species which have proved as a rich source of bioactive compounds. Calotropis

*Correspondence for Author Dr. Sardul S. Sandhu Fungal Biotechnology and

procera (Linn.) R.Br. a widely used medicinal plant in India, were exploited for endophytes as a possible source of bioactive secondary

Invertebrate Pathology

metabolites. Considering the importance of this plant, a study was

Laboratory, Department of

conducted to determine the colonization frequency of endophytic fungi

Biological Sciences, Rani

and to evaluate the antibacterial activity against Escherichia coli,

Durgawati University Jabalpur, (M.P.) India

Pseudomonas aeruginosa, Klebsiella pneumonae, Bacillus subtilis and Staphylococcus epidermidis of DMSO crude extracts of fungal endophytes from this plant. The endophytic fungi from different parts

of the plant (leaves, stems and roots) were isolated, pure cultures were raised and identified based on the morphology and characteristics of fungal spores. The highest colonization frequency of fungi was observed in leaf (Aspergillus niger, 33.33 %), stem (Altenaria alternate, 33.33%) and root (Curvularia lunata, 33.33%). The endophytic fungi that displayed broad spectrum antibacterial activity include: Fusarium solani, Cladosporium herbarum, Curvularia pallescens, Alternaria alternata and Drechslera nodulosa. Our preliminary results indicate that crude extracts of endophytic fungi of Calotropis procera (Linn.) R.Br. may possess some antibacterial bioactive compounds. Key Words: Antibacterial activity, Endophytic fungi, DMSO crude extract, Morphological study, Calotropis procera (Linn.) R.Br.

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INTRODUCTION The need for new bioactive compounds used in medicine, industry and agriculture has increased. While plants have been a major source of new compounds for drug discovery, attention has more recently turned to endophytes as these microorganisms demonstrate great potential sources for new bioactive compounds.[1] Endophytes are microbes which colonize living internal tissues of plants without causing any harm to their host.[2] Almost all groups of microorganisms have been found in endophytic association with Plants such as fungi, bacteria or actinomycetes. These Endophytes protect their hosts from infectious agents and adverse conditions by secreting bioactive secondary metabolites.[1] Endophytes, found ubiquitous in all plant species in the world, contribute to their host plants by producing plenty of substances that provide protection and ultimately survival value to the plant. Many researchers have proven that endophyte is a new and potential source of novel natural products for exploitation in modern medicine, agriculture and industry.[3] The endophytic fungi play important Physiological[4] and ecological[5][6] roles in their host life. The production of bioactive compounds by endophytes, especially those exclusive to their host plants, is not only important from an ecological perspective but also from a biochemical and molecular standpoint. Exciting possibilities exist for exploiting endophytic fungi for the production of a plethora of known and novel biologically active secondary metabolites. In the case of endophytes capable of producing host plant compounds, such production would then be independent of the variable quantities produced by plants influenced by environmental conditions. However, the practicality of commercial production of compounds by endophytic fungi still remains unproven. The reduction of secondary metabolite production on repeated sub-culturing under axenic monoculture conditions is one of the key challenges that need to be addressed in order to establish, restore and sustain the in vitro biosynthetic potential of endophytes.[7] They have the ability to produce a range of secondary metabolites, providing researchers with numerous leads for compounds of pharmaceutical significance and possible development as new drugs.[1] Many endophytic fungi have been reported to produce novel antibacterial, antifungal, antiviral, anti-inflammatory, antitumor and other compounds belonging to the alkaloids, steroid, flavenoid and terpenoids derivatives and other structure types.[8][3][9][10] The pharmaceutical and medical concerns of new drugs are the toxicity of these prospective drugs to human tissues. Since, the plant tissue where the endophytes exist is a eukaryotic system, it

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would appear that the secondary metabolites produced by the endophytes may have reduced cell toxicity; otherwise, death of the host tissue may occur. Thus, the host itself has naturally served as a selection system for microbes having bioactive molecules with reduced toxicity toward higher organisms.[1] Calotropis procera (Linn.) R.Br. (family Asclepiasaceae), also known as Giant Milkweed / Swallow Wort and Aak, Madaar of India. The plant is used in many Ayurvedic formulations like Arkelavana. The medicinal potential of Calotropis procera has been known to traditional systems of medicine for a while now with its leaves being widely used. The use of the plants, plant extracts, and pure compounds isolated from natural sources has always provided a foundation compounds.

for [11]

modern

pharmaceutical

Calotropis procera is a well

known plant and has been traditionally used for diarrhoea, stomatic, sinus fistula, and skin disease,[12][13] and the leaf part is used to treat jaundice. Calotropis gigantean (L) R.Br., a widely used medicinal plant in India, were exploited for endophytes as a possible source of bioactive compounds.[14]

Fig: Calotropis procera

The present study was carried out to isolate and identify endophytic fungi from the Calotropis procera (Linn.) R.Br. and study their antibacterial bioactive compounds produced by these fungi. MATERIALS AND METHODS Collection of plant Materials Tissue samples of leaves, Stems and roots were collected from healthy Calotropis procera (Linn.) R.Br. Shrub at the Rani Durgavati University, main Campus, Jabalpur, India. The plant material was brought to the laboratory in sterile bags and processed within a few hours after sampling. Fresh plant materials were used for isolation work to reduce the chance of contamination.

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Isolation of endophytic fungi from Calotropis procera Isolation of endophytic fungi from Calotropis procera was carried out using the protocol described by Strobel et al with slight modifications. The leaves, stems and roots were washed under running tap water for 1 hour, and the leaves, stem and root were cut into small pieces. The samples were surface sterilized by modified method of Dobranic et al.[15] Then the samples immersed in 70% ethanol for 1 minute, followed by 4% sodium hypochlorite for 2 minute and then rinsed in sterile distilled water for 1 minute. The excess moisture was blotted in a sterile whatman no. 1 filter paper. The surface sterilized 4 segments were placed in Petridishes containing Potato Dextrose Agar (PDA) supplemented with Tetracycline 100 units/ml concentration. The Petri-dishes were sealed using parafilm and incubated at 26±1°C for 7 days. The Petri-dishes were monitored every day to check the growth of endophytic fungal colonies from the segments. The isolates obtained were transferred on agar slants having PDA media for preservation of fungal isolates respectively, were stored at 4oC for further studies. Calculation of colonizing frequency Colonization frequency % (CF %) was calculated as described by Suryanarayanan et al. [16] Briefly, proper time of incubation was given for colonizing frequency counting. Colonization frequency (%) of an endophyte species was equal to the number of segments colonized by a single endophyte divided by the total number of segments observed x 100. Number of segment colonized by fungi Colonizing frequency %

=

X 100 Total number of segment observed

Identification of Endophytic Fungi The identification procedure of endophytic fungi was based on morphology. The seven isolated species were described according to their macroscopic features such as colour, shape and growth of cultured colonies, as well as microscopic characteristics like structure of hyphae, conidia and conidiophores. Obtained data then compared with the descriptions of endophytic fungi species present in the literature. When the morphological investigation fails to reveal the identity of the isolated fungus, the species is marked as ‘unknown’. Analysis of the antibacterial activity, was carried out on all species, identified and unidentified. Test organisms Total 5 test bacteria were selected for the study and among them 3 were gram negative and 2 were gram positive bacteria. The pathogenic bacteria Escherichia coli, Pseudomonas www.wjpps.com

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aeruginosa, Klebsiella pneumonae, Bacillus subtilis and Staphylococcus epidermidis were maintained on slants of nutrient agar medium and incubated at 37°C for 24 hour in bacteriological incubator. The bacterial cultures were obtained from Microbial Type Culture Collection (MTCC), Chandigarh, India. Fermentation and extraction Concerning the fermentation of the bioactive compounds using static flask culture, spores from 7 days old culture were transferred into 500 ml Erlenmeyer flask containing 250 ml of Potato Dextrose Broth (PDB). The seeded flasks were incubated at 28 ± 1°C for 14 days under stationary conditions. The cultures were harvested and filtered through Whatman no. 1 filter paper to give clear filtrate that was exposed to the extraction process. The culture filtrate was used for preliminary evaluation of antibacterial activity using an agar well diffusion method[15] against 5 pathogenic species of bacteria. The extract residue was dissolved in dimethyl sulfoxide (DMSO) and stored at 4°C to be used as stock solution for antibacterial activity. Antibacterial activity For antibacterial evaluation, agar well diffusion method was performed by standard method.[18] Potato Dextrose Agar plates were inoculated with overnight culture of each bacterial suspension, by evenly spreading out with sterile borosil glass spreader. The Agar wells were prepared by scooping out the media with a sterile cork borer (6 mm in diameter). The wells were then filled with 60 µl, of the fungal crude extract that was already dissolved in DMSO. The plates were then incubated at 37°C for 24 h and the zone of inhibition was recorded and compared with the control (i.e. a well filled with DMSO solution only). RESULTS Isolation of endophytic fungi from Calotropis procera The plant materials were collected from Rani Durgavati University campus, Jabalpur. About 36 segments (12 segments of leaf, 12 segments of stem and 12 segments of root) of Calotropis procera were processed for the isolation of endophytic fungus. A total of 12 fungus of which eight form of Ascomycetes, one form of Hyphomycetes, one form of Ulvophycetes and two form of fungi which do not produce any reproductive structures, as it produce sterile mycelia, was obtained.

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Table 1: Endophytic fungi isolated from different parts of Calotropis procera (Jan – Mar 2013) S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Plant Part Leaf Leaf Leaf Leaf Leaf Stem Stem Stem Stem Root Root Root

Name of Fungi Aspergillus niger Cladosporium herbarum Aspergillus tamari Drechslera nodulosa Fusarium solani Aspergillus japonicus Alternaria alternata Alternaria tenuissima Unidentified (CPS-3) Curvularia pallescens Curvularia lunata Unidentified (CPR-7)

Class Ascomycetes Ascomycetes Ascomycetes Ulvophycetes Hyphomycetes Ascomycetes Ascomycetes Ascomycetes Ascomycetes Ascomycetes -

Table 2: Name and colonizing frequency of Endophytic fungi isolate from Calotropis procera S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Name of Endophytic Fungi Aspergillus niger Cladosporium herbarum Aspergillus tamari Drechslera nodulosa Fusarium solani Aspergillus japonicus Alternaria alternata Alternaria tenuissima Unidentified (CPS-3) Curvularia pallescens Curvularia lunata Unidentified (CPR-7)

Isolate from Leaf Leaf Leaf Leaf Leaf Stem Stem Stem Stem Root Root Root

% Frequency of colonization 33.33 25.00 16.66 8.33 25.00 16.66 33.33 8.33 25.00 16.66 33.33 16.66

No. of isolates 4 3 2 1 3 2 4 1 3 2 4 2

Identification of endophytic fungi Cultivation of the different plant parts (leaf, stem and root) of Calotropis procera has led to the isolation of 12 species of endophytic fungi. Ten species were successfully identified as Aspergillus niger, Aspergillus tamari, Aspergillus japonicus, Cladosporium herbarum, Alternaria alternata, Alternaria tenuissima, Drechslera nodulosa, Fusarium solani, Curvularia pallescens, Curvularia lunata and two species remained unknown.

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Fig: 1 Colonization Frequency (%) of Endophytic Fungi isolated from the Calotropis procera Screening of endophytic fungi for antibacterial activity Screening of endophytic fungi to determine antibacterial activity by agar well diffusion method against 5 pathogenic bacterial strains is depicted in (Table – 3). Fusarium solani showed maximum antibacterial activity against Klebsiella pneumonae, Bacillus subtilis and Escherichia coli. Cladosporium herbarum showed maximum activity against Escherichia coli, Klebsiella pneumonae and Staphylococcus epidermidis. Similarly, Drechslera nodulosa showed antibacterial activity against Pseudomonas aeruginosa were observed minimum activity against Bacillus subtilis and respectively, Curvularia pallescens showed maximum antibacterial activity against Bacillus subtilis, Klebsiella pneumonae, Staphylococcus epidermidis and Escherichia coli. Alternaria alternata showed maximum antibacterial activity Klebsiella pneumonae, Staphylococcus epidermidis, Bacillus subtilis and Escherichia coli. Out of the two fungal strains Curvularia pallescens and Alternaria alternata was found to be most potent strain.

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Table 3: Screening of selected endophytic fungi against 5 pathogenic bacterial strains S.No.

1. 2. 3. 4. 5.

Name of Endophytic Name of bacterial strains Fungi Bacillus Staphylococcus Escheri Pseudomonas subtilis epidermidis chia coli aeruginosa Fusarium solani ++ +++ +++ + Cladosporium ++ +++ +++ ++ herbarum Drechslera nodulosa + + Curvularia pallescens +++ +++ +++ +++ Alternaria alternate +++ +++ +++ +++ +++ = very clear zone, ++ = clear zone, + = diffuse zone, - = no zone

Klebsiella pneumonae +++ +++ + +++ +++

Table 4: Broad spectrum activity of selected endophytic fungi against 5 pathogenic bacteria S.

Name of

No. Endophytic Fungi 1.

Fusarium solani

2.

Cladosporium herbarum

3.

Drechslera nodulosa

4.

Curvularia pallescens

5.

Alternaria alternata

Zone of inhibition (in mm) Bacillus Staphylococcus Escherichia Pseudomonas

Klebsiella

subtilis

epidermidis

coli

aeruginosa

pneumonae

19

32

31

15

31

20

30

32

18

31

9

11

10

12

11

27

24

24

21

25

25

26

25

22

29

Broad Spectrum Activity of endophytic fungi Broad spectrum activity of Endophytic fungi against 5 pathogenic bacterial strains is depicted in (Table. 4). Fusarium solani and Cladosporium herbarum produced the maximum inhibition zone of 32 mm against Staphylococcus epidermidis and Escherichia coli. Fusarium solani showed zone of inhibition of 32 mm, 31 mm, 19 mm,

against Staphylococcus

epidermidis, Escherichia coli, Klebsiella pneumonae and Bacillus subtilis respectively, Cladosporium herbarum showed 32 mm, 31 mm, 30 mm, and 20 mm zone against Escherichia coli, Klebsiella pneumonae, Staphylococcus epidermidis and Bacillus subtilis

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respectively, Alternaria alternata showed 29 mm 26 mm and 25 mm zone against Klebsiella pneumonae, Staphylococcus epidermidis, Bacillus subtilis and Escherichia coli and Curvularia pallescens showed 27 mm, 25 mm, 24 mm zone against Bacillus subtilis, Klebsiella pneumonae, Staphylococcus epidermidis and Escherichia coli. Respectively, Drechslera nodulosa showed minimum activity 12 mm against Pseudomonas aeruginosa..

Fig 2: Antibacterial activity of fungal extract isolated from Calotropis procera against five Pathogenic Bacteria

Bacillus subtilis

Staphylococcus epidermidis Fig 3: Zone of inhibition of fungal bioactive compounds against pathogenic bacteria strain by agar well diffusion method www.wjpps.com

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Escherichia coli

Pseudomonas aeruginosa

Klebsiella pneumonae Fig:4 Results of antibacterial activity of isolated Endophytic fungi against test organisms DISCUSSION Endophytes are those organisms that colonize the living internal tissues of their hosts without causing detectable symptoms. Several fungal endophytes have been isolated from a variety of plant species which have proved as a rich source of secondary metabolites.[19] In this present work a total of 12 fungal endophytes were isolated from Calotropis procera. Fusarium

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solani, Cladosporium herbarum, Curvularia pallescens, Alternaria alternate and Drechslera nodulosa was the main isolate and were screened for the antibacterial and production of secondary metabolites. Similarly, Idris et al,[20] were successfully isolated seven species of endophytic fung from Kigelia africana. In the present study 12 different species of endophytic fungi such as Aspergillus niger (33.33%), Aspergillus tamari (16.66%), Aspergillus japonicus (16.66%), Cladosporium herbarum (25.00%), Alternaria alternata (33.33%), Alternaria tenuissima (8.33%), Drechslera nodulosa (8.33%), Fusarium solani (25.00%), Curvularia pallescens (16.66%), Curvularia lunata (33.33%) and two unidentified (33.33%), were isolated from Calotropis procera. Similarly, Khan et al,[21] were also isolate endophytic fungi from Calotropis Procera and Withania somnifera.. Morphological investigations, using both macroscopic and microscopic features, have resulted in the identification of ten fungal species: Aspergillus niger, Aspergillus tamari, Aspergillus japonicus, Cladosporium herbarum, Alternaria alternata, Alternaria tenuissima, Drechslera nodulosa, Fusarium solani, Curvularia pallescens, Curvularia lunata. Two fungal species, although subjected to the same morphological investigations, remains unidentified. Similarly, Prabavathy et al.,[22] isolated fungi were identified as Aspergillus sp. based on morphological characters. In this study, an initial assessment was performed for the antibacterial activity of the isolated endophyte species. The crude extracts from the culture of endophytic fungi grown aerobically in PDA medium displayed antibacterial activity. Fungal extracts were effective against 5 bacterial strains included in the study. These results might be attributed either to the antimicrobial potency of the DMSO extract or to the high concentration of unidentified active principle in the extracts. The metabolite showed highest zone of inhibition against Staphylococcus epidermidis and Escherichia coli (32 mm) and lowest against Bacillus subtilis (9 mm). Recently, Bagyalakshmi et al.[23] isolated endophytic fungus Pestalotiopsis sp. from the leaves of Pinus caneriensis. The crude culture extract of hexane, ethyl acetate, dichloromethane and methanol were screened for antimicrobial activity. Other endophytic fungal extracts which showed low antimicrobial activity in the bioassay may have active compounds but probably in smaller amounts and/or the screened crude extracts could yield more potent compounds once they had undergone some purification.[24] In the present study, the DMSO extract of Fusarium solani and Cladosporium herbarum, Drechslera nodulosa, www.wjpps.com

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Curvularia pallescens and Alternaria alternata isolated from the Calotropis procera exhibited significant antibacterial activity against bacterial pathogens. The results revealed that the metabolites of all five isolated fungi are the potential source for the development of new antibacterial compounds. CONCLUSION In the present study, the endophytic fungi isolated from the parts (leaf, stem and root) of Calotropis procera showed promising high antibacterial activity against the five pathogenic bacteria. Endophytic fungi are a poorly investigated group of microorganisms that represent an abundant and dependable source of bioactive and chemically novel compounds with potential for exploitation in a wide variety of pharmaceutical and industrial areas. Hence, the isolation of endophytic fungi from medicinal plants for any bioactive compound may facilitate the new product discovery process. However there is a need of further in depth studies of these isolated endophytes. Further growing those on large scale, modifying culture conditions and supplying some stimulants might help in getting better production of particular bioactive compound. ACKNOWLEDGEMENT The authors wish to thank the Vice chancellor Prof. K.N. Singh Yadav, R.D. University, Jabalpur, India and the Head of the department of Biological Science, R.D. University, Prof. Y.K. Bansal for providing laboratory facility for this project. CONFLICT OF INTEREST There is no potential conflict of interest with reference to the current manuscript. All the authors have read the manuscript and agreed to submit the same for publication. REFERNCES 1. Strobel GA. Endophytes as sources of bioactive products. Microbes Infect, 2003; 5: 535544. 2. Bacon CW, White JF. Microbial endophytes. Marcel Decker, Inc., New York 2001. 3. Yu H, Zhang L, Li LZ, Guo C, Li L, Sun W, Qin L. Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res, 2010; 165: 437–449. 4. Malinowski DP, Belesky DP. Ecological importance of Neotyphodium spp. Grass endophytes in agroecosystems. Grassland Science, 2006; 52(1): 23-28.

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5. Tintjer T, Rudger AJ. Grass-herbivore interaction altered by strains of a native endophyte. New Phytologist, 2006; 170: 513-521. 6. Malinowski DP, Zuo H, Belesky DP, Alloush GA. Evidence for copper binding by extracellular root exudates of tall fescue but not perennial ryegradd infected with Neotyphodium spp., Endophytes. Plant and Soil, 2004; 267: 1-12. 7. Kusari S, Spiteller M. Are we ready for industrial production of bioactive plant secondary metabolites utilizing endophytes. Nat. Prod. Rep, 2011; 28: 1203-1207. 8. Guo B, Wang Y, Sun X, Tang K. Bioactive Natural Products from Endophytes: A Review. Appl Bioch Microbiol, 2008; 44: 136–142. 9. Aly AH, Debbab A, Proksch P. “Fungal endophytes: unique plant inhabitants with great promises. Appl Microbiol & Biotechnol; 2011; 90: 1829–1845. 10. Gutierrez RM, Gonzalez AM, Ramirez AM. Compounds derived from endophytes: A review of phyto-chemistry and pharmacology. current Medicinal Chemistry (under press) 2012. 11. Evans WC. Trease and Evans Pharmacognosy. Saunders an Imprint of Elsevier, 2005; pp. 41–7. 12. Alikhan I, Khanum A. Medicinal and Aromatic Plants of India. Ukaaz Publication, 2005; pp. 133–4. 13. Raghubir R, Rasik M, Gupta AJ. Healing potential of Calotropis procera on dermal wounds in guinea pigs. J Ethnopharmacol, 1999; 68: 261–6. 14. Selvanathan S, Indrakumar L, Johnpaul M. Biodiversity of the Endophytic Fungi Isolated from Calotropis gigantea (L.) R.BR. Recent Res Sci Technol, 2011; 3(4): 94-100. 15. Dobranic JK, Johnson JA, Alikhan QR. Isolation of endophytic fungi from eastern larch (Larix laricina) leaves from New Brunswick, Canada. Can. J. Microbiol, 1995; 41: 194198. 16. Suryanarayanan TS, Venkatesan G, Murali TS. Endophytic fungal communities in leaves of tropical forest trees: Diversity and distribution patterns. Current Science, 2003; 85(4): 489 - 492. 17. Lorian V. Antibiotics in laboratory medicine. Williams and Wilkins, Baltimore, 1996. 18. Newyork NY, Barnett HL, Hunter BB. Illustrated genera of imperfecti fungi, Burgers Company, Minneapolis; 1972. 19. Devi N, Shyamkeso N, Singh M. GC-MS analysis of metabolites from Endophytic fungus Collectotrichum gloeosporiodes isolated from Phlogacanthus thyrsiflorus. Int J Pharma Sci Review and Res, 2013; 23(2): 392-395. www.wjpps.com

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20. Idris A, Ietidal A, Eihab. Antibacterial activity of endophytic fungi extracts from the medicinal plant Kigelia africana. Egypt Acad J Biolog Sci, 2013; 5(1): 1-9. 21. Khan R, Saleem S, M. Iqbal C, Shekeel AK, Aqeel Ahma. Biodiversity of the endophytic fungi isolate from Calotropis Procera (AIT) R.BR. Pak J Bot, 2007; 39(6): 2233-2239. 22. Prabavathy D, Valli NC. Study on the antimicrobial activity of Aspergillus sp isolated from Justicia adathoda. Indian J Sci Technol, 2012; 5(9): 3317-3320. 23. Bagyalakshmi, Thalavaipandian A, Ramesh V, Arivudainambi USE, Rajendran A. A novel endophytic fungus Pestalotiopsis sp. Inhibiting Pinus caneriensis with antibacterial and antifungal potential. Int J Adva Lif Sci, 2012; 1: 1 – 7. 24. Fabry W, Okemo PO, Ansorg R. Antibacterial activity of East African medicinal plants. J Ethnopharmacol, 1998; 60: 79-84.

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