Introduction

The word endophyte means “in the plant” (endo-within, phyton-plant) [11]. Plants may serve as a reservoir of large numbers of microorganisms known as endophytes. Endophytes are microorganisms (mostly fungi and bacteria) that inhabit plant hosts for all or part of their life cycle. They colonize the internal plant tissues beneath the epidermal cell layers without causing any apparent harm or symptomatic infection to their host, living within the intercellular spaces of the tissues and its seems that they may penetrate the living cells. Endophytes form inconspicuous infections within tissues of healthy plants for all or nearly all their life cycle and their host tissues appear symptomless, and they remain asymptomatic for many years and only become parasitic when their hosts are stressed [10]. The term “Endophyte” originally coined by German scientist Heinrich Anton De Bray in 1884 [11]. Endophytic fungus was first identified by Freeman in 1904, and was isolated from Lolium persicum (Persian darnel) [9].

Approximately, there are near to 300,000 plant species on earth and each individual plant is the host to one or more endophytes, and many of them may colonize certain hosts. It has been estimated that there may be as many as one million different endophytic fungal taxa, thus endophytes may be hyperdiverse [10]. Endophytic fungal communities are influenced by many factors such as geographical location, climatic patterns, physiology and specificity of colonized tissue [11]. They are present in most plant parts, especially the leaves, where the tissue is apparently healthy [7]. Endophytes relationship with plant varies from symbiotic to pathogenic. Endophytes benefit host plant by preventing pathogenic organism from colonizing. It has been categorized into two main groups (Clavicipitaceous and non-clavicipitaceous) based on differences in evolution, taxonomy, plants host and ecological functions. Clavicipitaceous are able to infect only some species of grasses and nonclavicipitaceous are found in the asymptomatic tissues of other higher plants. It stimulates plants growth, increase disease resistance, improve the plants ability to withstand environmental stresses and recycle nutrients [14].

Isolation of endophytes is a critical step, because it requires sensitivity to recover a maximum number of colonized endophytes and should be accurate enough to eliminate the epiphytic microbes which are present on the plant surface. Endophytes can be isolated from various plant parts such as seeds, leaves and stems. The collected plants for studying endophytic communities should look apparently healthy and disease free plant, i.e. they do not display any visual symptoms of diseases, in order to minimize the presence of plant pathogenic and saprobic species, and to prevent the isolation of localized pathogenic endophytic microorganisms [10].

Material and methods

Plant Collection and Identification

Healthy (showing no visual disease) and mature leaves of ten medicinal plants (Table:1) were collected from adajan and athwalines area, in plastic bags and transported carefully to the laboratory. The collected plant material was authenticated at the Department of Botany, P.T.Science College, Athwalines, Surat. Fresh plant materials were used for isolation work to reduce the chance of contamination. The collected plant leaves were subjected to surface sterilization within a few hours after sampling.

Surface Sterilization

All the leaf samples were subjected to a three-step surface sterilization procedure according to the method described by Petrini. Initially all the leaves were washed in running tap water for 10 minutes to remove, soil particles and adhered debris, and finally washed with distilled water. This was followed by washing in 95% ethanol for 1 minute, in 2% sodium hypochlorite for 10 seconds and in 95% ethanol for 1 minute. Finally, the leaves were washed in sterile distilled water for 2 minutes [2]All the leaf samples were subjected to a three-step surface sterilization procedure according to the method described by Petrini. Initially all the leaves were washed in running tap water for 10 minutes to remove, soil particles and adhered debris, and finally washed with distilled water. This was followed by washing in 95% ethanol for 1 minute, in 2% sodium hypochlorite for 10 seconds and in 95% ethanol for 1 minute. Finally, the leaves were washed in sterile distilled water for 2 minutes [2]

Table 1: seleted medicinal plants for isolation of endophytic fungi

Sample no. Scientific name Local name
1 Ocimum tenuiflorum Tulsi
2 Epipremnum aureum Money plant
3 Azadirachta indica Neem
4 Piper betle Naagarvel paan
5 Catharanthus roseus Sadabahar
6 Ficus religiosa Pipdo
7 Musa acuminata Banana
8 Ficus Benghalensis Vad
9 Ficus racemosa Umaraa
10 Calotropis procera Aankado

Isolation of Endophytic Fungi from Plant Leaves

After surface sterilization, following two methods [2] were used for isolation of endophytes.

Method 1

The leaves were crushed with sterile distilled water using sterile mortar and pestle. About 1 ml of crushed sample was serially diluted up to 10-5 dilutions using 12.5 mM potassium phosphate buffer (pH 7.1). About 0.1-0.2 ml of aliquot from 10-2 to 10-5 dilutions were taken and spread on Potato dextrose agar media supplemented with chloramphenicol as an antibiotic (50µg/ml) and all the plates were incubated at 30°C [5]. Observation was carried out daily until the growth of endophytic fungi was observed.

Method 2

The outer tissues were removed from the leaves by crushing it with the application of minimal pressure using sterile mortar and pestle and the inner tissues were excised and about 2–3 segments of crushed leaves were placed onto petri plates containing Potato dextrose agar media supplemented with chloramphenicol as an antibiotic (50µg/ml) and incubated at 30°C. Observation was carried out daily until the growth of endophytic fungi was observed.

Purification, Selection and Preservation of Endophytic Fungi

After incubation, fungal colonies were selected and streaked on Potato dextrose agar plates and incubated at 30°C for about 72 hours.

Morphological and Microscopical Characterization of Endophytic Fungi

The identification procedure of endophytic fungi was based on morphology. The sample of fungal isolates were mounted on the sterile slides then it was stained with lactophenol cotton blue and examined in 40X light microscopy [15]. The twenty-five isolated species were described according to their macroscopic features (i.e. the topography, texture and colour) as well as microscopic characteristics (i.e. the structure of hyphae, conidia and conidiophores) [1]. Obtained data were then compared with the descriptions of fungi species in the identification of pathogenic fungi by Colin K. Campbell and matches were recorded [3][6][8][12]. Those cultures which failed to sporulate were grouped under mycelia sterilia. This is the common problem concerning with the identification of endophytes [13].

Results

A total of 25 morphologically distinct endophytic fungi were isolated. From the two methods used for the isolation of endophytic fungi, Method 2 showed better growth of endophytes compared to Method 1. The percentage occurrence of endophytic fungi varies with the host plants. (Fig. 1). The identification of fungal strains was done under the basis of macroscopic and microscopic observations of cultural characters (Fig. 2).

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(Figure:2) Morphotypic characterization of endophytic fungi by studying their Colony performance on the media PDA and by staining technique

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The Majority of the recovered endophtytes belong to the Ascomycota. These fungi belong to four Ascomycota classes. The Aspergillus, Trichophyton, Coccidioides species belong to Eurotiomycetes, Curvularia, Alternaria, Aureobasidium, Exserohilum, Bipolaris species belong to Dothideomycetes , Geotrichum, Candida species belong to Saccharomycetes and Fusarium species belong to Sordariomycetes class.(Fig.3).

Discussion

The plant tissues, specially leaves and stems are excellent reservoirs for endophytic fungi (Petrini, 1991; Bokhary et al., 2000). Environmental factors such as temperature, rainfall and atmospheric humidity and their effect on host plant made the variations in occurrence of endophytic fungi and their colonization frequency (Selvanathan et al., 2011). Therefore, in present survey of endophytic fungi isolated from ten medicinal plants from Gujarat region have variations in endophytes and their relative percentage occurrence.

K.P.Suradkar et al (2014) investigating Seasonal diversity of endophytic fungi. The leaf isolates mostly fell into Cladosporium spp., Verticillium spp., Arthrinium spp., Colletotrichum spp. and Aspergillus spp. from Ocimum tenuiflorum and Calotropis procera respectively. In our study we got Trichophyton spp., Bipolaris spp., Aspergillus spp., Geotrichum spp., Alternaria spp. and Candida spp. Fungal endophytes were isolated from leaves of Azadirachta indica and Piper betle in nashik (Sidhu et al 2015). Cladosporium spp., Curvularia spp. and Colletotrichum spp. were obtained as endophytic fungi while we found Fusarium spp., Trichophyton spp., Exserohilum spp and one mycelia sterilia from the same plants. V.H. Sunitha et al (2013) study the diversity of microbial community associated with healthy different plant parts of the Catharanthus roseus such as leaves, midrib, petiole and stems in Bangalore. They recovered and identified fungal endophytes, which were closely related to species of the following genera Colletotrichum spp., Drechsclera spp., Cladosporium spp., Myrothecium spp while we recovered only Aspergillus spp. Beatriz et al (2017) conducted endophytic fungi in economically important plants in Brasil. Xylaria spp., Colletotrichum spp. and Cordana spp. were isolated from Musa acuminata and we found one candida spp. from the same. Rasika et al (2016) isolate and identified endophytic fungi from selected medicinal plants like Ficus religiosa, Ficus benghalensis and Ficus racemosa in Akola. The isolates belonged to Alternaria spp., Rhizopus spp., Curvularia spp. and Trichoderma spp. In our study we got curvularia spp. as same but another two endophytes Aspergillus spp. and Coccidioides spp. were different. S. Shafique et al (2014) examined endophytic fungi from infected leaf of Epipremnum aureum showing necrotic spots in Punjab and isolted Alternaria ochroleuca and we found same Alternaria spp. and Aureobasidium spp as an endophytes from the healthy leaves of that plant. Most of these genera including Curvularia, Aspergillus and Fusarium are common as plant pathogenic fungi though non-pathogenic endophytic forms of these also exist. Some reports have stated that endophytes remain latent, with symptomless nature inside the host plant only until the environmental conditions are favourable for the fungus to turn into aggressive saprophytes or opportunistic pathogens. The fungi in the current study were isolated from a healthy, symptomless plant after thorough surface sterilization, which confirm the isolated fungi were in the endophytic form during the isolation period [4].

Conclusion

This investigation evaluated the diversity of endophytic fungi inhabiting various medicinal plant leaves. Twenty-five species of endophytic fungi were isolated and identified from leaves which belong to Alternaria spp., Trichophyton spp., Geotrichum spp., Candida albicans, Aspergillus spp., Aureobasidium spp., Fusarium spp., Exserohilum spp., Candida kefyr, Curvularia spp., Coccidioides spp., Bipolaris spp. One unidentified isolates were group to mycelia sterilia fungi which was morphological fungal types, but not forming true spores. Aspergillus had the highest frequency in this study and Fusarium was the second most frequent endophytic group. Almost fourteen isolates belong to the class Eurotiomycetes, six belong to Dothideomycetes, three belong to Saccharomycetes and one belong to Sordariomycetes. This study is based mainly on macroscopic and microscopic characterization of the isolated fungi which does not allow the identification of abundantly isolated sterile mycelia. In the future studies, we should adopt many molecular methods which help in identification of non-sporulating fungal isolates.