FUNGAL ENDOPHYTES IN THREE MEDICINAL PLANTS OF LAMIACEAE

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1 Acta Microbiologica et Immunologica Hungarica, 56 (3), pp (2009) DOI: /AMicr FUNGAL ENDOPHYTES IN THREE MEDICINAL PLANTS OF LAMIACEAE D. BANERJEE*, S. MANNA, S.MAHAPATRA and B. R. PATI Department of Microbiology, Vidyasagar University, Midnapore , West Bengal, India (Received: 10 April 2009; accepted: 16 June 2009) Three medicinal plants Ocimum sanctum, Ocimum bacilicum and Leucas aspera were screened to study endophytic diversity of the plants. Altogether 103 fungal endophytes belonging to fourteen genera were isolated. Leaves of all three medicinal plants were colonized by a great number of endophytic fungi. Leaves of O. sanctum were colonized by the most, that is, eleven endophytes. Highest Shannon-Wiener index (2.256) was exhibited by O. sanctum with the highest Simpson s diversity (0.8654) indicating great species specificity. O. bacilicum and L. aspera showed the highest similarity coefficient. Some fungal genera have been showed to be host specific. In the present study Curvularia sp., Hymenula sp., Tricoderma sp. and Tubercularia sp. exclusively colonized O. sanctum; whereas Alternaria sp. and Spicaria sp. colonized only L. aspera. Keywords: diversity, endophyte, Lamiaceae, Leucus, Ocimum Introduction Microbes that colonize internal plant tissues without causing any overt symptoms or apparent injury are called endophytes [1]. Both fungi and bacteria are common microbes that exist as endophytes, but the most frequently isolated endophytes are fungi [2]. Carroll and Carroll [3] reported that endophytes live asymptomatically and sometimes systematically within the plant tissues. * Corresponding author; db@vidyasagar.ac.in /$ Akadémiai Kiadó, Budapest

2 244 BANERJEE et al. Endophytes usually inhabit above ground plant tissues (leaves, stems, petioles and reproductive structures), and also some reports showed presence of endophytic fungi within the root. Some researchers isolated extremely abundant and very diverse types of endophytic fungi from plant tissues [4, 5]. Ganley et al. [6] reported that endophytes are the normal inhabitants of the plant tissues. They protect the plants from pests [7], other fungal pathogens [8] or enhance the defense of host plants against grazing animals [9] or increase host fitness against harsh environments [10]. Fungal endophytes studied in temperate regions [11] are more prevalent than tropical region [12 16]. Identification and monitoring of endophytic fungi can help in the development of agricultural production and management systems, which leads to the implementation of practical measures to minimize the disequilibrium between human and nature. Such practices are extremely important factors in determining the quality of endophytic microbes [17]. Endophytic fungi can be used as potential biological control agents. Endophytic fungi also constitute a valuable source of secondary metabolites for the discovery of new potential therapeutic drugs. The intent of this research was to provide an insight into the presence of endophytic fungal assemblage in different parts (leaf, stem and root) of three medicinal plants of the mint family (Lamiaceae). The medicinal plants were Ocimum sanctum, Ocimum bacilicum and Leucus aspera. These three medicinal plants were collected from different localities of Paschim Medinipur, West Bengal, India. The objective of this study was to explore the endophytic fungal biodiversity of the above-mentioned three medicinal plants. Collection of plant materials Materials and Methods Plant materials were collected from different locations of Paschim Medinipur, West Bengal, India (Latitude 22º 25 to 22º 57 North, Longitude 87º 11 East, Altitude 23 meters from mean sea level), having a tropical humid climate with a mean temperature of 32 ºC and an average annual rainfall of 1200 mm. Samples were collected between the months of August to October. The three herbal medicinal plants (Table I) chosen for fungal endophyte screening were Ocimum sanctum, Ocimum bacilicum and Leucas aspera, all belonging to the mint family (Lamiaceae). From mature, disease free plants, each sample type was collected from each location and from each plant species.

3 FUNGAL ENDOPHYTES OF LAMIACEAE 245 Table I Brief description of the medicinal plants Name of the plants Local name Brief description Usefulness Ocimum sanctum Kalatulsi or Tulshi Annual, cm long, much branched. Leaves by cm, elliptic oblong, acute, pubescent on both sides. Ocimum bacilicum Babuitulsi Erect branching herb, m high. Leaves cm or longer, ovate acute entire or more or less toothed or lobed. Leucas aspera Chotohalkusa A variable annual, cm high. Stem erect, usually much branched from below. Leaves by cm. Useful in disease of the heart and blood, leucoderma etc. Roots used in malarial fever. Leaves' juice is used in bronchitis and applied to ringworm and other skin disease. Useful in disease of the heart and brain, chronic pain in joints, asthma, inflammation and enlarged spleen. Leaves juice good for toothache, earache and headache. Leaves are useful in treatment of chronic rheumatism, juice used for the treatment of psoriasis and chronic skin eruption. Different plant parts selected for the identification of endophytic fungi Leaf: One leaf was collected randomly from each of fifteen plants of three plant species (total 45 plants) and two lamina segments of 0.5 cm 2 (including midrib) were selected from each leaf. Stem: From each plant of three plant species two stem segments were collected. The length of the stems was 1 cm in length and breadth was 0.3 cm. Root: Root segments (1 cm length) were selected randomly from each plant of the three plant species.

4 246 BANERJEE et al. Surface sterilization Samples were thoroughly washed under running tap water before surface sterilization, then the selected disease free plant parts were immersed in 95% ethanol for 1 minute. The plant parts were then immersed in 4% sodium hypochlorite (NaOCl) solution for 3 minutes (5 minutes for stem segments). Afterwards, the plant parts were dipped in 95% ethanol for 30 seconds [13]. The samples were then rinsed with sterile distilled water three times and allowed to surface dry under sterile conditions. Isolation of endophytes Surface sterile leaf, stem and root segments were plated on water agar (15 g/l) medium amended with streptomycin (100 mg/l) in 10 cm diameter Petri dishes. The Petri dishes were incubated in a light chamber for 1 2 weeks at 27 ºC [16]. After 7 15 days individual fungal colonies were picked from the edge with sterile, fine tipped needle and transferred to potato dextrose agar (PDA) medium. After 2 3 days of incubation, mycelial growth was observed and streaked on PDA slants and preserved at 4 ºC. Statistical analysis Different diversity parameters were calculated using PAST PAlaeontological Statistics software, ver available free from the internet. CF=(N COL /N t ) 100 N COL = number of segments colonized by each fungus; N t = total number of segments. Simpson s diversity (1-dominance), Shannon-Wiener index, and Evenness of the fungal endophytes were calculated using the following formulas: Simpson s diversity: D' =1 S (pi) 2 Pi = proportion of frequency of colonization of i th species in a medicinal plant. Shannon-Wiener index: H' = S s (pi log 2 pi) S = species richness, pi = proportion of frequency of colonization of i th species in a medicinal plant. Evenness: E =H/ln(S)

5 FUNGAL ENDOPHYTES OF LAMIACEAE 247 H = Shannon-Wiener index; S = species richness Jaccard s similarity coefficient was calculated for the host species based on the colonizing endophytic fungi using the formula: Similarity coefficient = C/ (A +B+C) A and B are the total numbers of endophytic fungal genera recovered from any two hosts and C is the number of the common fungal species. Results and Discussion All three medicinal plants had a huge number of fungal assemblages. Altogether 103 fungal endophytes were isolated from 135 segments of leaf, stem and root of the three medicinal plants. The 103 fungal endophytes belonged to fourteen genera (Table II). Twelve, six and ten different endophytic fungal genera colonized the three medicinal plants Ocimum sanctum, Ocimum bacilicum and Leucas aspera, respectively. Previous studies also indicated similar results, where different numbers of endophytic fungi were isolated from some medicinal plants [13]. The highest number of fungal endophytes was isolated from O. sanctum (12). Similarly, 13 genera of endophytic fungi were isolated from medicinal plant Table II Colonization frequency of endophytes from different segments of three medicinal plants Isolated fungal genera O. sanctum O. bacilicum L. aspera L S R L S R L S R Aspergillus sp Alternaria sp Aureobasidium sp Colletotrichum sp Curvularia sp Epicoccum sp Fusarium sp Hyalopus sp Hymenula sp Penicillium sp Phoma sp Spicaria sp Tricoderma sp Tubercularia sp Yeast Mycelia sterilia L Leaf; S Stem; R Root

6 248 BANERJEE et al. Spondias mombin [15], and eleven fungal genera were isolated from medicinal plant Callicarpa tomentosa [13]. Most of the endophytic fungi colonized the leaves of all three medicinal plants. Leaves of O. sanctum were colonized by the maximum number of endophytes (11). This is a clear evidence for the tissue specificity of endophytes. Previous researchers also observed tissue specificity of endophytes in their studies [13, 18, 19]. All the statistical analyses were done using the formulae stated by Raviraja [13] with the statistical software PAST. Species diversity of endophytic fungi was determined using the Shannon-Wiener index, Simpson s diversity and Fisher alpha etc. O. sanctum showed the highest Shannon-Wiener index (2.256 see Table III) with highest Fisher alpha (3.09), which is similar to that of the leaves of D. octopetela that had highest index (2.07) value shown by Fisher et al. [20]. O. sanctum (0.8654) had the highest Simpson s diversity indicating great species specificity. Table III Diversity indices, evenness, and species richness of endophytic fungi isolated from three medicinal plants Parameters O. sanctum O. bacilicum L. aspera Taxa_S Individuals Dominance_D Shannon_H Simpson_1-D Evenness_e^H/S Equitability_J Fisher_alpha Berger-Parker Similarity coefficient was calculated to determine the colonization similarity of fungal endophytes in different host plants. For all plant species pairs the similarity coefficient ranged between 28 60% (Table IV). O. bacilicum and L. aspera had the highest similarity coefficient (60%). Some fungal genera showed host specificity. In the present study Curvularia sp., Hymenula sp., Tricoderma sp. and Tubercularia sp. exclusively colonized O. sanctum; whereas Alternaria sp. and Spicaria sp. colonized only L. aspera. Previous studies also supported host specificity of fungal endophytes [13, 16, 21].

7 FUNGAL ENDOPHYTES OF LAMIACEAE 249 Table IV Similarity coefficient of three medicinal plants Ocimum sanctum Ocimum bacilicum Leucus aspera Ocimum sanctum Ocimum bacilicum Leucus aspera 100 Acknowledgement We are thankful to University Grants Commission (UGC), New Delhi, for financial assistance in the major research project (F No /2006). References 1. Bacon, C. W., White, J. F.: Microbial Endophytes. Marcel Dekcker Inc., New York (2000). 2. Strobel, G., Daisy, B.: Bioprospecting for microbial endophytes and their natural products. Microbiology and Molecular Biology Reviews 67, (2003). 3. Carroll, G. C., Carroll, F. E.: Studies on incidence of coniferous needle endophytes in the specific Northwest. Can J Bot 56, (1988). 4. Arnold, A. E., Maynard, Z., Gilbert, G. S., Coley, P. D., Kursar, T. A.: Are tropical fungal endophytes hyperdiverse? Ecology Letters 3, (2000). 5. Schweigicofler, W., Prillinger, H.: Analysis of endophyte and latent pathogenic fungi isolated from wooden parts of grapevine Vitis vinifera in Austria and Tyrol. Plant Pathology 47, (1997). 6. Ganley, R. J., Brunsfeld, S. J., Newcombe, G.: A community of unknown, endophytic fungi in western white pine. Proceedings of the National Academy of Science 101, (2004). 7. Azevedo, J. L., Maccheroni, W. J. R., Pereira, J. O., De Araujo, W.: Endophytic microorganisms: A review on insect control and recent advances on tropical plants. Electronic Journal of Biotechnology 3 (2000) Arnold, A. E., Mejia, L. C., Kyllo, D., Rojas, I., Robinson, N., Herre, E. A.: Fungal endophytes limit pathogen damage in a tropical tree. Proceeding of the National Academy of Science USA 100, (2003). 9. Clay, K.: Fungal endophytes of grasses: A defensive mutualism between between plants and fungi. Ecology 69, (1998). 10. Redman, R. S., Sheehan, K. B., Stout, R. G., Rodriguez, R. J., Hanson, J. M.: Thermotolarence generated by plant fungal symbiosis. Science 298, 1589 (2002). 11. Petrini, O.: Taxonomy of endophytic fungi of aerial plant tissues. In: Fokkema, N. J. Van Den Heuvel, J. (eds): Mcrobiology of the Phyllosphere. Cambridge University Press, Cambridge, UK, pp (1986). 12. Fisher, P. J., Sutton, B. C., Petrini, L. E., Petrini, O.: Fungal endophytes from Opuntina stricta: A first report. Nova Hedwigia 59, (1994).

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