1. Mycoendophytic Diversity Associated with Ethanomedicinal plants Ficus pumila Linn and Mirabilis jalapa Linn. Mycoendophytic Diversity Associated with Ethanomedicinal plants Ficus pumila Linn and Mirabilis jalapa Linn. Visiting Scholar Department of Plant Pathology University of Georgia Athens 30602, USA Ph:+1-706-715-1643 (M) e-mail: satish@uga.edu Department of Studies in Microbiology University of Mysore Manasagangotri Mysore, 570 006 Karnataka, India Ph:+91-821-2419734 (O) +91-9448323129 (M) e-mail: satish.micro@gmail.com Dr. S.Satish

2. Endophytes are the microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects Significance of endophytic Diversity - Endophytic mycobiome forms a significant part of the microbial diversity. - Endophytes comprise a large hidden component of fungal biodiversity (Arnold, 2007 and Rodriguez et al., 2009). - Taxol (Plaxitaxel): Produced by Taxomyces andreanae, endophytic fungus from Taxus brevifolia Unexplored or untapped source of novel bioactive metabolites. - Diverse biological activity due their unpresedented structural diversity (antibacterial, antifungal, antitumoral, antiviral, antioxidant, imunossupressor, insecticide) (Strobel et al., 2004) Endophytes

3. Endophytic fungi inhabit almost all variety of plant species. The diversity of fungal endophytes mainly depends on the type of plant species and geographical area. Plants with ethno botanical history form important criteria towards screening of endophytes which may lead to isolate endophyte with potent activity and few of the scientific reports suggests that plants growing in rich biodiversity lodge diverse group of endophytes with activity. Fungal diversity

4. Plant-associated microorganisms, especially endophytic fungi are largely underexplored in the discovery of natural products (Strobel and Daisy, 2003; Gunatilaka et al., 2006)

5. Figure : Habit of Mirabilis jalapa Linn. Botanical Name: Mirabilis jalapa Linn. Family: Nyctaginaceae (Bougainvillea family) Common name: Four O’ clock plant, Beauty-of- the-night, Marvel of Peru, Gulamaji (Local Native Name) Figure : Habit of Ficus pumila Linn. Botanical Name: Ficus pumila Linn. Family: Moraceae (Mulberry family) Common name: Creeping fig Medicinal plants selected for the isolation of endophytic fungi

6. Plant collection Plant materials of Ficus pumila and Mirabilis jalapa were collected during 2010-2011 in three different season i.e., summer, monsoon and winter in Mysore, KarntakaPlant materials of Ficus pumila and Mirabilis jalapa were collected during 2010-2011 in three different season i.e., summer, monsoon and winter in Mysore, Karntaka Isolation of endophytes Isolation of endophytic fungi was carried out according to the procedure of Schulz et al., (2003).Isolation of endophytic fungi was carried out according to the procedure of Schulz et al., (2003). Identificatio n of endophytes Identification of endophytic fungi was carried out based on the macroscopic and microscopic observations, following standard manuals (Gilman 1956; Wei, 1979; Sutton, 1980; Barnett and Hunter, 1998; Leslie and Summerelle, 2006) with some morphospeices (Xylaria) were identified by Molecular technique using PCR.Identification of endophytic fungi was carried out based on the macroscopic and microscopic observations, following standard manuals (Gilman 1956; Wei, 1979; Sutton, 1980; Barnett and Hunter, 1998; Leslie and Summerelle, 2006) with some morphospeices (Xylaria) were identified by Molecular technique using PCR. Data analyses To assess and quantify endophytic fungal diversity between two host plants following diversity indices were usedTo assess and quantify endophytic fungal diversity between two host plants following diversity indices were used Colonization rate (CR) (Photita et al., 2001)Colonization rate (CR) (Photita et al., 2001) Isolation rate (IR) (Maheshwari and Rajagopal, 2013)Isolation rate (IR) (Maheshwari and Rajagopal, 2013) Colonization frequency (CF) (Hata and Futai, 1995)Colonization frequency (CF) (Hata and Futai, 1995) Aim of the present investigation

7. Fig: Overview of the procedure of isolation, cultivation and identification of endophyte fungi

8.  3 Ascomycetes genera: Chaetomium sp., Sporomia sp. and Xylaria sp. (10.92%),  5 Coelomycetes genera: Colletotrichum sp., Pestalotiopsis sp., Phoma sp., Phomopsis sp. and Phyllosticta sp. (18.06%),  11 Hyphomycetes genera: Acremomium sp., Alternaria sp., Aspergillus sp., Cladosporium sp., Curvularia sp., Drechslera sp., Fusarium sp., Myrothecium sp., Nigrospora sp., Penicillium sp. and Trichoderma sp. (55.46%)  2 Zygomycetes genera : Mucor sp. and Rhizopus sp. (2.94%),  2 Morphospecies of Mycelia sterilia (8.4%). 1200 plant segments 238(19.83%)238(19.83%) 23 Taxa, comprising Results

9. Colonization and isolation rates  99 segments (50.51%) of the stem and 97 segments of (49.48%) leaf were colonized with endophytic fungi from both the plants.  Colonization rates (%) of endophytic fungi for both the tissues were found to be relatively homogeneous (14.33% and 14.0%) and (18.66% and 18.33%) for Mirabilis jalapa and Ficus pumila (Table 1 and 2 ).  Isolation rates of endophytic fungi in leaves of both plants were relative high when compared to stem in both plants, endophytic fungal isolates were prevalent on leaf tissue of plants (52.94%) than on stem tissue (47.0%) (Table 1 and 2 ).

10. Colonization frequency Aspergillus flavus and Xylaria sp., are dominant endophytic fungal genera in leaf while in stem species of Fusarium and Pestalotiopsis were dominant in Mirabilis jalapa and Ficus pumila respectively. Dominance and species richness  Despite the similar patterns of species accumulation, endophytes segregated with varying degree of colonization during three sampling season among different tissues of the two hosts.  Overall diversity indices values for foliar endophytes of Ficus pumila was relatively high when compared to leaf and stem tissues of Mirabilis jalapa.  But species richness was higher in stems of Mirabilis jalapa when compared with leaves and stems of Ficus pumila due the isolation 19 different endophytic fungal taxa.

11. Cladosporium sp. Colletotrichum sp. Curvularia sp. 1 Cladosporium sp. Colletotrichum sp. Curvularia sp. 1 Curvuilaria sp. 2 Dreschlera sp. Fusarium sp. 1 Curvuilaria sp. 2 Dreschlera sp. Fusarium sp. 1 Cultural, Morphological and microscopic Characteristics of Major endophytic fungal genera isolated in the present study

12. A. flavusA.niger Acermonium sp. A. flavusA.niger Acermonium sp. Alternaria sp. Chaetomium sp. 1 Chaetomium sp. 2 Alternaria sp. Chaetomium sp. 1 Chaetomium sp. 2

13. F. oxysporumFusarium sp. 2 Morphospecies sp. 1 F. oxysporumFusarium sp. 2 Morphospecies sp. 1 Morphospecies sp. 2 Morphospecies sp. 3 Morphospecies sp. 4 Morphospecies sp. 2 Morphospecies sp. 3 Morphospecies sp. 4

14. F. oxysporum Fusarium sp. 2 Morphospecies sp. 1 F. oxysporum Fusarium sp. 2 Morphospecies sp. 1 Morphospecies sp. 2 Morphospecies sp. 3 Morphospecies sp. 4

15. Pestalotiopsis sp.2 Phoma sp Phomopsis sp 1 Pestalotiopsis sp.2 Phoma sp Phomopsis sp 1 Phomopsis sp 2 Trichoderma sp Phomopsis sp 2 Trichoderma sp

16. StemLeafTotal No. of segments300 600 No. of segments yielding fungi21+12+10=4316+19+07=4285 No. of isolates26+14+10=5020+24+07=51101 Isolation rate0.160.170.16 Colonisation rate (%)14.331414.16 Table 1. Isolation and colonization and rate of endophytic fungi of Mirabilis jalapa Linn. StemLeafTotal No. of segments300 600 No. of segments yielding fungi15+21+20 =5618+19+18 =55111 No. of isolates 16+23 +23 =62 26 +25 +24=75137 Isolation rate0.200.250.22 Colonization rate (%)18.6618.330.18 Table 2. Isolation and colonization rate of endophytic fungi of Ficus pumila Linn.

17. ClassEndophyteStem CF (%) Leaf AscomycetesChaetomium sp.120.6610.33 Chaetomium sp.241.3-- Sporormia sp.--10.33 CoelomycetesPhoma sp.31.0-- Phomopsis sp.182.6-- Phomopsis sp.231.0-- HyphomycetesAcremonium sp.--10.33 Alternaria alternata--20.66 Aspergillus flavus--72.33 Aspergillus niger10.3351.66 Aspergillus sp.110.3320.66 Aspergillus sp.2--10.33 C. Cladosporioides--20.66 C. herbarum10.331 Cladosporium sp.--20.66 Curvularia sp.110.3331.0 Curvularia sp.210.33-- Drechslera sp.10.3331.0 Fusarium sp.182.6-- Fusarium sp.241.3-- Fusarium oxysporum--20.66 Penicillium sp.--20.66 Penicillium chrysogenum10.3331.0 Trichoderma sp.162.0-- Trichoderma sp.220.66-- ZygomycetesMucor sp.110.33-- Rhizopus sp.110.3320.66 Mycelia steriliaMorphospecies-110.3351.6 Morphospecies-2--62.0 Table 3. Diversity of endophytic fungi isolated from stem and leaf of Mirabilis jalapa Linn. during three different season (2010-2011)

18. ClassEndophyteStem CF (%) Leaf AscomycetesChaetomium sp.010.33051.66 Sporormia sp.--010.33 Xylaria sp.--113.66 CoelomycetesColletotrichum sp.020.66031.00 Pestalotiopsis sp.1072.33-- Pestalotiopsis sp.2072.33-- Phoma sp.062.00-- Phomopsis sp.1062.00-- Phomopsis sp.2062.00-- Phyllosticta sp.--020.66 HyphomycetesAcremonium sp.--031.00 Alternaria alternata--062.00 Alternaria sp.--010.33 Aspergillus flavus--041.33 Aspergillus niger--041.33 Aspergillus sp.--062.00 Cladosporium sp.--051.66 Curvularia sp.--072.33 Drechslera sp.--062.00 Fusarium sp.072.33 Myrothecium sp.062.0-- Nigrospora sp.041.3-- Penicillium sp.1--041.33 Trichoderma sp.062.0-- ZygomycetesRhizopus sp.020.66010.33 Mycelia SteriliaMorphospecies-1020.66041.33 Morphospecies-2--020.66 Table 4. Diversity of endophytic fungi isolated from stem and leaf tissue of Ficus pumila Linn.

19. Endophytes Tissue of the host plant SummerMonsoonWinter No. of endophytes AscomycetesStem033 Leaf200 CoelomycetesStem392 Leaf000 HyphomycetesStem7119 Leaf31617 ZygomycetesStem020 Leaf002 Mycelia steriliaStem010 Leaf245 Total174638 Table 5. Seasonal distribution of endophytic fungi in different tissues of Mirabilis jalapa Linn. during three different season (2010-2011).

20. Endophytes Tissue of the host plant SummerMonsoonWinter No. of endophytes AscomycetesStem00010 Leaf0665 CoelomycetesStem1012 Leaf0311 HyphomycetesStem04813 Leaf171611 ZygomycetesStem00020 Leaf0001 Mycelia steriliaStem02000 Leaf00024 Total424847 Table 7: Seasonal distribution of endophytic fungi in different tissues of Ficus pumila Linn. during three different season (2010-2011)

21. THANK YOU