Mycoendophytic Diversity Associated with Ethanomedicinal Mycoendophytic Diversity Associated with Ethanomedicinal plants plants Ficus pumilaFicus pumila Linn and Linn and Mirabilis jalapa Mirabilis jalapa Linn.Linn.

Visiting ScholarVisiting ScholarDepartment of Plant PathologyUniversity of GeorgiaAthens 30602, USAPh:+1-706-715-1643 (M) e-mail: satish@uga.edu

Department of Studies in MicrobiologyUniversity of MysoreManasagangotriMysore, 570 006Karnataka, IndiaPh:+91-821-2419734 (O) +91-9448323129 (M)e-mail: satish.micro@gmail.com

Dr. S.SatishDr. S.Satish

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)

EndophytesEndophytes

Endophytic fungi inhabit almost all variety of plant species. The diversity of fungal endophytes 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. mainly depends on the type of plant species and geographical area. Plants with ethno botanical Plants with ethno botanical

history form important criteria towards screening of endophytes which may lead to isolate 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 endophyte with potent activity and few of the scientific reports suggests that plants growing in

rich biodiversity lodge diverse group of endophytes with activity.rich biodiversity lodge diverse group of endophytes with activity.

Fungal diversityFungal diversity

Plant-associated microorganisms, especially endophytic fungi are largely underexplored in Plant-associated microorganisms, especially endophytic fungi are largely underexplored in

the discovery of natural products (Strobel and Daisy, 2003; Gunatilaka et al., 2006)the discovery of natural products (Strobel and Daisy, 2003; Gunatilaka et al., 2006)

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 fungiMedicinal plants selected for the isolation of endophytic fungi

Aim of the present investigationAim of the present investigation

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

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 1200 plant segments segments 1200 plant 1200 plant segments segments

238238(19.83%)(19.83%)

238238(19.83%)(19.83%)

23 Taxa, comprising

Results

Colonization and isolation ratesColonization 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 ).

Colonization frequencyColonization 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 richnessDominance 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.

Cladosporium Cladosporium sp. sp. Colletotrichum Colletotrichum sp. sp. Curvularia Curvularia sp. 1sp. 1

Curvuilaria Curvuilaria sp. 2 sp. 2 Dreschlera Dreschlera sp. sp. Fusarium Fusarium sp. 1sp. 1

Cultural , Morphological and microscopic Characteristics of Major Cultural , Morphological and microscopic Characteristics of Major endophytic fungal genera isolated in the present study endophytic fungal genera isolated in the present study

A. flavusA. flavus A.nigerA.niger Acermonium Acermonium sp.sp.

Alternaria Alternaria sp. sp. ChaetomiumChaetomium sp. 1 sp. 1 Chaetomium Chaetomium sp. 2sp. 2

F. oxysporumF. oxysporum Fusarium Fusarium sp. 2sp. 2 Morphospecies Morphospecies sp. 1sp. 1

Morphospecies Morphospecies sp. 2 sp. 2 Morphospecies Morphospecies sp. 3 sp. 3 Morphospecies Morphospecies sp. 4sp. 4

F. oxysporum F. oxysporum Fusarium Fusarium sp. 2sp. 2 Morphospecies Morphospecies sp. 1sp. 1

Morphospecies Morphospecies sp. 2 sp. 2 MorphospeciesMorphospecies sp. 3 sp. 3 MorphospeciesMorphospecies sp. 4 sp. 4

Pestalotiopsis Pestalotiopsis spsp..22 Phoma Phoma spsp Phomopsis Phomopsis sp 1sp 1

PhomopsisPhomopsis sp 2 sp 2 Trichoderma Trichoderma spsp

StemStem LeafLeaf TotalTotal

No. of segments 300 300 600

No. of segments yielding fungi 21+12+10=43 16+19+07=42 85

No. of isolates 26+14+10=50 20+24+07=51 101

Isolation rate 0.16 0.17 0.16

Colonisation rate (%) 14.33 14 14.16

Table 1. Isolation and colonization and rate of endophytic fungi of Table 1. Isolation and colonization and rate of endophytic fungi of Mirabilis jalapa Mirabilis jalapa Linn.Linn.

StemStem LeafLeaf TotalTotal

No. of segments 300 300 600

No. of segments yielding fungi 15+21+20 =56 18+19+18 =55 111

No. of isolates 16+23 +23 =62 26 +25 +24=75 137

Isolation rate 0.20 0.25 0.22

Colonization rate (%) 18.66 18.33 0.18

Table 2. Isolation and colonization rate of endophytic fungi of Table 2. Isolation and colonization rate of endophytic fungi of Ficus pumila Ficus pumila Linn.Linn.

ClassClass Endophyte Endophyte StemStem CF (%)CF (%) LeafLeaf CF (%)CF (%)Ascomycetes Chaetomium sp.1 2 0.66 1 0.33

Chaetomium sp.2 4 1.3 - -Sporormia sp. - - 1 0.33

Coelomycetes Phoma sp. 3 1.0 - -Phomopsis sp.1 8 2.6 - -

Phomopsis sp.2 3 1.0 - -Hyphomycetes Acremonium sp. - - 1 0.33

Alternaria alternata - - 2 0.66

Aspergillus flavus - - 7 2.33Aspergillus niger 1 0.33 5 1.66Aspergillus sp.1 1 0.33 2 0.66Aspergillus sp.2 - - 1 0.33

C. Cladosporioides - - 2 0.66

C. herbarum 1 0.33 1 0.33Cladosporium sp. - - 2 0.66

Curvularia sp.1 1 0.33 3 1.0Curvularia sp.2 1 0.33 - -Drechslera sp. 1 0.33 3 1.0Fusarium sp.1 8 2.6 - -

Fusarium sp.2 4 1.3 - -Fusarium oxysporum - - 2 0.66Penicillium sp. - - 2 0.66Penicillium chrysogenum 1 0.33 3 1.0

Trichoderma sp.1 6 2.0 - -Trichoderma sp.2 2 0.66 - -

Zygomycetes Mucor sp.1 1 0.33 - -Rhizopus sp.1 1 0.33 2 0.66

Mycelia sterilia Morphospecies-1 1 0.33 5 1.6Morphospecies-2 - - 6 2.0

Table 3. Diversity of endophytic fungi isolated from stem and leaf of Table 3. Diversity of endophytic fungi isolated from stem and leaf of Mirabilis jalapa Mirabilis jalapa Linn. during three different season (2010-2011)Linn. during three different season (2010-2011)

ClassClass Endophyte Endophyte StemStem CF (%)CF (%) LeafLeaf CF (%)CF (%)

Ascomycetes Chaetomium sp. 01 0.33 05 1.66

Sporormia sp. - - 01 0.33

Xylaria sp. - - 11 3.66

Coelomycetes Colletotrichum sp. 02 0.66 03 1.00

Pestalotiopsis sp.1 07 2.33 - -

Pestalotiopsis sp.2 07 2.33 - -

Phoma sp. 06 2.00 - -

Phomopsis sp.1 06 2.00 - -

Phomopsis sp.2 06 2.00 - -

Phyllosticta sp. - - 02 0.66

Hyphomycetes Acremonium sp. - - 03 1.00

Alternaria alternata - - 06 2.00

Alternaria sp. - - 01 0.33

Aspergillus flavus - - 04 1.33

Aspergillus niger - - 04 1.33

Aspergillus sp. - - 06 2.00

Cladosporium sp. - - 05 1.66

Curvularia sp. - - 07 2.33

Drechslera sp. - - 06 2.00

Fusarium sp. 07 2.33

Myrothecium sp. 06 2.0 - -

Nigrospora sp. 04 1.3 - -

Penicillium sp.1 - - 04 1.33

Trichoderma sp. 06 2.0 - -

Zygomycetes Rhizopus sp. 02 0.66 01 0.33

Mycelia Sterilia Morphospecies-1 02 0.66 04 1.33

Morphospecies-2 - - 02 0.66

Table 4. Diversity of endophytic fungi isolated from stem and leaf tissue of Table 4. Diversity of endophytic fungi isolated from stem and leaf tissue of Ficus pumilaFicus pumila Linn. Linn.

EndophytesEndophytes Tissue of the host Tissue of the host

plantplantSummerSummer MonsoonMonsoon WinterWinter

No. of endophytesNo. of endophytes No. of endophytesNo. of endophytes No. of endophytesNo. of endophytes

Ascomycetes Stem 0 3 3Leaf 2 0 0

Coelomycetes Stem 3 9 2Leaf 0 0 0

Hyphomycetes Stem 7 11 9Leaf 3 16 17

Zygomycetes Stem 0 2 0Leaf 0 0 2

Mycelia sterilia Stem 0 1 0Leaf 2 4 5

Total 17 46 38

Table 5. Seasonal distribution of endophytic fungi in different tissues of Table 5. Seasonal distribution of endophytic fungi in different tissues of Mirabilis jalapa Mirabilis jalapa Linn.Linn.during three different season (2010-2011).during three different season (2010-2011).

EndophytesEndophytes Tissue of the host Tissue of the host

plantplantSummerSummer MonsoonMonsoon WinterWinter

No. of endophytesNo. of endophytes No. of endophytesNo. of endophytes No. of endophytesNo. of endophytes

Ascomycetes Stem 00 01 0Leaf 06 6 5

Coelomycetes Stem 10 12 12Leaf 03 1 1

Hyphomycetes Stem 04 8 13Leaf 17 16 11

Zygomycetes Stem 00 02 0Leaf 00 0 1

Mycelia sterilia Stem 02 00 0Leaf 00 02 4

Total 42 48 47

Table 7: Seasonal distribution of endophytic fungi in different tissues of Table 7: Seasonal distribution of endophytic fungi in different tissues of Ficus pumila Ficus pumila Linn.Linn.during three different season (2010-2011)during three different season (2010-2011)

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