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

2.4. 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

H_S = ∑ (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

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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.

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

Figure 1: Morphology of Triphala tree species. [Click here to view]

Figure 2: Colony morphology and microphotographs of endophytic fungi. [Click here to view]

Figure 3: Relative frequencies of different endophytic fungal taxa isolated from Triphala trees. [Click here to view]

Figure 4: Isolation rate of fungal endophytes from P. emblica. [Click here to view]

Figure 5: Isolation rate of fungal endophytes from T. chebula. [Click here to view]

Figure 6: Isolation rate of fungal endophytes from T. bellirica. [Click here to view]

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.