1. INTRODUCTION:

About 100,000 species of fungi have been formally described by taxonomists, but the global biodiversity of the fungal kingdom is not fully understood. Several authors have suggested that the majority of undiscovered endophyte diversity occurs in leaves of tropical trees. Fungi are generally cosmopolitan therefore it can be present in everywhere on the earth but the composition of the fungal community usually differs between host species (Saikkonen, 2007), among the geographically separated individuals of the same host species (Collado et al. 1999), and also within the tissue or organs of a host plant (Kumar & Hyde 2004).Variation in the diversity of fungi may also be associated with location, climate and plant age (Petrini, 1991; Asai et al., 1998). The Endophytes are now considered as an important component of biodiversity. They can also represent a large reservoir of unexplored genetic diversity.

The evolutionary record of fungi said that the major lineage of fungi was believed to have first arisen about 1000 million or accordingly years ago, which was followed by land plants in approximately 700 million years ago (Heckman et al. 2001). From the biological and the environmental evolution perspective, fungi are one of the earliest eukaryotes to colonize the ancient earth (Gray and Shear, 1992; Horodyski and Knauth, 1994). Considering the harsh physical environments on the ancient earth, to ensure the chances of survival, fungi need to be more tolerant or resistant to adverse environmental factors than the latter appeared plants or animals. Indeed, within the last few decades a number of fungal species (halophile, xerophiles, or thermophile) that can live in a variety of extreme environments have been isolated.

2. TAXONOMY IMPORTANCE OF FUNGI:

The field of mycology has usually studied organisms of three separate Kingdoms, Protozoa, Straminipila, and Fungi (true fungi). The Kingdom Fungi (Bridge and Arora, 1998) (true fungi) include the four major phyla such as Chytridiomycota, Zygomycota, Ascomycota and the Basidiomycota. The deutiromycetes or “fungi imperfecti” are an additional group within the Kingdom Fungi, but they are not recognized as a formal phylum due to the absence of sexual spores although the only asexual spores present is observed. Without taxonomic knowledge of proper identification and preservation of fungal isolates, chemical investigations of fungi become difficult unless impossible to reproduce in lab. The proper identification and classification of fungi is critical to the study of natural products of fungi. Additionally, the proper characterizations of fungi combined with reported chemistry can provide additional tools for mycologists to classify fungi. Although fungi have historically been identified and classified primarily by morphological characteristics, mycologists now employ a number of techniques to help identify fungi and to organize fungal systematics. Morphology still plays a major role in taxonomy, but the additional use of molecular data is becoming more common. The following molecular data such as DNA GC content, Random Amplified Polymorphic DNA fingerprints (RAPD), Restriction Fragment Length Polymorphism analyses (RFLP), and the use of different DNA sequences are now common and combined with World Wide Web (WWW) searchable databases have made identification of known species more consistent, faster, and easier for the non-specialist (Takamatsu, 1998; Kiesling, 2002; Hibbett, 1992). Additionally to the identification of marine yeasts, DNA macroarray techniques have also been utilized(Peterson, 2000a). The use of ribosomal DNA (rDNA) sequences has become one of the most useful techniques to aid in fungal identification and to study phylogenetics. The ribosomal gene cluster is comprised of three regions coding for the 5.8S, 18S, and 28S ribosomal RNA genes. Although the rates of evolution vary within individual regions of the ribosomal RNA gene cluster. These genes are present as tandem repeats, they evolve as a single unit and therefore can be used to compare organisms at several levels (Peterson, 2000b). Since the ITS regions are fairly divergent and vary between species within a genus, they can be very useful in fungal taxonomy. Using ITS1 and ITS4 primers, the ITS1-5.8S-ITS2 region (∼550 base pairs) can be amplified by PCR and subsequently sequenced. The sequence can be compared to online GenBank databases and their taxonomic information obtained. Ribosomal gene sequence data have been used to study Fusarium sp, Aspergillus sp and Penicillium sp. as well as other groups. Additionally, details that describe the methods for PCR amplification of the nuclear and mitochondrial fungal ribosomal RNA genes have been summarized by White et al including a list of common primers (Nakagiri, 2002). Additional lists of primers have been published by S. W. Peterson. Although the ITS1-5.8SITS2 sequence is commonly used as a taxonomic tool, the IGS region is the most divergent and may be useful for closely related species that have identical or nearly identical ITS sequences (Leano, 2002).

3.TRADITIONAL TECHNIQUES USED IN ENDOPHYTIC FUNGI STUDIES:

Based on traditional methods, to study the endophytic fungi from inside plant tissues can be analyzed by two basic techniques namely direct observation and cultivation-dependent methods. In the direct observation method, endophytic fungal structures within living plant tissues are directly examined under a light and electron microscope, which can show all endophytic mycobiota within the plant tissue, particularly biotrophic fungi that cannot be cultured on standard growth media(Deckert et al. 2001; Lucero et al. 2011). However, due to lack of spore-producing structures and sexual or asexual spores in most endophytic fungi when present in within plant tissues, but have only a hyphal structure is observed and therefore cannot be identified to any taxonomic category according to morphology. In addition, endophytic isolates cannot be obtained as microbial resources for further use with the direct observation method. Therefore, this is not commonly used in endophyte diversity studies. In contrast to direct observation methods, cultivation dependent techniques have been routinely employed in endophyte diversity studies (e.g. Petrini et al. 1982; Rodrigues and Samuels, 1990; Guo et al. 2000; Sun et al. 2011; Vieira et al. 2011). Additionally, it is important to isolate endophytic fungi for further detailed studies include their biological and physicochemical characterization, population dynamics, species diversity, or as inocula to improve plant growth and health, or screening for novel biologically active secondary metabolites for biotechnological applications. With cultivation-dependent techniques, the isolation procedure is a critical and important step in working with endophytic fungi. The living plant tissues are subjected to a serial process of surface sterilisation to remove all organisms from the surface of the plant. Only internal fungi are isolated by means of incubation of the plant samples onto nutrient plates.

Cultivation-dependent techniques generally include the following steps (Hallmann et al. 2006). 1.Thorough washing the plant tissue under tap water to remove adhering soil particles, debris and major epiphytes. 2.Surface sterilisation of plant tissue to kill any microorganisms on the host surface by applying different sterilisation protocols to different tissue types. 3.Surface sterilized plant samples placed on nutrient agar containing plates to isolation of endophytic fungi growing out from them. 4.Purification and sporulation of endophytic isolates under various incubation conditions. 5.Identification of the endophytic fungi based on morphological characteristics of cultures.

The isolation technique is a method-dependent process, although it is valuable for quick recovery of a large number of endophytic fungal species from plant tissues. Endophytic fungus communities obtained from plants, however, are directly affected by surface sterilisation techniques, incubation conditions, and whether isolates sporulate. To be effective, the isolation procedure must be adapted to the respective plant species, tissues and fungi. In practice, the isolated strains can be assumed to be endophytic fungi when total surface sterilisation is confirmed, i.e. no fungal growth from imprinting the surface-sterilised plant tissues onto nutrient media or culturing aliquot of water from the last rinsing onto nutrient media. To observe the increase fungal diversity, the sterilised plant tissue is cut into small segments (5 mm diam), macerated or ground, then transferred onto nutrient agar for incubation, which may find out fast and slow-growing fungi. A number of different media should be used in the isolation procedure, such as a standard PDA (potato dextrose agar) and MEA (malt extract agar), as well as minimal media with plant tissue or extract. In addition, the tissue size and numbers of plant samples can affect the observed fungal diversity and community composition. For example, Gamboa et al. (2002) demonstrated that, in several tropic plants, the number of endophytic fungi obtained increases with the decreasing size of tissue fragments incubated. However, more endophytic taxa were recovered from the incubation of whole leaf (35 taxa) than leaf disks (5 mm diam., 9 taxa) of Acer truncatum (Sun et al. 2011).

4. MOLECULAR IDENTIFICATION OF ENDOPHYTIC FUNGI:

4.1.Molecular identification of sterile mycelia

The large number of sterile isolates poses a special problem, because they cannot be identified to any taxonomic category according to morphological characteristics. Therefore, various methods have to be employed to promote isolate sporulation, such as the use of different media and inclusion of sterile host tissue in cultures. For example, increased the initial 48% of sporulating isolates obtained from Livistona chinensis plants to 59.5% by the addition of sterile palm leaf tissue onto the agar surface, and further increased the number of identifiable (sporulating) isolates to 83.5% by inoculating the remaining unidentified isolates onto sterile petiole pieces in conical flasks at room temperature for 3 months. In addition, some fungi may be missed due to failure to grow or grow slowly, and are easily outcompeted by fast growing species under artificial conditions. The potential technical biases in traditional endophyte studies can be resolve by molecular techniques (Guo et al.,1998).

Based on the traditional cultivation-dependent processes, fungal isolates can be identified from morphological characteristics if they sporulate on the media. Despite the development of various methods to promote sporulation(Guo et al. 1998, 2000; Taylor et al. 1999), high numbers of isolates (up to 54% of the total) do not sporulate in cultures. Since the conventional classification of fungi, depending heavily on reproductive structures, hence, these non-sporulating strains cannot be provided with taxonomic names. To appreciate the considerable diversity of these Mycelia sterilia, they are generally categorised as ‘morphotype’ depending on similar cultural characteristics, such as colony colour, texture and growth rates(Guo et al. 2000, 2003; Wang et al. 2005; Sun et al. 2011). Although the arrangement of morphotypes into different taxa, however, it does not reflect species phylogeny, because morphotypes are not real taxonomic units. Molecular methods are, therefore, required for the identification and understanding of the diversity of endophytic mycelia sterilia. For example, in a survey of endophytic fungi from plants L. chinensis in Hong Kong, a large number of isolates nearly 16.5% of out of total isolates did not sporulate, remaining as Mycelia sterilia (Guo et al. 2000). These Mycelia sterilia isolates were grouped into 19 morphotypes based on their cultural morphology and identified as different genera: Diaporthe, Mycosphaerella and Xylaria, families: Pleosporaceae and Clypeosphaeriaceae and order: Xylariales based on ITS sequence analyses of rDNA isolated form mycelia sterilia (Guo et al., 2003).

4.2.Molecular identification of isolate of endophytic fungal community

Endophytic fungus communities consist of a wide range of fungal origins, such as Ascomycota, Basidiomycota and Zygomycota(Zheng and Jiang 1995; Sinclair and Cerkauskas 1996). Therefore, it is a tough task for mycologists to identify various endophytic fungi into genera or species based on morphological characteristics. Furthermore, it is very time-consuming to make a complete identification. Therefore, DNA sequencing analyses coupled with morphology have been widely used in the investigation of endophyte diversity, particularly for ecology studies. For example, a total of 27 fungal genera belonging to Ascomycota, Zygomycota and Basidiomycota were isolated and identified from roots of Pseudotsuga menziesii and Pinus ponderosa using a combination of morphology and ITS sequence data based on 90% and 95% sequence similarity (Hoff et al. 2004).

In endophytic fungi identification studies, 18S and 28S genes of rDNA have been employed in the identification of endophytic fungi at high taxonomic levels. For example, a total of 71 (of 257 strains) representative fungal strains isolated from bamboos Phyllostachy and Sasa species was placed in fungal groups (Sordariomycetes and Dothideomycetes) based on 18S gene sequence analyzes. These strains were further identified into lower taxonomic levels, according to ITS sequence data, and some strains may represent novel taxa. Fifty-nine of morphologically unidentifiable fungal strains isolated from healthy stems and pods of Theobroma cacao trees were identified based on the sequence analyses of the 28S gene. The majority of the isolates belonged to Basidiomycota, particularly to corticoid and polyporoid taxa (Crozier et al. 2006).

When endophytic fungi isolation from host plants is failed in cultivation technique, during that time molecular methods also permit the identification of fungi. In endophyte studies, ITS is the most widely used DNA barcode in molecular identification and shows potential in diversity and ecology studies, despite some limitations in species difference. When employing this approach, it is important to take into consideration that surface sterilization may not have denatured the DNA of epiphytes, though sodium hypochlorite is relatively effective for this purpose. In order to identify all the fungi actually colonizing a host, total DNA must be isolated from the environmental sample. It can then be directly amplified with fungal primers; denaturing gradient gel electrophoresis (DGGE) may then be used to separate the bands. Subsequent sequencing and phylogenetic analysis theoretically enables the identification of all the fungi colonizing a plant provided that the sequences found correspond to known sequences in the data banks (Kowalchuk et al., 1997). Primers for 28S rRNA region were found to be more specific than those for 18S rRNA region. On the other hand, in attempts to amplify plant DNA using ‘universal’ primers for the ITS and 5.8S regions of rDNA (Zhang et al. (1997), also amplified fungal DNA from the host plant, therefore, this is again emphasizing the importance of using specific primers (Guo et al. 2003; Murali et al. 2007; Promputtha et al. 2007; U’ren et al. 2009; Sun et al. 2011).

5. CONCLUSION:

Endophytic fungi documentation and continuous research will establish richness of endophytic fungi all over the world. For the future taxonomic details of endophytic fungi will be useful to derive their desired applications.

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Received on 20.07.2017 Modified on 17.08.2017

Research J. Pharm. and Tech. 2018; 11(1): 375-379.

DOI: 10.5958/0974-360X.2018.00068.9