Research Article | Volume: 8, Issue: 6, Nov-Dec, 2020

Distribution and diversity of endophytic fungi associated with three medicinal tree species from Eturnagaram Wildlife Sanctuary, TS, India

Bhavani Vemireddy Abhinesh Madasi Aruna Ajmeera Krishna Reddy Vanteru   

Open Access   

Published:  Nov 25, 2020

DOI: 10.7324/JABB.2020.80602
Abstract

Distribution and diversity of endophytic fungal species associated with three medicinal tree species Phyllanthus emblica Linn, Terminalia chebula Retz, and Terminalia bellirica Roxb, whose fruits constitute the triphala an Ayurvedic formulation was assessed, in terms of isolation rate (IR), colonization frequency (CF), absolute frequency (f), relative frequency (RF), and species diversity. Leaf, stem, and bark were selected for the isolation of endophytic fungi. A large number of fungal species representing Hyphomycetes, coelomycetes, and sterile mycelia were found to be associated with different plant parts. Hyphomycetes dominated in all the fungi. Maximum IR was recorded in bark. Diversity indices (Shannon–Wiener and Simpson) varied with the tree species. The diversity richness of fungal endophytes points out facts that these fungal endophytes may play a significant role in plant health, metabolism, and medicinal attributes.


Keyword:     Absolute frequency Colonization frequency Endophytic fungi Isolation rate Relative frequency Species diversity.


Citation:

Vemireddy B, Madasi A, Ajmeera A, Vanteru KR. Distribution and diversity of endophytic fungi associated with three medicinal tree species from Eturnagaram Wildlife Sanctuary, TS, India. J App Biol Biotech. 2020;8(6):7-12. https://dx.doi.org/10.7324/JABB.2020.80602

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. INTRODUCTION

Endophytic fungi colonize internal plant tissues without causing observable harm to their host. Fungal endophytes are a diverse group forming associations with almost all plants of the plant kingdom. They have been isolated from diverse plants including trees, fodders, vegetables, fruits, and medicinal plants [1]. Their biological diversity is enormous, especially in temperate and tropical vegetation’s. Fungal endophytes confer many benefits to host plants and enable them to withstand abiotic stresses such as drought [2] and metal contamination [3]. Endophytes may also benefit host plants by preventing the colonization of pathogenic and predatory organisms. They have been found to be repertoire of novel organic compounds with broad spectrum of biological activities. Their secondary metabolites are particularly active because of their interaction with host [4]. The metabolites include varied structural groups such as terpenoids, steroids, xanthones, chinones, phenols, isocoumarins, benzopyranones, tetralones, and enniatines [5] that are known to exhibit antibacterial, antiviral, antifungal, and anticancer activities [6].

Triphala is a popular, traditional Ayurvedic formulation consisting of equal parts of three myrobalans, namely, Amla (Phyllanthus emblica Linn.), Harada (Terminalia chebula Retz.), and Bahera (Terminalia bellirica Roxb.). Charaka Samhita, an authoritative treatise on Ayurvedic medicine, has mentioned the potential uses of Triphala to treat any kind of disease. In combination, the three tree myrobalans exhibit anti-cleansing property including blood, reduce blood pressure, decrease levels of cholesterol, and removes harmful toxins from liver [7]. In view of wide range of well know medicinal properties of these three plants, it is proposed to investigate the distribution and diversity of endophytic fungal flora of these plants and their possible role in imparting the medicinal attributes to these three medicinal plants.


2. MATERIALS AND METHODS

2.1. Plant Materials

The selected plants for the present study is Amla (Phyllanthus emblica Linn; family: Phyllanthaceae), Harada (Terminalia chebula Retz; family: Combretaceae), and Bahera (Terminalia bellirica Roxb; family: Combretaceae), whose fruits together constitute the Triphala.

2.2. Collection of Plant Materials

The different plant parts, namely, stem, bark, and leaves of P. emblica, T. chebula, and T. bellirica (Each plant 60 samples) were collected from different locations of Eturnagaram wildlife sanctuary, Mulugu district, Telangana state, India. The Eturnagaram Wildlife Sanctuary is basically a tropical dry deciduous forest. It is regarded as one of the rich species diversity terrestrial ecosystems. The entire area of the Eturnagaram sanctuary is about 812 sq. km and the geographical coordinates, that is, latitude 18.3378° N and longitude 80.4299°E with an average temperature 28.3°C and rainfall of 821 mm/32.3 in/year. From this thick, forest healthy and disease free plants were selected for sampling. The plant parts were carefully collected and brought to the laboratory in sterilized polythene bags and processed within 24 h.

2.3. Isolation and Morphological Identification of Endophytic Fungi

The collected samples were first cleaned thoroughly in gentle running tap water to remove dirt and debris [8]. The stem, bark, and leaves were cut into segments (0.5–1 cm) and surface sterilized by modified method of Dobranic et al. (1995) [9]. The samples were immersed in 75% ethanol for 60 s, followed by 4% sodium hypochlorite for 180 s and then again in 75% ethanol for 30 s. Later, the segments were washed with sterile distilled water for 3 times and the excess moisture was blotted on Whatman no.1 filter paper. The surface sterilized segments were seeded in Petri dishes containing potato dextrose agar (PDA) medium supplemented with streptomycin.

The seeded Petri dishes were incubated at 26 ± 1°C and maintained dark-light cycles for 5–7 days. Sterilization and inoculations were performed in laminar air flow.

The pure colonies of fungi growing out of the segments were sub cultured on separate PDA slants and stored at 4°C for further process. The fungi were identified based on the morphology of cultural characteristics and direct microscopic observations of hyphae, reproductive structures and with the help of standard manuals and monographs [10,11].

2.4. Data Analysis

2.4.1. Colonization frequency (CF)

The CF of endophytic fungi isolated from the three tree species was calculated by following formula suggested by Suryanarayana et al. (2003) [12] and expressed in percentage.



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2.4.2. Relative frequency (RF)

RF used to represent fungal density was calculated as described by Huang et al. (2008) [13].

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2.4.3. Isolation rate (IR)

IR, the measure of fungal richness of a sample was calculated as the number of isolates obtained from tissue segments divided by the total number of segments expressed, as fractions [14].



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2.4.4. Species diversity

Simpson’s Index of diversity was calculated using the following formula.


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Where,

n = The total number of organisms of a particular species

N = The total number of organisms of all species

Shannon–Wiener diversity index was calculated using the following formula.

S

HS = ∑ (Pi) (In Pi)

Where, i = 1

Hs: Symbol for the diversity in a sample of S species or kinds

S: The number of species in the sample

Pi: Relative abundance of ith species or kinds measures, = n/N

N: Total number of individuals of all kinds

ni: Number of individuals of ith species

In: Log to base 2


3. RESULTS AND DISCUSSION

The results obtained in the present investigations are presented in Tables 1 and 2, and Figures 3-6 A total of 348 fungal isolates were obtained from 540 segments of three tree species parts, namely, leaf, stem, bark of P. emblica, T. chebula, and T. bellirica [Table 1]. They represented to 38 species which were belonging to 16 genera, in which 27 hyphomycetes, four coelomycetes, one ascomycetes, and six non-sporulating sterile mycelia. In P. emblica, 25 species belongs to 12 genera whereas, in leaves highest CF was shown by Cladosporium herbarum (10%) and least was shown by Chaetomium globosum and Curvularia spicifera, Trichoderma harzianum (1.6%). CF of Colletotrichum falcatum (6.6%) was highest in stem and least was in sterile mycelium II (1.6%). In bark, highest CF was recorded by Trichoderma harzianum (10%) and least was in Aspergillus nidulans and sterile mycelium (1.6%). In T. chebula, 18 species belonged to 12 genera. In the leaves, highest CF was recorded for Phyllosticta vaccinii (16.6%) and least for Aspergillus terreus and Curvularia spicifera (3.3%) and CF of Aspergillus niger (5%) was highest in stem and least was shown by Penicillium chrysogenum and Colletotrichum falcatum (1.6%), whereas in bark, the highest CF was recorded for Fusarium dimerium (20%) and least was in Aspergillus nidulans and Phoma destructiva (1.6%). In T. bellirica 22 species belonged to 12 genera. In the leaves, the highest CF was recorded for sterile mycelium VI (11.6%) and least for Fusarium semitectum (1.6%) and CF of Penicillium chrysogenum (11.6%) was highest in stem and least for Curvularia lunata and Alternaria fasciculata (1.6%). In bark, the highest CF was recorded by Penicillium expansum (20%), followed by sterile mycelium VI (15%) and least was shown by Acremonium strictum, Aspergillus niger, Curvularia lunata, Cylindrophora apiculata, Drechslera spicifera, Fusarium semitectum, and Penicillium rubrum (1.6%), respectively [Table 1].

Table 1: Colonization frequency of endophytic fungal species isolated from Triphala tree species.

Endophytic fungi Phyllanthus emblica Terminalia chebulaTerminalia bellirica


LeafStemBarkLeafStemBarkLeafStemBark(f)RF%









NOICF%NOICF%NOICF%NOICF%NOICF%NOICF%NOICF%NOICF%NOICF%
Acremonium strictum------------35%--11.6%41.1
Alternaria alternata------58.3%--------23.3%72
A. fasciculata--------------11.6%23.3%30.8
Aspergillus flavus------------46.6%----41.1
A. fumigatus----46.6%--------23.3%--61.7
A. niger35%--46.6%610%35%--610%23.3%11.6%257.1
A. nidulans----11.6%----11.6%------20.5
A. terreus----23.3%23.3%--35%------72
Chaetomium globosum11.6%------------58.3%--61.7
Cladosporium cladosporioides58.3%23.3%23.3%------------92.5
C. herbarum610------23.3%23.3%------102.8
Colletotrichum falcatum46.6%46.6%----11.6%----58.3%--144
Curvularia lunata----------23.3%--11.6%11.6%41.1
C. spicifera11.6%----23.3%----------30.8
C. inaequalis----23.3%--23.3%--------20.5
Cylindrophora apiculata--------------23.3%11.6%30.8
Drechslera spicifera----23.3%------46.6%23.3%11.6%92.5
Fusarium dimerium----35%813.3%--1220%46.6%----277.7
F. semitectum23.3%23.3%23.3%------11.6%--11.6%82.2
F. sporotrichioides46.6%35%--------------72
F. solani------35%23.3%--------51.4
Nigrospora sphaerica------------23.3%----20.5
Penicillium chrysogenum23.3%--35%--11.6%46.6%46.6%711.6%--216
P. citrinum35.6%12.5%12.5%------------51.4
P. expansum--------------35%1220%154.3
P. notatum--------23.3%23.3%------41.1
P. rubrum----46.6%--------23.3%11.6%72
Trichoderma harzianum11.6%23.3%610%------------92.5
T. viride46.6%--46.6%------------82.2
Phoma destructiva--------23.3%11.6%------30.8
P. glomerata46.6%--46.6%------46.6%----123.4
Phyllosticta vaccinii--35%--1016.6%--35%46.6%23.3%--226.3
Sterile mycelium I58.3%23.3%23.3%------------92.5
Sterile mycelium II46.6%11.6%11.6%610----------123.4
Sterile mycelium III----------35%------30.8
Sterile mycelium IV----35%35%--23.3%------51.4
Sterile mycelium V46.6%--35%------58.3%35%46.6%195.4
Sterile mycelium VI------------711.6%--915%164.5
Total4788.2%2033.9%5290%5074.8%2124.7%3558%4979.4%3056.2%4459.4%348

540 segments were plated for frequency analysis: NOI: number of isolates; CF%: colonization frequency; f: absolute frequency: RF%: relative frequency.

Table 2: Diversity indices of endophytic fungi isolated from Triphala trees species.

IndicesPhyllanthus emblicaTerminalia chebulaTerminalia bellirica



LSBLSBLSB
Simpson’s dominance0.320.150.360.170.120.100.190.050.20
Simpson’s diversity0.920.820.980.830.780.820.860.770.88
Species richness169191089111012
Shannon-Wiener2.751.962.812.101.881.902.051.682.25

L: Leaf; S: Stem; B: Bark

Figure 1: Morphology of Triphala tree species.



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Figure 2: Colony morphology and microphotographs of endophytic fungi.



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Figure 3: Relative frequencies of different endophytic fungal taxa isolated from Triphala trees.



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Figure 4: Isolation rate of fungal endophytes from P. emblica.



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Figure 5: Isolation rate of fungal endophytes from T. chebula.



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Figure 6: Isolation rate of fungal endophytes from T. bellirica.



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Among three tree species of Triphala, the RF of endophytic fungal genera is highest in Fusarium dimerium (%RF = 7.7%), followed by Aspergillus niger (%RF = 7.1%) and the least percentage of frequency present in Aspergillus nidulans, Curvularia inaequalis, and Nigrospora sphaerica (%RF = 0.5%) [Figure 3].

In the plant parts, namely, leaves, stem, and bark of P. emblica maximum fungal endophytic IR were recorded in bark (0.86) and the lowest IR was recorded in stem (0.33). However, in T. chebula, the highest IR was observed in leaves (0.83), and lowest IR was recorded in stem (0.35). In T. bellirica, leaves (0.81) were occupied by highest IR and stem (0.5) was recorded with least IR [Figures 4-6].

Shannon–Wiener and Simpson diversity indices of fungal endophytes varied within three tree species. In P. emblica, high Shannon–Wiener diversity index was recorded in bark (H1 = 2.81), low indices were recorded in stem (H1 = 1.96), where Simpsons index was high in bark with a richness of 19 fungal species. For T. chebula high Shannon–Wiener diversity index was recorded in leaves (H1 = 2.10), low indices were recorded in stem (H1 = 1.88), and Simpsons index was high in leaves with a richness of ten fungal species. In T. bellirica, high Shannon–Wiener diversity index was recorded in bark (H1 = 2.25), low indices were recorded in stem (H1 = 1.68), and Simpsons index was high in bark with a richness of 121 fungal species [Table 2].

Triphala, an Ayurvedic herbal rasayana churna, is known as one of the oldest forms of health care, basically originated in India and it is called as a complete body cleanser. It also purifies blood, reduces blood pressure, and decreases level of cholesterol. Recent studies reveal that many tree species especially medicinal in nature are associated with many fungal endophytes. Endophytic fungi are one of the most unexplored and diverse group of organisms having symbiotic association with higher life forms and may produce beneficial substances to host [15]. The plant tissues, especially leaves and stems, are excellent reservoirs for endophytic fungi [16]. In this context, the present investigations aimed to isolate the endophytic fungi from Triphala tree species (P. emblica, T. chebula, and T. bellirica), to assess their possible role in imparting medicinal properties.

Three tree species were selected for the study from different location of Eturnagaram forest area, stretching over 25 km distance from sampling to sampling. The results of the present study reveal that three tree species of triphala plants are associated with different endophytic fungi belonging to diverse taxonomic groups. From 540 segments of plant material a total 348 isolates were obtained were belongs to 38 fungal taxa, several researchers working on different plants have also noted the association of endophytic fungi [1]. However, a wide variation has been observed in there CF between three plant segments, namely, leaves, stem, and bark. Where, Aspergillus, Fusarium, and Penicillium, Phyllosticta species, and sterile mycelium dominated. The IR also varied in these three plant parts. Diversity indices for fungal endophytes analyzed by Simpson and Shannon–Wiener indices indicated differences in endophytic fungal isolates and species richness [17]. Some of the endophytic fungi reported through this study may be explored for the secondary metabolites and used in pharma and agri-industries. The endophytic fungi isolated from three tree species of triphala from Eturnagaram forest area are rich with fungal diversity.


4. CONCLUSION

The present investigations reveal that the three medicinal plants are associated with large and diverse fungal endophytes in its different parts. The fungal species belonged to different taxonomic groups especially of hyphomycetes. The endophytic fungal distribution varied with the tree species. The contribution of these endophytes in the metabolism and health of the plant needs to be investigated in detail.


5. ACKNOWLEDGMENT

The authors express their sincere thanks to Head, Department of Botany, Kakatiya University, for encouragement and facilities. We are also thankful to Prof. S. Ram Reddy for constructive suggestions at various stages.


6. CONFLICTS OF INTEREST

All authors were declaring that there are no conflicts of interests regarding the publication of this paper.


7. Financial Support and sponsorship

None.

8. REFERENCES

1. Lu Y, Chen C, Zhang J, Chen W. Isolation and identification of endophytic fungi from Actinidia macrosperma investigation of their bioactivities. Evid Based Complement Alternat Med 2012;2012:1-8. [CrossRef]

2. Arechavaleta M, Bacon CW, Hoveland CS, Radcliffe DE. Effect of the tall fescue endophyte on plant response to environmental stress. Agron J 1989;81:83-90. [CrossRef]

3. Malinowski DP, Belesky DP. Adaptations of endophyte-infected cool-season grasses to environmental stresses. Mechanisms of drought and mineral stress tolerance. Crop Sci 2000;40:923-40. [CrossRef]

4. Giridharan P, Verekar SA, Khanna A, Mishra PD, Deshmukh SK. Anticancer activity of sclerotiorin, isolated from an endophytic fungus Cephalotheca faveolata Yaguchi, Nishim. and Udagawa. Indian J Exp Biol 2012;50:464-8.

5. Schulz B, Boyle C, Draeger S, Rommert AK. Endophytic fungi: A source of novel biologically active secondary metabolites. Mycol Res 2002;106:996-1004. [CrossRef]

6. Gunatilaka AA. Natural products from plant-associated microorganisms: Distribution, structural diversity, bioactivity, and implications of their occurrence. J Nat Prod 2006;69:509-26. [CrossRef]

7. Peterson CT, Denniston K, Chopra D. Therapeutic uses of Triphala in Ayurvedic medicine. J Altern Complement Med 2017;23:607-14. [CrossRef]

8. Selvanathan S, Indrakumar I, Johnpaul M. Biodiversity of the endophytic fungi isolated from Calotropis gigantea L. Recent Res Sci Technol 2011;3:94-100.

9. Dobranic JK, Johnson IA, Alikhan QR. Isolation of endophytic fungi from larch (Larix laricina) leaves from New Brunswick, Canada. Can J Microbiol 1995;41:194-8. [CrossRef]

10. Barnett HL, Hunter BB. Illustrated Genera of Imperfect Fungi. New York: Macmillan Publishing Company; 1987.

11. Duong LM, Jeewon R, Lumyong S, Hyde KD. DGGE coupled with ribosomal DNA phylogenies reveal uncharacterized fungal phylotypes on living leaves of Magnolia liliifera. Fungal Divers 2006;23:121-38.

12. Suryanarayanan TS, Venkatesan G, Murali TS. Endophytic fungal communities in leaves of tropical forests trees: Diversity and distribution pattern. Curr Sci 2003;85:489-93.

13. Huang WY, Cai YZ, Hyde KD, Corke H, Sun M. Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Divers 2008;33:61-75.

14. Photia W, Taylor PW, Ford R, Hyde KD, Lumyong S. Morphological and molecular characterization of Colletotrichum species from herbaceous plants in Thailand. Fungal Divers 2005;18:117-33.

15. Weber J. A natural biological control of Dutch elm disease. Nature 1981;292:449-51. [CrossRef]

16. Petrini O. Fungal endophytes of tree leaves. In: Andrews JH, Hirano S, editors. Microbial Ecology of Leaves. New York: Springer; 1991. p. 179-97. [CrossRef]

17. Brower J, Zar J, Ende CV. Field and Laboratory Method for General Ecology. Iowa: Brown Publishers; 2004.

Reference

1. Lu Y, Chen C, Zhang J, Chen W. Isolation and identification of endophytic fungi from Actinidia macrosperma investigation of their bioactivities. Evid Based Complement Alternat Med 2012;2012:1-8. https://doi.org/10.1155/2012/485042

2. Arechavaleta M, Bacon CW, Hoveland CS, Radcliffe DE. Effect of the tall fescue endophyte on plant response to environmental stress. Agron J 1989;81:83-90. https://doi.org/10.2134/agronj1989.00021962008100010015x

3. Malinowski DP, Belesky DP. Adaptations of endophyte-infected cool-season grasses to environmental stresses. Mechanisms of drought and mineral stress tolerance. Crop Sci 2000;40:923-40. https://doi.org/10.2135/cropsci2000.404923x

4. Giridharan P, Verekar SA, Khanna A, Mishra PD, Deshmukh SK. Anticancer activity of sclerotiorin, isolated from an endophytic fungus Cephalotheca faveolata Yaguchi, Nishim. and Udagawa. Indian J Exp Biol 2012;50:464-8.

5. Schulz B, Boyle C, Draeger S, Rommert AK. Endophytic fungi: A source of novel biologically active secondary metabolites. Mycol Res 2002;106:996-1004. https://doi.org/10.1017/S0953756202006342

6. Gunatilaka AA. Natural products from plant-associated microorganisms: Distribution, structural diversity, bioactivity, and implications of their occurrence. J Nat Prod 2006;69:509-26. https://doi.org/10.1021/np058128n

7. Peterson CT, Denniston K, Chopra D. Therapeutic uses of Triphala in Ayurvedic medicine. J Altern Complement Med 2017;23:607-14. https://doi.org/10.1089/acm.2017.0083

8. Selvanathan S, Indrakumar I, Johnpaul M. Biodiversity of the endophytic fungi isolated from Calotropis gigantea L. Recent Res Sci Technol 2011;3:94-100.

9. Dobranic JK, Johnson IA, Alikhan QR. Isolation of endophytic fungi from larch (Larix laricina) leaves from New Brunswick, Canada. Can J Microbiol 1995;41:194-8. https://doi.org/10.1139/m95-026

10. Barnett HL, Hunter BB. Illustrated Genera of Imperfect Fungi. New York: Macmillan Publishing Company; 1987.

11. Duong LM, Jeewon R, Lumyong S, Hyde KD. DGGE coupled with ribosomal DNA phylogenies reveal uncharacterized fungal phylotypes on living leaves of Magnolia liliifera. Fungal Divers 2006;23:121-38.

12. Suryanarayanan TS, Venkatesan G, Murali TS. Endophytic fungal communities in leaves of tropical forests trees: Diversity and distribution pattern. Curr Sci 2003;85:489-93.

13. Huang WY, Cai YZ, Hyde KD, Corke H, Sun M. Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Divers 2008;33:61-75.

14. Photia W, Taylor PW, Ford R, Hyde KD, Lumyong S. Morphological and molecular characterization of Colletotrichum species from herbaceous plants in Thailand. Fungal Divers 2005;18:117-33.

15. Weber J. A natural biological control of Dutch elm disease. Nature 1981;292:449-51. https://doi.org/10.1038/292449a0

16. Petrini O. Fungal endophytes of tree leaves. In: Andrews JH, Hirano S, editors. Microbial Ecology of Leaves. New York: Springer; 1991. p. 179-97. https://doi.org/10.1007/978-1-4612-3168-4_9

17. Brower J, Zar J, Ende CV. Field and Laboratory Method for General Ecology. Iowa: Brown Publishers; 2004.

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