Published:  Nov 25, 2020DOI: 10.7324/JABB.2020.80602
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.
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
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 . 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  and metal contamination . 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 . The metabolites include varied structural groups such as terpenoids, steroids, xanthones, chinones, phenols, isocoumarins, benzopyranones, tetralones, and enniatines  that are known to exhibit antibacterial, antiviral, antifungal, and anticancer activities .
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 . 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.
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.
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.
The collected samples were first cleaned thoroughly in gentle running tap water to remove dirt and debris . The stem, bark, and leaves were cut into segments (0.5–1 cm) and surface sterilized by modified method of Dobranic et al. (1995) . 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].
The CF of endophytic fungi isolated from the three tree species was calculated by following formula suggested by Suryanarayana et al. (2003)  and expressed in percentage.
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RF used to represent fungal density was calculated as described by Huang et al. (2008) .
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 .
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Simpson’s Index of diversity was calculated using the following formula.
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.
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
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 chebula||Terminalia bellirica|
|Sterile mycelium I||5||8.3%||2||3.3%||2||3.3%||-||-||-||-||-||-||-||-||-||-||-||-||9||2.5|
|Sterile mycelium II||4||6.6%||1||1.6%||1||1.6%||6||10||-||-||-||-||-||-||-||-||-||-||12||3.4|
|Sterile mycelium III||-||-||-||-||-||-||-||-||-||-||3||5%||-||-||-||-||-||-||3||0.8|
|Sterile mycelium IV||-||-||-||-||3||5%||3||5%||-||-||2||3.3%||-||-||-||-||-||-||5||1.4|
|Sterile mycelium V||4||6.6%||-||-||3||5%||-||-||-||-||-||-||5||8.3%||3||5%||4||6.6%||19||5.4|
|Sterile mycelium VI||-||-||-||-||-||-||-||-||-||-||-||-||7||11.6%||-||-||9||15%||16||4.5|
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.
|Indices||Phyllanthus emblica||Terminalia chebula||Terminalia bellirica|
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 . The plant tissues, especially leaves and stems, are excellent reservoirs for endophytic fungi . 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 . 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 . 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.
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.
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.
All authors were declaring that there are no conflicts of interests regarding the publication of this paper.
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