International Journal of Pharmaceutical Sciences and Nanotechnology 566 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 • Issue 2 • July - September 2009 Volume 2 • Issue 2 • July – September 2009 Research Paper Preliminary Screening of Endophytic Fungi from Medicinal Plants in India for Antimicrobial and Antitumor Activity Gani Sharmila Banu1, Ganesan Kumar2,* 1 Centre for Biotechnology, Muthayammal College of Arts and Sciences, Kakkaveri, Rasipuram, Namakkal, Tamilnadu, India-637408. 2 Department of Biochemistry, Selvamm Arts and Science College, Namakkal (DT)-637 003. Tamilnadu, India. ABSTRACT: The screening of antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi was carried out on isopropanol extracts prepared from 121 isolates of endophytic fungi isolated from medicinal plants in India. Sensitivity was found to vary among the microorganisms. Bacillus subtilis, Saccharomyces cerevisiae and Alternaria sp. were susceptible to extracts from three, two and two isolates of endophytic fungi, respectively. None was found effective against Salmonella typhimurium. Sixteen endophytic fungal isolates tested were also found to exhibit antitumor activity in the yeast cell-based assay. KEYWORDS: Medicinal plants; Endophytic fungi; Antimicrobial; Antitumor Introduction Materials and Methods The nature and biological role of endophytic fungi in their plant host is variable. Endophytic fungi are known to have Isolation of endophytic fungi mutualistic relations to their isolation of endophytic fungi A random sample from each plant consisting of hosts, often protecting plants against herbivore, insect asymptomatic leaves and branches was taken. The leaves attack or tissue invading pathogens (Siegel et al., 1985; and branches were thoroughly washed in running tap Clay, 1986; Yang et al., 1994) and in some instances the water, after which they were surface sterilized by endophyte may survive as a latent pathogen, causing or submerging them in 75% ethanol for 2 min. The branch portions were further sterilized sequentially in 5.3% quiescent infections for a long period and symptoms sodium hypochlorite solution for 5 min, and 75% ethanol appear only when physiological or ecological conditions for 0.5 min. After drying, each leaf was divided into three favors virulence (Carroll, 1986; Bettucci, and Saravay, segments and placed on potato dextrose agar (PDA) 1993). In India, extract from many types of local plants are supplemented with 50-mg/l chloramphenicol to suppress used in traditional manner for treatments of various bacterial growth. Branch portions were cut to expose their ailments (Beulah Jerlin et al., 2004; Rajasekara Pandian et inner tissue and placed on the same medium. All the plates al., 2006). The question is whether they are produced by were incubated at 270C for up to 3 weeks. Emerging fungi the plant itself or as a consequence of a mutual relationship were transferred to fresh PDA plates, incubated for 1 week with beneficial organisms in their tissue. Many reports and periodically checked for purity. showed that in a microbe-plant relationship, endophytes Tests for Antimicrobial and antitumor activities contribute substances that possess various types of bioactivity, such as antibacterial and antifungal. Thus in The endophytic fungi were grown at 270C with shaking in this study, we focus on the isolation of endophytic fungi 5 ml F-4 medium (Glycerol 40 g/l, Soya bean meal 25 g/l, Yeast extract 5 g/l, Corn steep liquor 1 g/l, NaCl 0.5 g/l) and screening them for bioactivity. and PD-Y medium (Potato dextrose broth 24 g/l, Yeast extract 2 g/l) for 5 days. For extraction, an equal volume of * For correspondence: Ganesan Kumar, isopropanol was added to the culture broth and vortexed E-mail: firstname.lastname@example.org vigorously for 1 min followed by a centrifugation at 3000 rpm for 10 min. 566 Gani Sharmila Banu et al. : Preliminary Screening of Endophytic Fungi from Medicinal Plant in India for… 567 About 80 ml of supernatant was applied per sterile synthetic chemical also have long been used as active paper disc (5 mm diameter). After drying, the extract agents in reducing the incidence of plant, animal and impregnated discs were used in a disc diffusion assays human diseases, they are costly, have potentially harmful using Alternaria sp. in potato dextrose agar (PDA), effect on the environment and may induce pathogen Bacillus substilis, Salmonella typhimurium in PMg agar, resistance. Thus, biological controls or the use of and Saccharomyces cerevisiae in YPG agar as test microorganisms or their secretions to prevent diseases microorganisms for antimicrobial activity. Five millilitre of offer an attractive alternative or supplement to disease spore suspension of Alternaria sp. grown in vegetable juice management without the negative impact of chemical (tomato juice, 200 ml CaCO3, 4.5 g agar, 3.0 g in a total control. vol. of 300 ml) were used. S. cerevisiae was grown in YPG In natural product discovery programs, typical broth (yeast extract, 20 g/l; peptone 20 g/l; glucose, 20 g/l), procedures included isolating microorganisms from and B. subtilis and S. typhimurium were grown in PMg samples, growing them at various temperatures in a variety broth (peptone 10 g/l; MgSO4.7H2O, 2 g/l). of selective or nonselective media and testing the extracts Chloramphenicol (50-mg/ ml) and nystatin (100 mg/ml) in a spectrum of targeted screens for activity for potential were used as positive controls. industrial or pharmaceutical applications. For a successful To prepare assay plate in a 21.5 cm X 21.5 cm square fungal screening, a varied and novel repertoire of either plate, Yeast Nitrogen Base (YNB) broth (1.4 g) and Bacto well-known or unexplored fungi is desirable. The most agar (2.4 g) were dissolved in 150 ml of sterile distilled promising trend in isolating new fungi is the move towards water. The pH was adjusted to 6.5 prior to autoclaving at investigating novel endophytes, with the idea that unusual 1210C for 15 min. When the agar is about 420C, the endophytes may produce untapped natural products. following components were added: 20 ml of 50% A total of 121 endophyte isolates were obtained from galactose, 2 ml of 20% sucrose, 10 ml of 20X concentrated 62 of 72 (86.1%) different types of medicinal plants used adenine (0.5 mg/ml), 2 ml of 100X concentrated histidine by the local population in India (Table 1). The results of (2 mg/ml), 2 ml of 100X concentrated tryptophan (2 this study showed that endophyte fungi were more mg/ml), 2 ml of 100X concentrated uracil (2 mg/ml) or 2 prevalent in the leaves (110/121 or 90.9%) than in the ml of 100X concentrated leucine (2 mg/ml) when using the branches. Further, more intensive samplings are necessary test strain UCK or UCS, respectively; 10x concentrated 4 to clarify the fungal assemblages of the leaves and dropout amino acid (containing each at 10x concentrated branches, as in traditional practice; the local population of arginine at 240 mg, methionine at 240 mg, tyrosine at used mostly the extract from the leaves of the plants (Ong 360 mg, isoleucine at 360 mg, lysine at 360 mg, and Nordiana, 1998; 1999). phenylalanine at 600 mg, aspartic acid at 1000 mg, valine at 1500 mg and thymine at 2000 mg per 1000 ml sterile Though there are still a lot of subjects to be explained distilled water) and yeast glycerol (30-40%) stock of the in the mutualistic association of endophytic fungi and their yeast test strain W303-1AY18 containing either the plant host, more reports indicated the occurrence of plasmids pMR438-CyclinA D 24-62 (UCK) or Yep51- endophytes in plants especially in relation to the possible SRX5 src (UCS) (Sikder et al., 1997), and poured into the origin of the plant metabolites detected (Strobel et al., square plate on a horizontal place. The 5 mm paper discs 1996; Cacabuono and Pomilio, 1997; Rizzo et al., 1997). impregnated with the supernatant described above were The culture residue of the isopropanol extract of the placed on the agar plate and incubated at 300C for 3-4 endophytes cultures 12L and 22L in F-4 medium and 1B, days. The growth circle around the disk that indicates 19L, 21L2 and 27L1 in PD-Y medium yielded impressive positive results for anti-tumor activity was measured. anti-fungal, anti-bacterial or anti-yeast activities. However, extracts from endophytic fungal cultures of 5L, 24L2, 37L, Glucose (50%) was used as a positive control. All the 41L1, 50ML, b34L1, b53L, b20L, b69L2, b30L, b9l, screening procedures were performed twice in duplicates. b70L2, b14L, b49L and b29L in PD-Y broth showed Inhibition zone (for antimicrobial test) or growth zone (for positive activity for anti-tumor in the UCK/UCS yeast cell- anti-tumor test) around the disk of 6 mm or more were based assay (Table 2). The basis of the anti-tumor defined as positive for biological activity. screening using a yeast cell-based assay was that the hyper activation of cylcin-dependent-kinase (CDK) resulted in Results and Discussion growth arrest of the yeast harboring the genetically Plants have long provided mankind with a source of engineered recombinant plasmids, and a compound from medicinal agents, with natural products once serving as the extract that can rescue the cyclinA1-induced growth source of all drugs (Balandrin et al., 1993). Though arrest is viewed as a potential anti-tumor candidate. 568 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 • Issue 2 • July - September 2009 Table 1 Endophytic fungi isolated from medicinal plants. No Local name Scientific name First isolation Second isolation 1. Kuppai meni Acalypha indica - b1L 2. Vasambu Acorus calamus 2L, 2ML b2L 3. Nayuruvi Acyranthes aspera - b3L 4. sirupulai Aerva lanata 4L b4L 5. Vellai poondu Alium sativum - - 6. Kumari Aloe vera - - 7. Malai vembu Andrographis paniculata 7L - 8. Karuvel Areca catechu - - 9. Kaduku Brassica juncea 9L, 9B b9L 10. Usihthakarai Cassia tora 10L, 10ML b10L 11. Nithya kalyani Catharantus roseus - b11L 12. Valuluvai Celastrus paniculatus 12L b12L1, b12L2 13. Ilavangam Cinnamomum zeylanicum 13L b13L 14. Athondai Capparis zeylanica - b14L1, b14L2 15. Nallavelai Cleome gynandra - b15L1, b15L2 16. Kattukodi Cocculus hirsutus - b16L 17. Omavalli Coleus aromaticus 17L b17L 18. Kanavalai Commelina bengalensis 18L/B - 19. Agasa karudan Corallocarpus epigaeus 19L - 20. Naruvili Cordia oblique - - 21. Kothamalli Coriandrum sativum - b21L, b21B 22. Kottam Costus speciosus - b22L 23. Nilappanaik-kilangu Curculigo orchioides 23L b23L1, b23L2 24. Manjai inji Curcuma amada - b24L 25. Kua Curcuma angustifolia - b25L 26. Kasthuri manjal Curcuma aromatica 26L, 26B b26L1, b26L2 27. Karuumathai Datura metal 27L - 28. Karisilankanni Eclipta prostrata 28L, 28ML - 29. Anashovadi Elephantopus scaber - b29L 30. Kirambu Eugenia caryophyllata - b30L1, b30L2 31. Palperukki Euphorbia heterophylla 31L1, 31L2, 31ML b31L1, b31L2 32. Amman pacharisi Euphorbia hirta - - 33. Athi Ficus racemosa - b33L 34. Karunochi Gendarussa vulgaris 34L1. 34L2, 34B1, 34B2 b34L 35. athumathuram Glycyrrhiza glabra - b35L1, b35L2 36. Nannari Hemidesmus indicus 36L b36L 37. Kasarai Hibiscus cannabinus - b37L Table 1 Contd… Gani Sharmila Banu et al. : Preliminary Screening of Endophytic Fungi from Medicinal Plant in India for… 569 38. Narunthali Ipomoea obscura - b38L 39. Mallai Jasminum sambac 39L, 39B 40. Adalai Jatropha glandulifera 40L b40L 41. Kattamanakku Jatropha gossypifolia 41L b41L 42. Kacholum Kaempferia angustifolia - - 43. Tindiyam Lagerstroemia indica 43L1, 43L2 b43L1, b43L2 44. Eluthani poondu Launaea sarmentosa 44L1, 44L2, 44B b44L, b44b 45. Maruthontri Lawsonia inermis - b45B 46. Arai Marsilea quadrifolia - b46L 47. Elikathilai Merremia emarginata 47L1, 47L2 b47L1, b47L2 48. Sathikkay Myristica fragrans - b48L 49. Karuveppilai Murraya koenigii - b49L, b49B 50. Sadamanjil Nardostachys jatamansi - b50L 51. Thulasi Ocimum basilicum 51B - 52. Inbura Oldenlania umbellata - b52L 53. Anai nerunchil Padalium murex - b53L 54. Uppilankodi Pentatropis capensis - b54L, b54B 55. Sathakuppai Peucedanum grande b55L 56. Thippili Piper longum 56L, 56B1, 56B2 b56L 57. Milagu Piper nigrum - - 58. Ilanthalari Plumeria rubra 58L1, 58L2 b58L 59. Kodi veli Plumbago zeylanica 59L1, 59L2 b59L1, b59L2 60. Narumunnai Premna corymbosa 60L1, 60L2, 60ML b60L1, b60L2 61. Nagamalli Rhinacanthus nasutus - b61L1, b61L2 62. Alingi Rhododendron arboreum 62L1, 62L2 b62L1, b62L2 63. Amanakku Ricinus communis 63ML - 64. Rosa Rosa centifolia - b64L1, b64L2 65. Asogam Saraca indica 65L b65L 66. Nathai suri Spermacoce hispida 66L b66L1, b66L2 67. Puli Tamarindus indica 67B b67L 68. Kadukkay Termanalia chebula - b68L 69. Manakkarai Vangueria spinosa b69L 70. Amukkiran Withania somnifera - b70L 71. Iratchai Zanthoxylum budrunga 71L, 72ML - 72. Inji Zingziber officinale - b72L 73. Ilanthai Zizyphus jujuba - b73L 74. Korgodi Zizyphus nummularia 74L b74L 75. Suraimullu Zizyphusoenoplia 75L, 75ML b75L - No endophytic fungi isolated 570 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 • Issue 2 • July - September 2009 Table 2 Endophytic fungal isolates showing biological-activity against test organisms. Antimicrobial activity (mm) a Antitumor activity (mm) b Endophytes Bs St Sc Al UCS UCK 12L 15 - - - - - 19L 19.5 - - 20 - - 22L 19.2 - - - - - 21L2 - - 13.2 - - - 27L1 - - 13.5 - - - 1B - - - 21 - - 5L - - - - 8 8.5 24L2 - - - - 8 8 37L - - - - 9.2 9.1 41L1 - - - - 8.3 8 50ML - - - - 10 10.3 b34L - - - - 7.2 7.1 b53L - - - - 8 8.1 b20L - - - - 7.5 7.5 b69L2 - - - - 8.1 8 b9L - - - - 10 10.3 b30L - - -- - 9 9 b7L - - - - 9.3 9.2 b70L2 - - - - 8.6 8.3 b14L - - - - 8.2 8.2 b49L - - - - 8.7 8.5 b29L - - - - 8.3 8.1 a Test microorganisms: Bs- Bacillus subtilis; St- Salmonella typhimurium; Sc- Saccharomyces cerevisiae; and Al- Alternaria sp. b Yeast test strain W303-1AY18 containing: plasmid pMR438-CyclinA D 24-62 (UCK) or Yep51-SRX5 src (UCS). (-) None detected. In this study, we demonstrated that crude extracts from Also extracts which showed no anti-microbial or anti- the culture broth of endophytic fungi grown, aerobically in tumor activity in the disc-diffusion bioassay might be PD-Y or F4 medium displayed anti-bacterial, anti-fungal, active against other microbes, which were not tested. anti-yeast or anti-tumor activity. These results suggest the Looking at the differing activity of test results obtained, presence of either good antimicrobial potency of the additional modes of action should be explored for those extract or a high concentration of an active principle in the isolates that do not have antimicrobial activity, as it is extracts of strains showing positive biological activities. possible that some of these endophytes may produce Other endophytic fungal extracts, which showed low anti- substances that may ward off microbial infections by microbial or anti-tumor activity in the bioassay, may have stimulating the host immune system rather than by active compounds but probably in smaller amounts and/or antimicrobial activity. the screened crude extracts could yield more potent In addition, there is also the possibility that substances compounds once they had undergone some purification present in the extract can stimulate the growth of the (Fabry et al., 1998). microorganisms, as was evident by several isolates Gani Sharmila Banu et al. : Preliminary Screening of Endophytic Fungi from Medicinal Plant in India for… 571 showing good bacterial growth forming wide zone of Carroll GC. 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