Keywords

1 Introduction

1.1 What Are Endophytes?

Endophytic microbes are those microbes that at some time in their life cycles colonize internal plant tissues without causing apparent harm to the host (Petrini 1991). Endophytic microbes provide hosts with protection against an array of biotic and abiotic stresses (Bacon and White 2000; Omacini et al. 2001; Redman et al. 2002). Endophytic microbes are ubiquitous and have been reported from all plants investigated (Petrini et al. 1982; Carroll 1988). They have been reported in algae (Hawksworth 1987), lichens (Petrini et al. 1990; Li et al. 2007), mosses (Schulz et al. 1993), conifers (Carroll and Carroll 1978) and angiosperms (Hyde et al. 1997; Verma et al. 2007, 2014; Gond et al. 2011), including parasitic plants (Suryanarayanan et al. 2000). Many investigations from the last few decades suggest that endophytes are promising sources of bioactive natural products (Kusari and Spiteller 2011; Verma et al. 2009a; Strobel and Daisy 2003; Stierle et al. 1993; Xu et al. 2014). However, commercialization of natural products from endophytes is not yet widespread. Endophytic microbes include fungi (Mishra et al. 2012; Verma et al. 2014) and bacteria (Gond et al. 2015a; Kumar et al. 2016). Ecophysiological roles of most endophytes are not well understood but are assumed to vary according to microbe, plants and environmental conditions (Carroll 1995; Blodgett et al. 2007; Rodriguez et al. 2009).

1.2 Challenges in the Study of Endophytes

Studying endophytic microbes is often challenging to investigators because often microbes go unobserved in plants, many cannot be isolated, and it is difficult to conduct experiments where endophytes cannot be cultured or plants free of endophytes cannot be obtained. Further, methods used to study endophytes are still imperfect, and additional methodologies and techniques are needed to better understand endophytes and their potential applications (Gamboa et al. 2002; Rodriguez et al. 2009). In general, research on endophytic microbes has three major foci, including to assess diversity, explain plant-microbe interactions and assess biotechnological potentials (Fig. 4.1). The present review discusses the approaches, techniques and their limitations that may be employed to confront the challenges to the study of endophytes.

Fig. 4.1
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Foci of endophyte research

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2 Isolation of Endophytic Microbes

Isolation of endophytic microorganisms is an important step in exploring the phylogeny, diversity, interactions with plants and applications as bio-inoculants for improvement of fitness of agricultural plants and for examining sources of biologically active molecules of industrial or medicinal importance (Hallmann et al. 2006; Strobel and Daisy 2003; Verma et al. 2009a; Kharwar et al. 2011b). Isolation procedures should be good enough to recover multiple endophytes and at the same time thorough enough to eliminate saprophytes and casual associates of plants (Hallmann et al. 2006). The procedure for isolation of endophytes is more or less the same for all kinds of endophytic microbes, including fungi and bacteria. Isolation involves surface sterilization, plating tissue pieces or crushed plant tissues on different media and then purification of microbes that grow from tissues (Verma et al. 2009b; Kharwar et al. 2011a; Mishra et al. 2012).

Since endophytes are present in many organs, they can be isolated from different parts of the plant, including leaves, stem twigs, bark, roots, fruits and seeds (Bacon and White 1994; Ghimire and Hyde 2004). In general, it is suggested that the plant parts collected should be healthy and young in order to minimize unwanted opportunistic plant pathogens and saprobic species (Ghimire and Hyde 2004; Kharwar et al. 2011a; Verma et al. 2007). Endophytes are sometimes slow growing, and often faster-growing saprobes make it difficult to isolate endophytes (Bacon and White 1994). Plant parts should be kept in a refrigerator and processed in the shortest time possible after collection, if possible within 24 h. The samples for investigation should be cut into small pieces to facilitate sterilization and isolation processes. Most common surface sterilization techniques comprise dipping plant tissues in 75–90% ethanol for 1–2 min, then in 4% aqueous sodium hypochlorite for 5–10 min and finally in 90% ethanol for 30 s followed by rinsing with deionized sterile water at least three times to remove sterilizing agents (Petrini et al. 1992; Bills 1996; Verma et al. 2014; Stone et al. 2004; Kumar et al. 2016) (Table 4.1). Effectiveness of surface sterilization may be checked by making imprint of sterilized tissue sample (Schulz et al. 1998). Since different tissues of plants differ in their nature, thickness, roughness and permeability, therefore steps and concentrations of sterilizing agents may vary from plant to plant and tissue to tissue. Some work is thus necessary to optimize the surface sterilization procedure before isolation of endophytes (Hyde and Soytong 2008). Preferred isolation media differ depending on the kind of endophytes (fungi, bacteria and actinomycetes) to be isolated.

Table 4.1 List of the most commonly used surface sterilization agents with their concentrations and sterilization times
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2.1 Media for Isolation of Fungal Endophytes

Bills and Polishook (1992) tested several media for endophytic fungal isolation and observed that malt-yeast extract media (1% malt extract, 0.2% yeast extract) gave the maximum species richness. Verma et al. (2011) used four media PDA (potato dextrose agar), MYA (malt-yeast extract agar), MCA (mycological agar: 1% papaic digest of soybean meal, 1% dextrose, 1.5% agar) and nutrient agar (0.5% peptone, 0.3% beef extract, 0.5% NaCl, 1.5% agar) for the isolation of endophytic fungi from the root and fruits of Azadirachta indica and suggested PDA and MCA as better isolation media for fungal endophytes than the others. Several researchers have used diverse isolation media to analyse endophytic fungal diversity but have also suggested using WA (water agar) for isolation to reduce contamination (Stone et al. 2004). Colony-limiting agents and antibiotics are recommended for primary isolations of endophytic fungi (Table 4.2).

Table 4.2 Some of the common media and antimicrobial agents used for the isolation of endophytes
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2.2 Media for Isolation of Bacterial Endophytes

Procedures for the isolation of bacterial endophytes were described in detail by Hallmann et al. (2006). Crushed tissue samples in sterile water may be spread onto solid agar media of various types. Common media used for the isolation of bacterial endophytes are LBA (Luria-Bertani agar), TSA (tryptic soy agar; 10%), YESA (yeast extract sucrose agar) and nutrient agar. The use of antifungals like cycloheximide may also be used in media for the isolation bacterial endophytes (Table 4.2). Starch casein agar (SCA) with slight modification in ingredients is most commonly used for actinomycete isolation (Kutser and Williams 1964; Kumar et al. 2016). Media should be amended with antifungal agents like cycloheximide or nalidixic acid to restrict fungal growth (Verma et al. 2009b) (Table 4.2).

3 Identification of Endophytes

After isolation and purification of endophytes, the next important steps are to identify microbes and to assess species diversity (Sun and Guo 2012). Endophytes may be identified through culture-dependent methods or directly from the tissues using culture-independent methods (Sun and Guo 2012). The most common method to identify fungal endophytes is the cultivation-dependent approach which is frequently employed in assessing endophyte species diversity (e.g. Petrini and Fisher 1988; Fisher et al. 1986; Guo et al. 1998, 2003; Taylor et al. 1999; Sun et al. 2008; Kharwar et al. 2011a; Mishra et al. 2012; Verma et al. 2014), in the bioprospection of endophytes for bioactive metabolite production (e.g. Wang et al. 2006; Li et al. 2008; Kharwar et al. 2011a; Xu et al. 2014) and as mediators of host defence against biotic and abiotic stresses (e.g. Omacini et al. 2001; Redman et al. 2002, 2011; Dai et al. 2008; Gao et al. 2010; Gond et al. 2015a). This method involves isolation and purification of endophytic microbes from healthy tissues onto culture media (as previously described) followed by their identification. Endophytic fungi can sometimes be identified by their structural features in culture, i.e. growth patterns, colony colour, texture and fruiting structures, including spores, conidia, conidiophores, sporangiophores or sexual spores. This requires taxonomical expertise and references linking structural features of fungi to their identities. Some of the most routinely used standard taxonomic manuals for fungi are Illustrated Genera of Imperfect Fungi by Barnett and Hunter (1998), The Genera of Fungi Sporulating in Pure Culture by Von Arx (1978), More Dematiaceous Hyphomycetes by Ellis (1976) and The Fungi: An Advanced Treatise by Ainsworth et al. (1973). A most frequently encountered problem in fungal endophyte studies is the presence of mycelia sterilia (fungi which do not produce sexual or asexual spores), making their identification difficult (Ghimire and Hyde 2004; Sun and Guo 2012). Many times taxonomic manuals fail to provide enough information on spore-producing fungi to identify them to species level. In a study of fungal endophytes of Mahua plants (M. longifolia) from India, investigators were able to identify 28 species by morphological characters from 40 different morphotypes (Verma et al. 2014), in Adenocalymma alliaceum 14 species out of 17 morphotypes (Kharwar et al. 2011a) and in Tinospora cordifolia 24 out of 29 morphotypes (Mishra et al. 2012), and in Nyctanthes arbor-tristis 17 out of 19 morphotypes were identified by morphological characters (Gond et al. 2011). Due to the absence of differentiating morphological features, bacterial species generally cannot be distinguished using morphology alone.

3.1 Molecular Tools to Identify Endophytes

The morphological method of identification of endophytes may be useful for many endophytes (Kumar and Hyde 2004; Verma et al. 2014). However, a substantial number of isolates are reported as Mycelia sterilia due to failure to sporulate in culture. The number of Mycelia sterilia ranges significantly among studies of endophytes; in a study of endophytes in palm (Trachycarpus fortunei), 11% were reported as Mycelia sterilia, while in a study of endophytes of Quercus ilex in Switzerland, 54% were reported as Mycelia sterilia (Fisher et al. 1994; Frohlich et al. 2000; Ghimire and Hyde 2004). Some workers tried to improve sporulation in Mycelia sterilia in culture by changing the nutrient composition of the medium without much success (Guo et al. 1998; Frohlich et al. 2000; Kumaresan and Suryanarayanan 2002; Sun and Guo 2012). Even some of the spore-producing fungi are difficult to identify to correct taxonomic units because of the lack of literature (Verma et al. 2014). Further, some taxonomists believe that morphological characters are not reliable taxonomic features or sufficient to distinguish some taxa (Sun and Guo 2012). Molecular techniques therefore could be the best alternative to identify unidentified endophytic microbes. Molecular approaches have been used to determine the taxonomic status of fungi isolated from many habitats (White et al. 1990; Ma et al. 1997; Zhang et al. 1997; Ranghoo et al. 1999). Molecular identification requires isolation of total genomic DNA of all the recovered isolates from the host plant, PCR amplification of conserved sequences of DNA (rDNA) using specific or universal primers, sequencing of PCR products, BLAST options in GenBank or other DNA databanks, retrieval of related sequences from the databank and construction of phylogenetic trees using molecular software (Saitou and Nei 1987). Genotype-based identification of microbes is believed to be more reliable because some portions of nucleic acid sequences are highly stable and conserved in a wide range of organisms (Lindahl et al. 2013). Certain locations of ribosomal DNA (rDNA) sequences have been shown to have the highest accuracy for identification of bacteria and fungi up to species level and further may be used in phylogenetic studies (Sugita and Nishikawa 2003).

3.2 Markers and Primers for Endophyte Identification

The fungal nuclear rRNA operon (rDNA) in multiple copies in fungal genomes is a continuous sequence made up of the 18S, ITS1, 5.8S, ITS2 and 28s subunit regions (Iwen et al. 2002). The gene for the smaller subunit is 18S (SSU:18S), and large subunit is 28S (LSU:28S) separated by the internal transcribed spacer (ITS1 and ITS2) regions. ITS1 and ITS2 are highly variable spacers and intercalated by another 5.8S gene (Fig. 4.2). Each rRNA gene is separated by two intergeneric spacers (IGS1 and IGS2) with intercalated another 5S gene. For good marker highly conserved regions of rDNA and its less variation across related taxa are needed, but a useful marker should also be able to differentiate among closely related taxa (Lindahl et al. 2013). Because of high interspecies variation and low intraspecies variation, nuclear rDNA genes have been exploited as an ideal marker for identification of fungi (Lindahl et al. 2013). Since the length of the amplified fragment is critical for the phylogenetic analysis, therefore primers should also amplify enough length which could require for correct analysis. ITS1, ITS2 and ITS4 are frequently used primers in fungal endophyte identification (Mishra et al. 2012; Verma et al. 2014). ITS1 (F) and ITS4 (R) primers amplify partial sequences of 18S, complete ITS1, ITS2 and 5.8S and partial sequences of 28S rRNA genes. Some authors argue to use NL1 and NL2 primers to amplify D1 and D2 region of 28S rRNA because it gives more resolution than ITS regions (Khot et al. 2009). Likewise many bacterial endophytes may be identified using the 16S rDNA gene since this gene is highly conserved and gives good resolution of species in most bacterial groups. Researchers have used a variety of primers to amplify 16S rDNA genes, amplifying around 1000–1500 base pairs of rDNA, for example, 8F, UnF, 11F, 27F, 6R, UnR, 907R, 1492R and 1525R (Barghouthi 2011; Gond et al. 2015a). Although 16S rDNA gene sequences allow bacterial identification that is usually more robust, reproducible and accurate than that obtained by phenotypic testing, however, due to limited variability of the 16S rRNA gene in some groups and limited representation in some databases, it is often difficult to identify species in some bacterial groups (Clarridge 2004).

Fig. 4.2
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Schematic representation of fungal rRNA gene structure with position of different primers used for its amplifications

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4 Techniques to Evaluate Endophyte Distribution in Plants

Schulz et al. (1998) introduced a tissue blotting technique to check surface sterilization for the isolation of endophytes from the interior of healthy plant tissues. This technique may also be used to determine whether the hypothesized endophytes are also superficial on plant tissues. Endophytism can be further evaluated by doing microdissection and histological visualization of plant tissues through light, confocal or electron microscopy (Stone et al. 2004; Lucero et al. 2011; Sun and Guo 2012). Histological investigation of tissue is a method to observe all types of endophytes, including bacteria and fungi, within living host tissues. This enables us to see endophyte growth patterns, e.g. intra- or intercellular nature, and endophyte density in tissue and identify the part of tissue in which the endophyte is more concentrated. Most endophytes within plant tissues cannot be identified to any taxonomic category because they do not produce any identifying characters in vivo. For microscopic investigations of endophytes, the tissues must be stained with fungal specific or stain combination and mounted. Stone et al. (2004) have described a detailed protocol for clearing tissues for direct visualization of endophytic fungi in host tissues. The staining procedures must be optimized according to plant tissue and endophytic microbes (Hood and Shew 1996), described as follows.

4.1 Hood and Shew Staining Protocol

Leaf tissues are cut into small pieces (0.5 cm2) in size and washed thoroughly with running tap water; thereafter, tissues are surface sterilized followed by triple rinse in deionized water. Then leaf segments are treated with 2.5% KOH and heated at 40–60 °C to remove chlorophylls. This process is repeated several days until the leaf becomes straw coloured. The KOH solution is changed daily 2–3 times for 5–10 days. The cleared leaf tissues are rinsed in distilled water three times and bleached 3 min in 4% NaOCl and finally rinsed in distilled water twice. For staining KOH-aniline blue, solutions are prepared at least 2–3 h prior to use as 0.05% aniline blue dye in 0.067 M K2HPO4 at pH 9.0. Leaf sections are mounted on glass slides in small drops of stain solution and visualized under a compound light microscope (Fig. 4.3).

Fig. 4.3
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Histological visualization of endophytic fungal hyphae and spores in healthy leaf tissues of M. longifolia (a, b, c, d) and in tall fescue leaf (e)

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4.2 Fluorescent Probes for Localization of Bacterial and Fungal Endophytes

Fluorescent probing to determine internal endophyte distribution (intra- or intercellular) and density of endophytic microbes in different tissues of the plant has now become a popular method in the study of endophyte-plant interactions. There are two categories of fluorescent probes: one is green fluorescent protein (GFP) that autofluoresces, and the other is chemically linked fluorescent compounds that bind to proteins and nucleic acids or react with enzymes (Thirugnanasambandam et al. 2011; Card et al. 2013). Tagging bacteria with GFP using compatible vector has now become popular to track the endophytic bacteria location inside plant tissues. However tagging with GFP is most crucial step in that. Mousa et al. (2016) have successfully tagged the endophyte Enterobacter sp. with GFP using vector (pDSK-GFPuv) and visualized endophytic bacteria inside root tissues of maize, wheat and millets using confocal scanning microscopy. Several fluorescein-based compounds or dyes, including fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), SYTO9 (S9), propidium iodide (PI), diamidino-2-phenylindole (DAPI), etc., have been used to observe in vivo tissue colonization by endophytic bacteria and fungi. FDA, CFDA and CMFDA fluorescence are activated by a cellular esterase enzyme which is widely found in plants and microbes. Card et al. (2013) evaluated FDA, CFDA, CMFDA and calcofluor white fluorescent compounds to visualize Epichloë coenophiala in tissues of tall fescue grass (Pooideae) and suggested that CMFDA is more a suitable stain in comparison to others; it produced better contrast between host tissues and endophytes. SYTO9, SYTO13, propidium iodide and diamidino-2-phenylindole are nucleic acid-staining dyes (Thomas and Reddy 2013). SYTO9 and SYTO13 have been shown very useful to stain and visualize living bacterial endophytes in different plant tissues (White et al. 2012; Thomas and Reddy 2013). Thomas and Reddy (2013) have compared SYTO9, PI and DAPI for the staining of endophytic bacteria in banana shoot tips and found that SYTO9 stained living bacterial cells; however propidium iodide (PI) and diamidino-2-phenylindole (DAPI) stained dead bacteria or bacteria within damaged plant tissues. White and his group also have used SYTO9 and SYTO13 to locate the bacterial endophyte in several plant tissues including vanilla, cotton, turf grasses, Amaranthus, etc. (Fig. 4.4). SYTO9 has excitation/emission band at 485/498 nm for DNA and 486/501 nm for RNA. For the staining of endophytic bacteria in vitro, surface-sterilized plant tissues were cut into fine sections and then stained with SYTO9 or SYTO13 (10–20 μM) in water for 5–10 min and observed using a fluorescent microscope. Endophytic bacteria around root cells and root hairs of seedlings can be visualized directly by staining without sectioning (White et al. 2012).

Fig. 4.4
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SYTO@13 fluorescence staining of endophytic bacteria, (a) and (b) in root cells and root hairs of fescue grass, (c) in Amaranthus leaf, (d) intracellular localization of bacteria in leaf cells of vanilla orchid; red arrows indicate presence of bacteria (White et al. 2014b)

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4.3 ROS Staining to Study Bacterial Endophytes

Reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide (•O−2) and hydroxyl radical (•OH) are recognized as important signalling molecules in various metabolic and developmental processes in plants. H2O2 is the most stable ROS in tissue, and H2O2 levels are found to be increased under biotic and abiotic stresses (Choudhury et al. 2013). It has been found that invasion of plant cells by many bacterial endophytes is associated with secretion of hydrogen peroxide in cells in the vicinity of intracellular bacteria (White et al. 2014a). White et al. (2014a) reported that 3,3′-diaminobenzidine tetrachloride (DAB), which has been used to stain cellular production of hydrogen peroxide in many plants tissues and organs (Daudi and O’Brien 2012), is a good staining agent for bacterial endophytes. DAB with counterstain aniline blue/lactophenol was found suitable to visualize endophytic (intra- and intercellular) bacteria as well as epiphytic colonization of bacteria in several vascular plant species through light microscopy (White et al. 2014a, 2015). ROS staining steps include (1) washing the roots properly with sterilized water, (2) putting the root of seedlings into DAB solution (2.5 mM) overnight, (3) washing with deionized water, (4) counterstaining with aniline blue and mounting on slide and (5) observing under a light microscope. In this staining procedure, bacteria on the surface are generally stained with aniline blue, and endophytic bacteria stained with DAB are dark brown in colour (Fig. 4.5). This staining method is selective to stain bacteria with their localization inside and outside the cell. DAB-aniline blue stain combination also permits the visualization of deformation and lysis of bacteria in plant root cells. Several different shapes and swollen endophytic bacteria (L-forms) have been observed in root cells of many plants (White et al. 2014a, 2015). White and his group presently are working with this staining procedure to visualize the endophytic bacterial community of many vascular plants including Cynodon sp., Phragmites sp., Daucus carota, Oryza sativa, Triticum aestivum, etc. with satisfactory results (Fig. 4.5). However this staining procedure is not effective to visualize endophytes in shoot systems due to failure of the stain to penetrate the waxy cuticle and pigmentation.

Fig. 4.5
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ROS staining by DAB-aniline stains to visualize endophytic bacteria: clusters of endophytic bacteria in the root hairs (a) in root parenchyma cells (b) of Cynodon sp. and in the root hairs (c) root parenchyma cells (d) of rice seedlings

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5 Endophyte Modulation of Seedling Development

One of the more exciting discoveries regarding endophytes is the extent to which they play roles in modulation of host development. In experiments using grasses, it has been shown that root hair development is a function of the presence of bacteria that become intracellular in root cells and trigger hair formation, and they exit hairs at the hair tip where the cell wall is thinnest (White et al. 2015, 2017). The rate of seedling development in some plants is being found to largely depend on activities of microbial endophytes. Without intracellular bacterial endophytes, seedlings do not produce root hairs, and seedling growth is repressed. To assess endophyte modulation of development, we have conducted experiments on seedlings. Typically we used seedlings where we surface-sterilized seeds and germinated them on water agarose media. In these experiments we used plants: Bermuda grass (Cynodon dactylon) and annual bluegrass (Poa annua). These seeds could be surface-sterilized for approximately 40 min with continuous agitation in 4% sodium hypochlorite solution to remove all surface microbes (White et al. 2015, 2017). Seeds were then plated onto 0.7% agarose media in Petri dishes without nutrient additives. Bacterial aqueous suspensions were inoculated onto seeds and plates incubated at room temperature for 6–7 days. Seedlings were assessed for gravitropic affects, root hair development and root length extension. Without endophytic bacteria most of the seedling roots fail to show gravitropic growth; instead they remain on the surface of agarose without vertical growth. For the few roots that penetrate, the agarose root hairs do not form, and root length is often reduced. Bacteria may be visualized within seedling root cells by flooding plates bearing seedlings with DAB and incubating overnight at laboratory ambient temperature (White et al. 2014a). In all probability a similar in vitro assay system could be developed for testing effects of endophytes on seedling development in other species of plants.

5.1 Examining Modulation of Seedling Development Where Endophytes Are Not Culturable

In some cases endophytes are not isolatable, and it may be difficult to remove endophytes from seedlings if endophytes are vectored within seeds. One instance where we encountered this situation is with a bacterial endophyte in tomato (Solanum lycospersicum). Through microscopic examination of DAB-stained (White et al. 2014a) seedlings derived from surface-sterilized seeds, we saw evidence of an intracellular bacterial endophyte that we could not isolate. Through the use of a combination of rigorous surface disinfection using 4% sodium hypochlorite for 40 min to remove all surface bacteria, followed by an overnight soak in a 100–200 mg/l solution of streptomycin sulphate to reduce or remove internal bacteria, we were able to suppress development of the bacterial endophyte in seedlings germinated on agarose (Verma et al. 2017). Suppression of the bacterial endophyte in tomato seedlings resulted in suppression of root hairs on tomato roots. We also found similar effect in rice seedling experiments (Verma et al. 2017).

6 Application of Butyric Acid to Regulate Bacterial Entry into Plant Root Cells

In plant roots bacteria enter root cells at the root meristem tips where cell walls are thin. As root cells differentiate, bacteria in the epidermal cells stimulate root hair development and exit through the hair tip. We have actively been looking for ways to control the entry of bacteria into root meristem cells. Recently we have identified butyric acid as a regulatory molecule for plant intracellular invasion by bacteria. By incorporation of butyric acid (0–1 mM concentration) into agarose in which grass seedlings (Poa annua) bearing a Pseudomonas endophyte, we were able to regulate the entry of bacteria into cells of the grass. In agarose that did not contain butyric, we saw the highest level of bacterial entry into plant cells (with the longest root hair development); at the 1 mM concentration of butyric acid, bacteria did not enter into plant cells (and root hairs did not form); and intermediate level of butyric acid (0.5 mM) showed some entry of bacteria into meristematic cells (and short root hairs formed). Butyric acid may be useful to regulate intracellular invasion of meristematic cells and evaluate endophyte effects on growth and development of many plant species.

7 Use of Surrogate Hosts

In some cases experiments cannot be conducted using the host of the endophyte due to inability to obtain sufficient plant material, failure of seeds to germinate or slowness of plant growth under laboratory conditions. In several studies we have had success in using surrogate hosts to conduct experiments to determine the likely effects of endophytes on its host plant. For assessing the effects of endophytes from grass hosts, we use turfgrass species Poa annua (cool-season grass) and Cynodon dactylon (warm-season grass) to conduct in vitro experiments. Seeds of these species are readily purchased, and many of their seed-transmitted microbes may be removed by vigorous surface disinfection (40 min in 4% sodium hypochlorite). These species also readily germinate in agarose so that observations may be made on developing seedlings. Because some endophytes may be adapted to a particular host species, in selection of surrogate plants, it is probably advisable to select surrogate test plants that are taxonomically close to the original host of the endophyte.

8 Analysis of Endophyte Diversity

Diversity of isolated endophytic microbes on different culture media can be studied through nonmolecular methods. There are several ways to assess the diversity of isolated microbes, i.e. colonization frequency, isolation frequency, alpha diversity indices (Simpson’s, Shannon-Wiener diversity indices), species richness-evenness, etc. (Hata and Futai 1995; Mishra et al. 2012; Verma et al. 2007, 2014). The colonization frequency (%CF) of endophytic fungi may be calculated manually using the formula given by Hata and Futai (1995). %CF = Ncol / Nt × 100, where Ncol = number of segments colonized by each fungus and Nt = total number of segments studied.

Diversity indices: Simpson’s and Shannon-Wiener diversity indices and species richness may be calculated manually or by several programs available including PAST, BioDiversity Pro and Origin software (Verma et al. 2014; Orduna et al. 2011) based on the formula of Simpson’s diversity = 1 − ∑(pi)2 and Shannon-Wiener diversity = − ∑ s (pi log pi), where pi = proportion of frequency of the ith species in a sample. Species evenness can be calculated as evenness (E) = H/log(S), where H = Shannon-Wiener diversity and S = species richness (i.e. total number of species). Statistical verification can be done by ANOVA by SPSS 16.0 or a newer version of software to assess significant differences among mean diversity indices of samples collected from different locations and tissues (to check the temporal and spatial variation in diversity). Kharwar and his co-workers used the same approach to analyse the diversity of endophytic fungi of more than ten medicinal plants of Northern India.

To further determine the interrelationship and tissue and site specificity of endophytic microbial populations among different tissue samples and sites, ecological associations among endophytes with the different tissues and different localities of the host, principal component analysis (PCA) may be performed (Orduña et al. 2011; Verma et al. 2014).

8.1 Non-culture Methods

There is a difference between the number of microbes isolated into pure culture and numbers present as endophytes in plants (as few as 1–5% of the microbes may be isolated from the natural habitats) (Stewart 2012; Staley and Konopka 1985). It is likely that numerous microbes are never isolated and a majority of endophytic diversity, like other microbial habitats, are missed by cultivation-based methods (Hugenholtz et al. 1998). This is because many microbes do not grow under the conditions currently being employed to isolate them. In the past few years, molecular approaches have been employed to elucidate diversity of microbes within host tissues (Guo et al. 2001). Guo et al. (2001) amplified the 18S rRNA gene from the total DNA extracted from frond tissues of Livistona chinensis followed by cloning, sequencing and phylogenetic analysis to elucidate fungal endophyte diversity. They recovered some novel fungal endophytes that had not previously been isolated by culture methods. Technology has advanced the study of molecular diversity and phylogeny of endophytes (Fig. 4.6) (De Hoog et al. 2005; Green et al. 2004; Duong et al. 2006). Denaturing gradient gel electrophoresis (DGGE) has been employed successfully in studying direct endophyte diversity in leaves of Magnolia liliifera (Duong et al. 2006).

Fig. 4.6
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Schematic way for the steps of analysis of diversity and phylogeny of endophytes

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8.2 Metagenomics and Pyrosequencing

Metagenomics (environmental genomics) involves the study of all genetic material recovered directly from any environmental samples, i.e. water, soil, plant tissue and animal tissue samples. This technique offers a powerful tool for surveying microbes and has been employed in the study of the plethora of endophytes in several studies (Guo et al. 2001; Duong et al. 2006; Marco 2011). Metagenomics is not restricted to phylogenetic description based on the 16S or 18S rRNA genes but provides much important information about functional genes and their diversity composition in communities.

Pyrosequencing has been applied to the study of the composition of endophytic community diversity in several host plants (Romero et al. 2014; Manter et al. 2010). High-throughput sequencing technologies (pyrosequencing) or next-generation sequencing (NSG) does not require a cloning step, and greater yields of sequence data can be obtained (Mardis 2008; Segata et al. 2013). Advancement in next-generation sequencing has minimized the cost of analysis of complex metagenomic sequences. It has revolutionized the metagenomic study. Shotgun metagenomics and pyrosequencing provide information about organism’s diversity and their theoretical metabolic functional role in host plant (Kaul et al. 2014; Tian et al. 2015). This could be very important in the study of functional endophytism. Recently metagenomics and pyrosequencing technologies have been applied successfully to study the functional metabolic role of endophytes in several host plants along with their structural diversity (e.g. Sessitsch et al. 2012; Tian et al. 2015). Sessitsch et al. (2012) reported functional roles of the endophytic community of rice roots, and their data suggested that endophytes are involved in promoting plant growth and enhancing stress tolerance against biotic and abiotic stresses. They also found that endophytes might play important roles in the entire nitrogen cycle including nitrogen fixation, denitrification and nitrification. Tian et al. (2015) investigated and compared functional genomics of healthy and nematode-infected tomatoes root-endophyte microbiomes. They showed that the bacterial community was involved in nematode pathogenesis. Using 454 pyrosequencing, Jumpponen and Jones (2009) analysed fungal communities in the phyllosphere of Quercus macrocarpa and compared fungal diversity and distribution among trees located in different locations. Using 454 pyrosequencing techniques, Toju et al. (2013) also described the association of both endophytic fungi and mycorrhizal fungi in roots of different plant species. Metagenomics and NGS technologies have been employed extensively in biological science in the past few decades; however its application in revealing endophyte functional diversity and their composition in plant is limited to few studies. Some limitations of the techniques include datasets that are very complex and comprehensive requiring novel tools, statistical software, extensive storage, visualization and analysis with existing datasets (Thomas et al. 2012). Further, there is insufficient annotation of submitted sequences in public databases, and this may be the one major limitation of metagenomic approaches.

8.3 Microarray: Gene Chips to Study the Expression and Mechanisms of Interaction

DNA microarray (commonly known as DNA chip or gene chip) along with real-time PCR (RT-qPCR) analysis is one of the fastest-growing new molecular techniques. RT-PCR is now very common to study the gene expression in host plant tissue, and it has been now widely applied in enabling endophyte role in modulation of gene expression in host plant (Gond et al. 2015a). However it is limited to few gene studies at a time. DNA microarray has the advantage of studying whole-gene expression altogether in the organism, and it has been used for the study of differential gene expression in biological systems. This important technology may be used to understand the molecular mechanisms of plant-endophyte interactions (Dinkins et al. 2010; Kaul et al. 2014). A diagrammatic representation of DNA microarray is shown in Fig. 4.7. Since very little information about the molecular mechanisms of the plant-endophyte interactions are known, therefore microarray study followed by RT-PCR may put forth more light on that aspect. Some studies have sought to analyse differential gene expression in endophyte-free and endophyte-containing plants using DNA chip techniques (Johnson et al. 2003; Felitti et al. 2004, 2006; Dinkins et al. 2010). Commercial gene chips are available for many crop and medicinal plants. There is an opportunity for researchers to analyse and differentiate the relative expression of a large number of genes simultaneously for at least those plants for whom microarray platforms are available or with their close relatives (Ciannamea et al. 2006; Sawbridge et al. 2003; Dinkins et al. 2010). Microarray analysis of transcriptomes of Arabidopsis-Pseudomonas fluorescens (endophyte) interactions showed the up-regulation of many genes involved in critical metabolism, signal transduction and stress management. Significantly putative auxin- and nodulin-related genes were up-regulated, and ethylene-responsive genes were downregulated (Wang et al. 2005). Dinkins et al. (2010), with some success, studied the differential gene expression in tall fescue with their obligate endophyte Epichloë coenophiala using the Affymetrix Wheat Genome Array GeneChip® and Barley1 Genome Array GeneChip®. Irizarry and White have also applied Affymetrix cotton GeneChip to study the role Bacillus sp. (endophyte) in cotton seedling differential gene expression. They found that endophytes up-regulated some key genes related with nitrogen metabolism and auxin synthesis (not published). They have also observed the phenotypic effect of the same bacteria on the development of better root architecture than control (Irizarry and White 2017).

Fig. 4.7
figure 7

Schematic representation of steps to study gene expression during plant-endophyte interaction

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Phenotypic effects of endophytes on hosts include growth promotion, stress tolerance, enhanced ability to acquire mineral from the soils and improved nitrogen utilization (Malinowski and Belesky 2000). Microarray analysis could be used to understand the molecular mechanisms of beneficial impacts of endophytes on plants. A major limitation of microarray analysis is the lack of availability of gene chip platforms for specific plants and compatible reference datasets for statistical analysis.

9 Techniques for Bioactive Metabolite Analysis

Many endophytic microbes, particularly fungi, synthesize bioactive metabolites that may be candidates for treating many current and newly emerging diseases in humans, plants and animals (Strobel and Daisy 2003; Kusari and Spiteller 2011, 2012). Many endophytes have been found to produce metabolites similar to those produced by host plants. Some examples are paclitaxel (Taxol), podophyllotoxin, deoxypodophyllotoxin, camptothecin, hypericin, pipericin and emodin (Chithra et al. 2014; Gond et al. 2014a, b; Shweta et al. 2010; Tan and Zou 2001) and azadirachtin (Kusari et al. 2012). There is potential to discover novel and useful molecules from endophytes by manipulating culture conditions. Cultural conditions include media types, source of nitrogen, temperature, etc. (Kusari et al. 2012). A procedure for isolating bioactive molecules is shown in Fig. 4.8.

Fig. 4.8
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Schematic showing steps in extraction and identification of bioactive molecules from endophytic microbes

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Steps in metabolite isolation include (1) culture of endophytic isolates in broth media (different types of broth media may be tested); (2) extraction of secondary metabolites from the medium at different time intervals using different organic solvents (e.g. hexane, ethyl acetate, chloroform, etc.) followed by drying of the crude extract (Duarte et al. 2012); (3) screening (e.g. antimicrobial, anticancer, antioxidant, antimalarial, antiviral test, etc.) of bioactivity of the crude extract; (4) purification of pure bioactive compounds from the active crude extract by (4a) thin-layer chromatography (TLC), (4b) column chromatography, (4c) high-performance liquid chromatography (HPLC) and (4d) gas chromatography; and (5) identification of structure of pure compound by (5a) nuclear magnetic resonance (NMR), (5b) LCMS, (5c) GCMS and (5d) X-ray crystallography (Fig. 4.8).

Several forms of nuclear magnetic resonance (1D and 2D NMR) spectroscopy are frequently applied techniques for structural elucidation of bioactive compounds isolated from endophytes (Strobel et al. 1999, 2002; Kusari et al. 2009). Several forms of 2D proton NMR like correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) help in the determination of the correct conformation of molecules (Duarte et al. 2012). LCMS and HPLC and other mass-based techniques are generally used for the identification of already known compounds (Stierle et al. 1993; Liu et al. 2007; Kusari et al. 2008). GCMS analysis is frequently successful for the isolation and identification of oily or volatile compounds (Gond et al. 2014a, b). In some cases pure compounds may crystallize, and structures may be elucidated by crystallography (Kharwar et al. 2009; Strobel et al. 2002). Purification and identification steps are critical and may require optimization and the expertise of an organic analytical chemist for deduction of correct structures. A large number of bioactive compounds have been reported from endophytic fungi in the past few decades using the above methods. Many good reviews are available on endophytic fungal metabolites, their sources and molecular structures (Tan and Zou 2001; Schulz et al. 2002; Strobel and Daisy 2003; Gunatilaka 2006; Kusari and Spiteller 2011, 2012; Kharwar et al. 2011b; Xu et al. 2014; Gond et al. 2014a, b).

10 Conclusions

Endophytic microbes are now recognized as important components of microbial communities of plants. However, we still do not understand the extent to which endophytes and hosts have come to be interdependent. In the extreme case of interdependency, we ask the question: Can plants survive or seedlings develop without involvement of endophytic microbes? This question has not been answered, but it is increasingly looking relevant. From a less extreme perspective, endophytes may be probiotics of plants. Here too, it is important to work out the mechanisms involved in plant-endophyte interactions at the molecular level. New technologies including transcriptomics (RT-qPCR), DNA microarray analysis, high-throughput sequencing (NGS) and protein-based array analysis may help to develop an understanding of the interaction mechanisms and functional roles. Conducting relevant experiments is the most important approach to determine the extent of the interdependency of endophytes and hosts. Once the mechanisms of interactions and functional roles are understood, we may understand why plants maintain endophytic microbial communities. Developing a better understanding of endophytes and their effects on hosts and host ecology will also enable us to find applications for endophytes in agriculture and medicine.