Keywords

1 Introduction

Orchidaceae is one of the largest angiospermic plant families with roughly around 28,484 species (Govaerts et al. 2017). A majority of the orchids are narrowly distributed in a particular region (Lozano et al. 1996) and are highly affected by habitat clearance compared to other plant species (Jacquemyn et al. 2007). The mycorrhizal fungi symbiotically associate with orchids and serve as an important source of nutrients and water to the orchids (Zettler 1997a, b). The specialization in pollination and mycorrhizal association is linked to the size and diversity of taxa in Orchidaceae (Stewart and Kane 2007; Cozzolino and Widmer 2005; Taylor et al. 2003). The floral beauty and attraction of orchids directly contribute to their mass decline in the wild. But, mycorrhizal associations have enabled orchids to survive in diverse habitats around the world. In nature, orchid seeds fail to germinate in the absence of mycorrhizal fungi (Fig. 13.1). In vitro culture of orchids was successful using both seeds (Knudson 1922) and explants (Arditti 1984). Many studies have focused on asymbiotic propagation of orchids from seeds (Arditti 1967; Sheehan 1983). As symbiotic propagation and restoration of orchid population in the wild are either totally neglected or not much emphasized, Rasmussen (1995) suggested that a worldwide support and importance should be given to investigations involving symbiotic seed germination. Orchids are economically important as Vanillin from Vanilla planifolia is used as flavoring agent for food and drink, and tissues of Gastrodia are used in the preparations of natural medicine (Griesbach 2000). Anthropogenic activities including theft of attractive individuals have driven many of the aesthetic orchid species in nature to extinction. Therefore, conservation efforts for these orchid species should involve a thorough understanding of their biology (Dearnaley 2007). In this chapter we discuss not only the structure, diversity, and significance of the mycorrhizal fungi in orchid lifecycle, but also the importance of these fungi in orchid conservation.

Fig. 13.1
figure 1

Different stages in the lifecycle of an orchid. Mycorrhizal fungi are mandatory for seed germination and seedling development. Adult green photosynthetic orchid can acquire nutrients through mycorrhizal fungi and even supplement carbon through mixotrophy

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2 Types of Orchid Roots and Velamen

The root system of orchid life-forms differs in several aspects (Rasmussen 1995). The roots of epiphytic and lithophytic orchids are ecologically similar as the roots are exposed to light and air (Fig. 13.2). The aerial roots of epiphytic and lithophytic orchids are perennial, photosynthetic with a fairly constant growth throughout the year (Dressler 1993; Muthukumar and Shenbagam 2018). In contrast, roots of terrestrial orchids are usually non-photosynthetic, have a limited life span of up to 3 years, and show seasonal changes in growth and architecture (Bayman and Otero 2006).

Fig. 13.2
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(a)–(l) Morphology and mycorrhizal anatomy of orchid roots. (a), Velamentous roots of Dendrobium sp. (a) and Acampe praemorsa (b); (b), Velamen (vel) and exodermis (ex) in aerial roots of A. praemorsa; (c), Bilayered velamen (vel) and exodermis (ex) in Spathoglottis plicata;(d), Root hairs of S. plicata with fungal hyphae (black single arrow head) and moniliform cells (black double arrow head); (e) Chlamydospore-like cells (c) in root hair of S. plicata; (f), Fungal hyphae (black arrow heads) transversing the velamen in Dendrobium sp.; (g), Fungal hyphae (black arrow head) in the velamen (vel) and passage cell (pc) of the exodermis (ex) (white arrow heads) in Dendrobium sp. root; (h), Transverse section (T.S) of Dendrobium sp. substrate root with patchy mycorrhizal colonized region (mcr). The root portion attached to the substrate is indicated by black arrow heads; (i), T.S. of Eulophia epidendraea terrestrial root with diffused mycorrhizal colonized regions (mcr) throughout the root cortex; (j), Intact pelotons (ip) in cortical cells of Luisia pulniana; (k), Intact (ip) and degenerating (dp) pelotons in Vanda spathulata; (l), Degenerated peloton (dp) with fungal hyphal remnants (black arrow head) in cortical cell of L. pulniana. Scale bars: A = 2 cm; B–L = 50 μm

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Roots of terrestrial orchids grow in soil or litter, and certain terrestrial orchids have two distinct types of roots, i.e., mycorrhizal and non-mycorrhizal (Rasmussen 1995). Non-mycorrhizal roots have more xylem and higher amyloplast than mycorrhizal roots (Bayman and Otero 2006). Nevertheless, most of the terrestrial orchids possess mycorrhizal fungi in their roots even in the adult stage of their life cycle (Rasmussen 1995). Compared to the obligate mycorrhizal nature of the terrestrial orchids, roots of most of the epiphytic and lithophytic orchids are facultatively mycorrhizal with frequency of colonization exhibiting variation to certain extent (Zelmer et al. 1996; Bayman et al. 1997; Otero et al. 2002; Rasmussen 2002).

Orchids have multiple epidermis consisting of one to several layers of thin-walled cells called velamen (Porembski and Barthlott 1988). The velamen helps the roots to trap water and nutrients (Dressler 1990; Rasmussen 1995). Roots of epiphytic orchids have more layers of velamen than terrestrial roots (Dressler 1990). In addition, epiphytic orchids growing in exposed or dry microenvironment like the Acampe tend to have multilayered velamen compared to those like Bulbophyllum which grow in more humid microenvironment (Muthukumar and Kowsalya 2017; Muthukumar and Shenbagam 2018). Based on their observations of orchids from exposed habitats of dry and humid environments, Sanford and Adanlawo (1973) suggested that the size of velamen may be indicative of the prevailing environmental factors such as moisture and temperature of the habitats. Oliveira and Sajo (1999) indicated that the velamen cells with suberized and lignified thickenings provided mechanical support to avoid cellular collapse during dehydration.

3 Nature of the Endophytic Fungi

In nature, orchid roots are colonized by diverse group of fungi, some of which may not be of true mycorrhizal in nature (Warcup 1981; Bayman et al. 1997). The endophytic orchid mycorrhizal fungi (OMF) of true mycorrhizal nature should have the potential to stimulate seed germination, enhance protocorms, and/or early seedling development and subsequently improve the growth and reproduction of the adult plants (Liu et al. 2010). OMF forms pelotons in root cortical cells and in seeds and the limitation of the OMF colonization to suspensor cells in the embryo and the epidermal hairs in germinating seeds clearly shows that the colonization process is dictated by the orchid and the symbiosis is adapted to this control (Hadley 1982).

The one way, through which the orchids obtain nutrients from the fungus, is through the digestion of the fungal hyphae. In orchids, the digestion of the pelotons takes place either by tolypophagy or phytophagy. In tolypophagy the complete digestion of pelotons occurs, whereas in phytophagy the fungal cell contents are released into the plant fungus interface through the lysis of the fungal tips (Burgeff 1959). Therefore, at any given point of time, orchid roots contain different proportions of intact and lysed pelotons (Fig. 13.2). Varying levels of intact to lysed or lysing pelotons have been reported in several orchids growing in the Western Ghats of south India (Muthukumar and Sathiyadash 2009; Muthukumar et al. 2011; Sathiyadash et al. 2012). Some components derived during this hyphal digestion play the role of fungal elicitors, which make easier the further colonization of the OMF (Liu et al. 2010). Further, the fungal elicitors of OMF have been shown to promote the development of protocorms and the development of seedling growth in orchids (Dong et al. 2008; Zhao and Liu 2008).

Several fungi that exhibit differential responses as parasites and pathogens are also shown to be symbionts of orchids. For example, Ceratobasidium stimulate the germination and seedling growth of Ionopsis sp. (Otero et al. 2004), but the same fungal isolate was shown to be pathogenic to Dendrobium mericlones (Porras-Alfaro 2004). Similarly, studies have also shown that the fungal symbionts of orchids could cause root rots in pea, soy bean, pine and spruce (Hietala et al. 2001; Yang et al. 2005). Thanatephorus isolated from the orchid Pterostylis acuminata was moderately pathogenic on lettuce and severely pathogenic on cauliflower and radish (Carling et al. 1999). Rhizoctonia solani which act as mycorrhizal symbionts in V. planifolia and Vanilla phaeantha also cause pathogenic lesions on the same root (Alconero 1969).

4 Orchid Mcyorrhizal Fungi (OMF)

4.1 Colonization Patterns of OMF in Orchid Roots

The fungal colonization patterns significantly differ both in epiphytic or lithophytic and ground orchids. In all these types the fungus enters the root primarily through the root hairs and occasionally directly through the epidermal cells (Sathiyadash et al. 2012). In epiphytic forms, root portions that are only attached to the substrate like the tree bark contain mycorrhizal colonization and those that are not attached to any substrate are free from fungal colonization (Fig. 13.2). Nevertheless, the free aerial root upon entry into soil develops mycorrhizal colonization (Muthukumar and Kowsalya 2017). The fungal colonization therefore is patchy in epiphytic and lithophytic orchids, whereas it is diffused throughout in ground orchids (Sathiyadash et al. 2012; Muthukumar and Kowsalya 2017). The fungal hyphae transverse the velamen tissue and enter the cortex through the passage cells of the exodermis. From the exodermis the fungus spreads through the root cortex intracellularly forming a highly coiled structure called pelotons (Fig. 13.2). The size of the pelotons varies with the orchid species and is determined by the cortical cell size as evidenced by a strong correlation between the peloton and cortical cell dimensions (Sathiyadash et al. 2012). The nucleus of the cortical cells is pushed to the periphery in the invaded cells and like in other mycorrhizal types, the fungus never trespass the endodermis. In the cells of the root cortex and in root hairs chlamydospore-like structures or moniliform cells are present (Sathiyadash et al. 2012).

The relationship between orchid and the mycorrhizal fungi is unique in the plant kingdom. Orchid mycorrhizae may at times involve mycobionts that are pathogenic to other plant species as shown above, suggesting the orchid’s specific ability to neutralize the possible virulence pathways of the pathogens (Watkinson 2002). These factors render orchids as an excellent model to study plant-fungal interactions. The establishment of the symbiotic interaction between the mycorrhizal fungi and the process of fungal colonization encompasses a wide range of similarities and differences with pathogenesis. This knowledge could provide an idea in developing new strategies to overcome or reduce the severity of the pathogenic interactions in non-orchidaceous plant species (Watkinson 2002). In the symbiosis between the orchid and the fungi, orchid is a dominant partner which helps to predict the evolution of plant-fungal interactions.

An increased survival of the orchid seedlings was often possible when the seedlings are mycorrhizal (Anderson 1991). Seeds of certain orchids like Encyclia tampensis, Liparis liliifolia, and Taeniophyllum obtusum germinate only in the presence of OMF under in vitro conditions (Irawati 1993; Rasmussen and Whigham 1998; Zettler et al. 1999). Likewise, protocorms of Eulophia alta develop more rapidly when mycorrhizal under in vitro conditions (Johnson et al. 2007). Symbiotically raised orchid seedlings can serve as both plant material as well as the source for mycorrhizal inoculums in conservation efforts (Batty et al. 2006).

4.2 Nutrient Transfer by OMF

Most of the orchids depend on mycorrhizal partners for their nutrients during seed germination and early developmental stages (Rasmussen 1995; Cameron et al. 2006; Rasmussen and Rasmussen 2009; Wu et al. 2013) (Fig. 13.1). The limited food resources in the seeds and the inefficiency of orchids to acquire nutrients from the substrates render orchids highly dependent on mycorrhizal fungi (Leake 1994). The OMF transfer nutrients from different substrates to their symbiotic orchid host (Dearnaley and Cameron 2017). Numerous studies have demonstrated the mycorrhizal fungi mediated enhancement of nutrient acquisition by orchids in the native ecosystems (Alexander 2007; Smith and Read 2008 and references there in). In mixotrophic orchids, OMF transfer carbon (C), nitrogen (N), phosphorus (P), and other minerals to the adult photosynthesizing green plants (Zimmer et al. 2007). In epiphytic orchids, the OMF mycelium decompose organic matter such as bark and make available the nutrients to the orchids (Zhang et al. 2018). Nevertheless, at times the transfer of nutrients could be unidirectional (Hadley 1989), bidirectional (Cameron et al. 2006) or even mycorrhizal independent (Purves and Hadley 1975).

Carbon Transfer

Most of the adult green orchids are autotrophic and fulfill their C requirement by photosynthesis. In addition to these, the orchid might also supplement its C requirement through the transfer organic C from the OMF (Gebauer and Meyer 2003). No difference in the C fixation or distribution was evident among the mycorrhizal and non-mycorrhizal Goodyera repens plants exposed to 14CO2 (Hadley and Purves 1974). Moreover, the fungal mycelium originating from the colonized roots had no measurable radioactivity. So it could be inferred that Rhizoctonia goodyerae-repentis fail to utilize the photosynthates from the host orchids (Hadley and Purves 1974). The absence of C translocation from the host to the fungus could be either due to the inability of the fungus to obtain the host metabolites, or may also be due to the adequate amount of C in the growing media (Hadley and Purves 1974). In Ceratobasidium cornigerum, 14C supplied as glucose peaked in the external mycelium within 48 hours, while its accumulation took several days in G. repens protocorms (Hadley 1984). Further, when the protocorms, plantlets and plants of G. repens were fed with 14C through the external mycelium of the fungus, the plants failed to take up the C even under stressed conditions (Alexander and Hadley 1985). This suggests that the translocation of C movement mostly was unidirectional from the fungus to the host and the translocation of C ceased when the orchids reached certain stage of development (Alexander and Hadley 1985). In contrast to these, the C uptake rate by mycorrhizal Dactylorhiza purpurella and Cymbidium protocorms was higher than non-mycorrhizal protocorms (Hadley 1984). In the same study, the C uptake by the mycorrhizal G. repens protocorms was 50% higher than the non-mycorrhizal protocorms (Hadley 1984). In addition to the mycorrhizal mycelium of Ceratobasidium cornigerum transferring C to roots, rhizomes and green shoots of the G. repens plants, the C fed to shoots as 14CO2 was readily assimilated and transferred to the rhizome and onwards to the extending external mycelium of the OMF (Cameron et al. 2006). This confirms the bidirectional flow of C and the true mutualistic nature of OMF.

Nitrogen Transfer

The plants absorb the N in the form of NO3− and NH4+ (Zhang et al. 2018). Both terrestrial and epiphytic orchid life forms absorb NO3− and NH4+ from the substrates, but terrestrial orchids tend to absorb more NO3− than NH4+. In addition, NO3− concentration plays a critical role in flower and flower bud formation in Cymbidium sinense, whereas NH4+ concentration fails to produce any such effects (Pan and Chen 1994). This clearly shows the orchids preference for different N forms. The inability of the orchids to utilize organic N emphasizes the importance of the mycorrhizal symbiosis in orchid seed germination and development of the seedlings in the natural habitats (Dijk and Eck 1995). In addition, the N nutrition of orchids gets even more complicated as the N sources vary depending on the orchids’ requirement and life stages (Dijk and Eck 1995). The mycorrhizal seedlings of Cymbidium goeringii also differed in its uptake of N forms from the soil at different depths due to the variation in the distribution patterns of the Rhizoctonia spp., hyphae in the soil. Further, the fungal hyphae present in the upper and deeper layers of the soil are responsible for NH4+ and organic N uptake respectively (Wu et al. 2013). The transfer of N from the fungus to the orchid was first demonstrated by Cameron et al. (2006) in G. repens. Significant amounts of glycine labeled N (15N) was transferred to the roots (2%) and shoots (20%) of the terrestrial orchid G. repens by the mycorrhizal fungus (Cameron et al. 2006). This suggests that the rate N transfer from the fungus to the plant is likely to be dependent on the activity and nature of the plant-fungal interfaces in the roots (Cameron et al. 2006).

The foliar concentrations of isotope labeled N (15N) both in green (Cephalanthera damasonium) and achlorophyllous (Dactylorhiza sambucina) orchids growing in forest and grassland sites in Europe was significantly higher than the co-occurring herbs and trees suggesting the significance of the fungal symbiosis (Gebauer and Meyer 2003). Earlier studies have demonstrated an improved N nutrition in certain autotrophic plants, when glycine and other amino compounds were provided to its mycorrhizal fungal partner (Taylor et al. 2004).

Phosphorus Transfer

The P uptake of adult G. repens plants colonized by R. goodyerae-repentis and grown in different P levels was 100 times more than its non-mycorrhizal counterparts because of OMF mediated P uptake like in other mycorrhizal systems (Alexander and Hadley 1984). Radioactivity could be detected in the protocorms of D. purpurella inoculated with orchid endophytes and fed with radioactive P orthophosphate suggesting the transfer of P from the fungus to the host plant (Smith 1966). The translocation of 32P via the fungal hyphae occurs up to a distance of 9 cm from the root, with optimum distance being 2–3 cm (Alexander and Hadley 1984). However, the density of the fungal hyphae decreases with an increasing distance leading to a delay of phosphate reaching the nearest root (Alexander and Hadley 1984). Although the lack of a significant positive correlation between root length and 32P content in mycorrhizal G. repens (Alexander and Hadley 1984) casts doubt on the role of OMF in P nutrition, later studies have shown that OMF mediated P transfer. For example, the external mycelium of C. cornigerum colonizing G. repens was capable of assimilating and transporting the 33P orthophosphate into the plant (Cameron et al. 2007). Further, 7 days after 33P exposure, 6.3% of the 10% of the P transferred over diffusion barrier were detected in the shoots of G. repens indicating that the OMF could uptake and translocate significant amount of P to its partner (Cameron et al. 2007).

4.3 Plant Growth Stimulation by OMF

Colonization by OMF enhances both the orchids vegetative and reproductive growth, ex vitro survival rates, induces early flowering, improves flower quality, and reduces disease severity in the seedlings (Chang 2008). Similarly, all the Acampe praemorsa in vitro raised seedlings inoculated with OMF survived under ex vitro conditions (Sathiyadash et al. 2013). Colonization of roots by Rhizoctonia sp., increases plant height, biomass, root formation, and root length in Cymbidium kanran (Lee et al. 2003), Cymbidium goeringii (Wu et al. 2013), and Cymbidium sp. (Wu et al. 2010). However, colonization by Tulasnella repens only enhanced the plant biomass in these orchids. Similar effects were also reported for Dendrobium nobile, D. loddigesii (Zaiqi and Yin 2008), Dendrobium officinale (Yang et al. 2008a), Haemaria discolor (Chang and Chou 2001), and Anoectochilus formosanus (Chang and Chou 2007) colonized by different OMF. Orchids like Anoectochilus roxburghii (Dan et al. 2012a), Dendrobium candidum and D. nobile colonized by Epulorhiza sp., Mycena dendrobii, Moniliopsis sp., Gliocladium sp., Mycena anoectochila were taller and had higher biomass (Dan et al. 2012b). Cymbidium colonized by six strains of unidentified OMF endophytes, as well as Doritaenopsis and Phalaenopsis colonized by Rhizoctonia and Ceratobasidium had higher biomass than un-inoculated seedlings (Fang et al. 2008; Wu et al. 2009, 2011). The in vitro raised Guarianthe skinneri when acclimatized with Trichoderma harzianum were taller and had more leaf and shoot numbers (Gutierrez-Miceli et al. 2008). Ex vitro raised Cattleya aurantiaca and Brassavola nodasa colonized by Epulorhiza were taller and heavier than their non-mycorrhizal counterparts (Ovando et al. 2005). Colonization of Dendrobium officinale roots by Mycena sp., increased plant height, biomass, and the number of new buds by two- four folds (Zhang et al. 2012). Further, increment in plant height and biomass are often considered as reliable parameters for the successful establishment of the symbiotic relationship (Jin et al. 2009a, b).

4.4 Phytohormone Production by OMF

The beneficial plant growth promoting microorganisms produce phytohormones, which are utilized by the host plants and it is one of the mechanisms for plant growth promotion by the microorganisms (Van Loon 2007; Shoresh et al. 2010). The bioactive compounds like indole-acetic acid (IAA), gibberellic acid (GA), and naphthalene acetic acid (NAA) produced by OMF are suggested to stimulate the development of D. candidum and D. nobile plantlets (Dan et al. 2012b). Gibberellic acid and NAA synthesized by OMF can also promote the elongation of stems and roots of D. huoshanense (Zhang et al. 1999). Isolates of Tulasnella, Epulorhiza and an unusual orchid endophyte Colletotrichum gloeosporioides has been shown to produce IAA under in vitro culture conditions (Robinson et al. 1998; Chung et al. 2003). The significant amounts of IAA detected both in the fungal mycelium as well as in the culture medium may be transferred to the orchid hosts which may influence the colonization process (Barroso et al. 1986). In addition, the detection of the presence of indole-3-ethanol (IEt) in the Ophrys lutea culture medium indicates its synthesis and this IEt could act as a precursor for IAA synthesis in the host orchids (Barroso et al. 1986).

Earlier studies have shown that OMF are capable of secreting gibberellins, heteroauxin, dormin, zeatin and zeatin riboside (Wu and Zheng 1994; Wu et al. 2002) and these plant hormones were shown to improve the growth of orchids (Yang et al. 2008a, b). In Gastrodia elata, seed germination and cell differentiation process are stimulated by plant hormones or fungal metabolic products (Guo and Xu 1990; Xu 1993). In Dendrobium hancockii, the germination of seeds was stimulated by the presence of OMF extracts obtained from the protocorms of other orchids like Liparis nervosa and G. elata (Guo and Xu 1990). Trichoderma sp., can either synthesis phytohormones or alter the internal phytohormone homeostasis of the host plant (Shoresh et al. 2010; Salas-Marina et al. 2011). Extracted compounds and mixtures of fungi, filter concentrates, mycelia extracts, and hydrolytic products of cell walls, peptides and proteins are shown to act as fungal elicitors (Smith 1996; Hahn 1996).

4.5 Role of OMF in Disease Resistance

One of the major bottlenecks in commercial orchid cultivation is the disease outbreak, as bacteria, fungi or viruses affect the quality of plants by leaving brown spots or scars, and when the disease is not controlled or eradicated it may cause huge economic loss in orchid production (Wu et al. 2011). Soft rot diseases caused by Erwinia spp. is the most devastating disease in commercial orchid production (Liau et al. 2003). Phalaenopsis inoculated with R. solani and Ceratobasidium strains reduced the severity of soft rot, and a correlation also exists between disease symptom reduction and plant growth (Wu et al. 2011). In Vanilla, colonization by Ceratobasidium was shown to control the root rot caused by Fusarium (Bayman et al. 2011). OMF directly inhibit the pathogens through competition for space or nutrients. Further, OMF provides growth promoters or systemic-induced resistance to strengthen the plant defense mechanisms (Burns and Benson 2000; González et al. 2002). The improved nutritional status of orchids due to OMF makes them less susceptible to pathogens (Bayman et al. 2011).

Ceratobasidium isolated from orchids could also protect other type of plants from pathogens (Bayman et al. 2011). Hypovirulent and non-pathogenic Ceratobasidium present in soil protect the seedlings of cucumber and other crops from damping-off disease (Sneh et al. 2004; Ichielevich-Auster et al. 1985). Ceratobasidium isolated from Cranichis  sp., and Maxillaria sp., decreased the severity of sheath blight in rice caused by R. solani (Mosquera-Espinosa et al. 2013).

5 OMF Diversity

The presence of compatible mycobionts and different environmental factors plays a major role in the growth and recruitment of orchids in nature (McCormick et al. 2004). The unique feature of orchidaceae as well as its wide diversity may be attributed to its distinctive relationship with the mycorrhizal fungi (Zettler et al. 2004). The OMF diversity could be separated based on the ecology and photosynthetic ability of the orchid hosts (Taylor et al. 2002). However, information is limited on the role of fungal diversity in orchid distribution, population size, and genetic diversity (McCormick et al. 2004).

5.1 OMF Diversity in Terrestrial Photosynthetic Orchids

Terrestrial photosynthetic orchids are generally colonized by fungi belonging to five groups such as Heterobasidiomycetes, Hericianae, Hymenocaetanae, Thelephoranae, and Agaricanae (Rasmussen 2002) (Table 13.1 and Fig. 13.3). Earlier records mainly focused on the saprophytic fungi involved in the mycorrhizal association of orchids, but later both ectomycorrhizal (ECM) and parasitic fungi were also shown to associate with orchids in a mutualistic manner (Rasmussen 2002). Isolation of the ECM fungi belonging to Russulaceae from Corallorhiza spp. (Taylor and Bruns 1997) and the colonization of field-grown seedlings of Corallorhiza trifida with Thelephora–Tomentella complex of Thelephoriaceae, suggests that the ECM fungi can symbiotically associate with orchids (McKendrick et al. 2000). The ECM fungal genera associating with the non-photosynthetic Lecanorchis spp. include Lactarius, Russula, Atheliaceae, and Sebacina, of which Lactarius and Russula were found to be dominant (Okayama et al. 2012). Colonization of the achlorophyllous Epipogium roseum by the saprotrophic members of Coprinaceae suggest that the decaying wood materials would be used as a large and persistent C source for the growth of this orchid (Yamato et al. 2005). Mycobionts of Hexalectris spp. were found to be members of Sebacinaceae, Ceratobasidiaceae, Russulaceae, and Thelephoraceae (Kennedy et al. 2011). It is hypothesized that achlorophyllous orchids obligately associated with fungi have access to a large and persistent C source supplied by ECM fungi (Taylor and Bruns 1997; Leake 2005), as these fungi are likely to have access to such C supply (Kennedy et al. 2011).

Table 13.1 The diversity of fungal taxa involved in mycorrhizal association with different orchid life-forms
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Fig. 13.3
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(a)–(b) Morphology of orchid mycorrhizal fungi isolated from roots of the terrestrial orchid Tropidia thwaitesii and cultured in potato dextrose agar medium. (a), Hyphae and moniliform cells of Tulasnella (a) and Euphlorhiza (b). Scale bars = 20 μm

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An investigation on the mycobionts of Caladenia yielded 103 fungal isolates, most of which belonged to Sebacina vermifera and one isolate belonged to Tulasnella calospora. In an allied Caladenia species, 94% of the 33 isolates belonged to S. vermifera and a few to T. calospora, indicating that these orchids were associated with specific OMF (Warcup 1971). Further, investigations on mycobionts of terrestrial orchids have resulted in the discovery of new fungal taxa. For example, two new mycorrhizal endophytes, Ceratorhiza pernacatena and Epulorhiza calendulina, were characterized and identified from the roots of Platanthera praeclara and Amerorachis rotundifolia, respectively, from Canada (Zelmer and Currah 1995). A phylogenetic analysis of the mycorrhizal diversity from a single peloton of Disabracteata (a South African orchid) and Pyrorchis nigricans showed the presence of the fungi belonging to distinct groups of the Rhizoctonia alliance, like Epulorhiza, Ceratobasidium, and Sebacina (Bonnardeaux et al. 2007). Nevertheless, composite peloton formation by multiple endophytes within a cell further complicates the nutritional basis of OMF relationships (Rasmussen 2002). Further, the simultaneous presence of both intact and degraded patches of pelotons in root cortex may represent individual colonization events as adjacent patches may be formed by different fungi (Zelmer et al. 1996). If the nutritional concept is considered over the lifetime of an orchid, different types of fungi could be involved in utilizing different substrates during different periods (Rasmussen 2002). For example, the development of G. elata seedlings associated with Mycena osmundicola was dependent on its successive association with the parasitic Armllaria mellea (Xu and Mu 1990).

The saprotrophs were possibly over represented in previous studies on OMF diversity because of their ability to establish and grow in pure cultures more readily (Rasmussen 2002). Nevertheless, the development of DNA based approaches has widened the range of endophytic fungi with different nutritional strategies associating with orchids (Rasmussen 2002). In the view of Rasmussen (2002), majority of orchid mycobionts are basidiomycetes. In contrast, 78% of the photosynthetic and non-photosynthetic roots of Epipactis microphylla were found to be colonized by the Tuber sp., with the remaining roots also being colonized by other ascomycetes and a few basidiomycetes fungi (Selosse et al. 2004). The other potential ascomycetes mycorrhizal endophytes of Epipactis spp. in addition to Tuber sp., include Wilcoxina and Phialophora (Bidartondo et al. 2004).

The diversity of OMF associated with photosynthetic terrestrial orchids is generally low due to their high host-mycobiont specificity (McCormick et al. 2004). A phylogenetic analysis of the mycorrhizal association in Cypripedium elucidated that, most of the Cypripedium fungi formed narrow clades within the family Tulasnellaceae, whereas other root endophytes from Sebacinaceae, Ceratobasidiaceae, and Phialophora rarely support the high specificity in photosynthetic orchids (Shefferson et al. 2005). Narrow mycorrhizal specificity was seen in Stigmatodactylus sikokianus for Sebacina spp. even when the orchid roots were collected from different geographical regions (Yagame and Yamato 2008). The frequent colonization of roots in Paphiopedilum micranthum, Paphiopedilum ameniacum, Paphiopedilum dianthum, Cypripedium flavum, Cypripedium guttatum, and Cypripedium tibeticum, mainly by members of Tulasnellaceae, is a clear proof for the narrow phylogenetic range of mycorrhizal taxa in terrestrial photosynthetic orchids (Yuan et al. 2010). Further, the diverse OMF isolates colonizing these orchids belonged to the four discrete groups of the genus Epulorhiza. However, P. nigricans was colonized by the widest diversity of fungi including isolates of Epulorhiza and Ceratobasidium suggesting that this orchid could potentially benefit from this diverse association (Bonnardeaux et al. 2007).

The simultaneous colonization of D. officinale by Tulasnella spp., Epulorhiza sp., and Plateus seticeps and association of Dendrobium fimbriatum with Tulasnella sp., in certain orchid populations (Xing et al. 2013) suggest that co-occurring orchid species could benefit from different mycorrhizal partners (Waterman et al. 2011). However, this needs further confirmation as the small sample size of Dendrobium could have led to an underestimation of the fungal diversity (Xing et al. 2013). Twenty seven isolates of Tulasnella calospora, Tulasnella pruinosa, Tulasnella asymmetrica, Tulasnella irregularis, Epulorhiza sp., Tulasnella violea, and uncultured Tulasnella were associated with the orchid genera Paphiopedilum, Dendrobium, and Cymbidium (Nontachaiyapoom et al. 2010). In a study on the mycorrhizal diversity of Coppensia doniana, all the isolated OMF belonged to the genus Ceratobasidium which explains the wide potential habitats of this orchid, as these fungi can gain energy from organic matter in soil, litter, tree barks, and rock surfaces (Valadares et al. 2011).

The diverse group of fungal associates, such as typical mycorrhizal fungi from Tulasnellaceae, Ceratobasidiaceae, and ECM taxa of Pezizales colonizing the terrestrial orchid Gymnadenia conopsea contributes to its ability to colonize different habitat types (Stark et al. 2009). Edaphic conditions are also known to play an important role in the distribution of OMF colonizing roots of terrestrial orchids. For example, Tulasnella prima is the major mycobiont colonizing roots of several Chiloglottis species growing in soil and sphagnum hammocks over a range of more than 1000 km in southeastern Australia (Ruibal et al. 2017). In contrast Tulasnella sphagneti associated with Chiloglottis is rather restricted to the wet conditions of alpine sphagnum hammocks (Ruibal et al. 2017).

5.2 OMF Diversity Epiphytic Photosynthetic Orchid

In epiphytic orchids, low levels of patchy mycorrhizal colonization could be seen in roots attached to substratum (Bermudes and Benzing 1989; Pereira et al. 2005). Twenty-four Epulorhiza isolates were obtained from the roots and protocorms of Oncidium, Vanda, Arachnis, Dendrobium, Arundina, Diplocaulobium, and Spathoglottis examined from different sites in Singapore (Ma et al. 2003). The Andean epiphytic orchids like Stelis concinna, Stelis hallii, Stelis superbiens, and Pleurothallis lilijae were found to be colonized by Tulasnella spp. based on the septal ultrastructure, whereas the molecular analysis of the roots yielded seven distinct Tulasnella clades (Suarez et al. 2006). All the Tulasnella sequences were distinct from the already known sequences of mycobionts in certain terrestrial orchids and this indicated the adaptation of these fungi to tree stems and also its importance in orchid growth (Suarez et al. 2006).

Twenty-six isolates of OMF were identified from the tropical epiphytic orchids which formed two fungal lineages, related to Ceratobasidium spp., of which majority of orchids hosted more than one fungal lineage suggesting the variation in mycobiont association within related orchid species (Otero et al. 2002). Mycobionts from Tolumnia variegata clade with four fungal lineages of which only one lineage include fungi from Ionopsis utricularioides (Otero et al. 2002) and in a later study the same orchid was seen colonized with Ceratobasidium suggesting that I. utricularioides is specialized in effectively exploiting a specific fungal clade (Otero et al. 2004). The broad mycobiont specificity of I. utricularioides explains its survival in a broad geographical range and also its large population size (Otero et al. 2004). The rare epiphytic orchid Sarcochilus weinthalii was seen associated with single species of Ceratobasidium and direct sequencing of colonized root fragments and culture dependent methods also indicated its narrow specificity (Graham and Dearnaley 2012). In China, four epiphytic endemic orchids (Laelia autumnalis, Laelia speciosa, Euchile citrina, and Prosthechea squalida) harbored 71 isolates of fungal endophytes belonging to 20 genera of basidiomycetes and ascomycetes indicating the rich diversity of OMF association in these orchid hosts (Beltrán-Nambo et al. 2018). In south Ecuador, the epiphytic orchids colonized by 115 fungal isolates of 49 fungal operational taxonomic units (OTUs,) including four mycorrhizal OTUs belonging to Ceratobasidiaceae and Tulasnellaceae revealed high diversity of fungi colonized with orchid roots (Novotna et al. 2018).

6 Taxonomy of OMF

In the past, majority of the peloton forming fungi were classified as a member of anamorphic form-genus Rhizoctonia based on morphological characteristics (Athipunyakom et al. 2004b). The genus concept in Rhizoctonia was first established by De Candolle (1815). Earlier the form-genus Rhizoctonia consisted of a heterogenous assemblage of filamentous fungal taxa that failed to produce sexual spores and only share limited common features in their anamorphic stage (Garcia et al. 2006). However, new morphological criteria include nuclear number, septal nature, and parenthesome perforation, as well as teleomorphic stage for OMF (Moore 1987). Based on these, three new genera such as Ceratorhiza, Epulorhiza, and Moniliopsis were separated from Rhizoctonia. Ma et al. (2003) characterized the Rhizoctonia isolates using different media and colony hyphal characteristics, and identified them as species belonging to Epulorhiza. Basidial morphology was considered to be a reliable criterion for characterizing Rhizoctonia species at morphospecies level (Warcup and Talbot 1966, 1971, 1980), as fungal isolates from orchid roots only occasionally forms fruiting bodies in pure culture (Currah et al. 1987, 1990; Milligan and Williams 1988). In addition, the septal ultrastructure characteristics are also considered important for distinguishing the Rhizoctonia taxa (Khan and Kimbrough 1982; Marchisio et al. 1985; Currah and Sherburne 1992).

A system of anastomosis grouping based on hyphal fusion was widely adopted until last decade, as the basis for recognizing groups and taxa among the several fungi that constitute form-genus (Sneh et al. 1991). Currently, there are several accepted classifications based on the anastomosis group concept for both multinucleate (Ceratobasidium) taxa within the Rhizoctonia species complex (Carling 1996). Rhizoctonia-like mycobionts from Australian orchids though formed five anastomosing groups; the molecular sequencing showed that most of the anastomosing groups were monophyletic (Ramsay et al. 1987). Therefore, hyphal anastomosis behavior may not be the best indicator of evolutionary relationships between different intraspecific groups (Vilgalys and Cubeta 1994).

Later, the developments of biochemical methods were thought to be better to infer the phylogenetic relationships of fungi in Rhizoctonia species complex (Jabaji-Hare 1996). The biochemical methods for characterizing Rhizoctonia species include soluble protein patterns, zymograms and isoenzyme profiles which were employed to identify and study the genetic relationships among members of the form-genus (Jabaji-Hare 1996). Damaj et al. (1993) studied the relationship between binucleate Rhizoctonia isolates by isoenzyme electrophoresis. Grouping and identification of binucleate Rhizoctonia by pectic zymograms distinguished five zymographic groups in Ceratobasidium cornigerum (MacNish et al. 1993).

As induction of teleomorphic stages under laboratory conditions has been difficult for Rhizoctonia, their characterization is primarily based on the comparison of a limited number of anamorphic features and cytological probes (Moore 1987; Andersen and Stalpers 1994; Roberts 1999). Recently, molecular identification of fungi involving polymerase chain reaction (PCR) amplification of the nuclear ribosomal internal transcribed spacer (ITS) has revolutionized the identification of OMF (Currah et al. 1995; Gardes and Bruns 1993; Redecker 2000; Vralstad et al. 2002). Internal transcribed spacer region is the most effective single loci for the identification of fungi from species to genus levels (Bruns 2001; Seifert et al. 2007). Gardes and Bruns (1993) suggested that ITS region has certain advantages in fungi, as the whole ITS region has 600–800 bp and could be easily amplified with universal primers (White et al. 1990). Amplification of ITS gene from small, dilute or highly degraded samples is easy because of its multicopy nature of the rDNA repeats. Earlier studies (Gardes and Bruns 1991; Chen et al. 1992; Lee and Taylor 1992) demonstrated that the ITS region is often variable among morphologically distinct fungal species. Gardes and Bruns (1993) also developed a taxon specific primer for amplification of ITS rRNA gene. Based on these developments, the presence of Tulasnella and Laccaria in roots of Dactylorhiza majalis was detected by amplifying the mitochondrial large subunit (Mt-Lt) RNA gene (Kristiansen et al. 2001). The endophytes colonizing Neuwiedia vetrifolia roots were identified as belonging to Tulasnellales and Ceratobasidiales by amplification and sequencing of the mitochondrial ribosomal large subunit DNA (Kristiansen et al. 2004). The OMF endophytes of Acianthus, Caladania, and Pterostylis were identified using ITS-restriction fragment length polymorphism (RFLP) analysis from Queensland (Bougoure et al. 2005). Pereira et al. (2005) suggested that random amplification of polymorphic DNA (RAPD) analysis might reveal higher polymorphism between Epulorhiza epiphytica and Epulorhiza repens than found in the PCR-RFLP analysis. Furthermore, RAPD and morphological analysis indicated a degree of relatedness among the Ceratorhiza isolates obtained from the roots of different Oncidium species (Pereira et al. 2005).

Though, the presence of Epulorhiza in the roots of several tropical orchids was confirmed by Ma et al. (2003) using amplification of rDNA repeat from 3΄ end of the 18S rDNA gene and 5′ end of the 28S rDNA gene, the use of PCR based approaches for the amplification of OMF endophyte has certain limitations. For example, taxa belonging to Tulasnellaceae are difficult to characterize using standard PCR primer sets in spite of their common presence in orchid roots. This shortfall arises from the high evolution of the nuclear ribosomal operon (Binder et al. 2005; Moncalvo et al. 2006) and subsequent mutations of the conserved regions to which the primers hybridize (Taylor et al. 2002). Electron microscopic examination of the fungal pelotons of several epiphytic Pleurothalline orchids growing in the Andes revealed the predominance of the fungi belonging to Tulasnellaceae (Suarez et al. 2006). Nevertheless, only few of these fungi could be amplified using standard primers. But, true mycorrhizal symbionts of these orchids started to emerge only when nested PCR and several Tulasnella specific primers were used. Therefore, Taylor and McCormick (2008) developed a new set of selective primers with certain advantages like, low amplification of plant sequences and improved amplification of all tested Basidiomycota nuclear ribosomal DNA, including Tulasnella. Since then, specific primers have been routinely used to identify the mycobionts of orchids like Cypripedium, Papilopedium, Dactylorhiza, and Dendrobium. These types of sequencing analyses have revealed the association of a wide range of fungi belonging to Tulasnellaceae, Pluteaceae, and Ceratobasidiaceae with orchids (Shimura et al. 2009; Yuan et al. 2010; Valadares et al. 2011; Pellegrino and Bellusci 2009; Xing et al. 2013). The various mycorrhizal fungi isolated from the orchid roots along with their origin are shown in Table 13.1.

7 Symbiotic Seed Germination

7.1 Fungal Preference

Orchids have been well known for their exclusive association with fungi. Generally, orchids have minute seeds with a very small embryo without an endosperm. Therefore association with fungi is a prerequisite for orchid seed germination, as the germinating seeds are incapable of obtaining nutrients successfully independent of fungi (Arditti 1992; Uetake et al. 1992). Bernard (1899) first described the role of mycobionts in orchid seed germination. Different isolates of fungi are known to differ in their potential to stimulate the germination of orchid seeds which tempted Warcup (1973) to suggest that the most efficient fungal isolates need not have its origin from the same host. This suggestion was also supported by the study of Stewart and Zettler (2002) where the terrestrial orchid Habenaria macroceratitis was non-specific in its requirement for the fungal mycobiont. Nevertheless, Stewart and Kane (2006) in a later study speculated that H. macroceratitis could have a certain degree of fungal preference as the germination percentage of the seeds was higher in the presence of fungi isolated from orchid populations from which the seeds were collected compared to those isolated from other orchid populations. The existence of fungal preference is also evident in another study (Stewart and Zettler 2002) where Epulorhiza strains isolated from Habenaria quinqueseta promoted seed germination of Habenaria repens better than those isolated from Epidendrum conspseum, Spiranthes brevilabris, and H. macroceratitis. In the same study, Stewart and Zettler (2002) also showed that the in vitro raised seedlings of H. repens growing in different potting media and colonized by Epulorhiza strains isolated from S. brevilabris survived better compared to those colonized with Epulorhiza strains isolated from Epidendrum conopseum. Similarly, Cymbidium aloifolium inoculated with OMF Ceratobasidium strain RR isolated from distant orchid taxa (Rhynchostylis retusa) promoted the number, length, leaf thickness, root number and length, and biomass (Hossain et al. 2013). In addition, Epulorhiza sp., isolated from terrestrial orchid Eulophia epidendraea promoted the seed germination and seedling development of epiphytic endemic orchid Coelogyne nervosa (Sathiyadash et al. 2014). In a recent study Ceratobasidium species isolated from the roots of adult ghost orchid Dendrophylax lindenii was able to promote seed germination and seedling development of the same orchid (Mújica et al. 2018).

7.2 Fungal Specificity

An understanding on the specificity of OMF-orchid interactions is of crucial importance both for ecology and conservation of orchids. Seed germination, protocorm development, and seedling growth are stimulated by digestion of orchid mycobionts and subsequent uptake of the released nutrients by the immature orchid embryo (Clements 1988; Rasmussen 1995). Widely distributed orchids are expected to be either general in their preferences for mycorrhizal fungi or could be specific in associating with a broadly distributed fungus, as in many mutualistic relationships (Bascompte et al. 2003; Vazquez and Aizen 2003). In contrast, a narrow OMF specificity could be the reason for rarity and vulnerability of certain orchid species. Orchid-mycobiont specificity was considered controversial for many years. Earlier researchers thought that the orchid-fungus relationship to be quite natural and nonspecific both under in vitro and in situ conditions (Knudson 1922; Curtis 1939; Hadley 1970; Masuhara and Katsuya 1989; Masuhara et al. 1993).

To assess the orchid-mycobiont specificity under in vitro conditions, symbiotic seed germination techniques are often considered to be more useful (Dixon 1987; Zettler 1997a, b; Stewart and Kane 2006). The seed germination efficiency of terrestrial orchids are often low (Stewart et al. 2003; Zettler et al. 2005; Stewart and Kane 2006) compared to asymbiotic seed germination for the same taxa. This low seed germination efficiency is often attributed to the existence of certain degree of orchid-mycobiont specificity as the mycobionts colonizing terrestrial orchids during seed germination and later stages could be entirely different (Stewart and Kane 2007).

Spiranthes brevilabris although non-specific for mycobionts, fungal isolates Econ 242 (Epidendrum magnoliae) and Sbrev-266 (S. brevilabris) induced seed germination and development of the seedlings better than the several isolates tested (Stewart et al. 2003). Spiranthes cernua seeds successfully developed into leaf bearing stage when colonized by the mycobionts originating from Platanthera ciliaris suggesting that the mycobiont specificity could rarely be species-specific (Zettler and McInnis 1993). In contrast, in vitro seed germination of Bipinnula fimbriata showed the lack of specificity for mycobionts, as the seed germination was stimulated by all the Rhizoctonia-like mycobionts isolated from the adult orchids in its habitat (Steinfort et al. 2010). Similarly, Ceratobasidium isolated from Chloraea crispa of different habitat promoted protocorm development in B. fimbriata (Steinfort et al. 2010). Hadley (1970) also observed that the four isolates of Tulasnella calospora originating from wide geographical regions exhibited almost similar patterns of symbiotic response in Coeloglossum, Dactylorhiza, Goodyera, Cymbidium, Epidendrum, Laeliocattleya, and Spathoglottis (Hadley 1970). Nevertheless, Thanatephorus orchidicola originating from Dactylorhiza elata and Coeloglossum viride failed to form symbiosis with any of the orchids tested (Hadley 1970). As the mycobionts isolated from particular host was equally symbiotic with other orchid hosts, further, the orchid-fungus relationship does not appear to be also completely random, as certain orchids tend to be more receptive to certain endophytes than others (Hadley 1970).

Isolates of Sebacina vermifera and Tulasnella calospora originating from Caladenia and Diuris stimulated seed germination of the same host expressing the existence of a genus level specificity (Warcup 1971). Similarly, Ceratobasidium sp., (strain VT3) isolated from epiphytic orchid Vanda thwaitesii efficiently promoted seed germination, protocorm, leaf, and up to root formation stage in same taxa (Decruse et al. 2018). In addition, the three co-occurring orchid species Anacamptis morio, Gymnadenia conopsea, and Orchis mascula shared a low number of OMF OTUs, explaining the high specificity in OMF associations (Waud et al. 2016). Further, although S. vermifera stimulated the seed germination of Cladenia, Glossodia and Elythranthera (sub tribe Caladeniinae) failed to stimulat seed germination in Eriochilus, Leporella, Acianthus sp., Microtis, and Parsophyllum, thereby exhibiting specificity at subtribe level (Warcup 1981). The failure of the mycobionts isolated from Spiranthes floridana to support the seed germination and seedling development of S. brevilabris suggests that the genus Spiranthes failed to share the mycobionts during seed germination (Stewart and Kane 2007). Shefferson et al. (2005) also observed high mycobiont specificity at genus level occurring in the terrestrial orchid Cypripedium. Similar observation was made in the non-photosynthetic Hexalectris spp. colonized by S. vermifera (Taylor et al. 2003).

7.3 Cold Treatment

A delay in germination due to polymorphism may be an inherent characteristic of orchid seeds of temperate origin preventing the simultaneous germination of the seeds (Baskin and Baskin 1998). In natural habitats, seeds of Platanthera praeclara germinate during the first spring following dehiscence of capsule and seed dispersal, but further development of the protocorms were delayed until exposed to one or more winters (Sharma et al. 2003). So seeds subjected to cold treatment appear to be necessary to break seed dormancy in orchids.

The duration of the cold stratification may vary within a genus and may be species specific. Terrestrial orchids may require 3–9 months of cold stratification which may be species specific (Rasmussen 1995). Seeds of P. leucophaea require a cold stratification of 2–11 months for germination and the protocorms required chilling to transform into leaf-bearing seedlings (Stoutamire 1996; Zettler et al. 2001). In addition, cold stratification for 4 months combined with colonization by Ceratorhiza or Ceratorhiza and Epulorhiza resulted in higher percentage of protocorms advancing to the next stage (stage 3, top shaped protocorm and appearance of leaf) and proceed further. However, cold treatment for 6 months along with mycorrhization resulted in higher percentage seedlings transforming from stage 3 to 4 (elongation of the leaf promordium and protocorm enlargement) (Sharma et al. 2003). Higher seed germination percentage was observed in Cypripedium macranthos inoculated with mycorrhizal fungi after cold treatment (Shimura and Koda 2005).

7.4 Light

Orchid seeds possess a hydrophobic testa which maintains the seeds even the soil surface and exposing them to sunlight (Stewart and Kane 2006). Light exposure often initiates nutrient mobilization and therefore may not be directly involved in seed germination (Rasmussen and Rasmussen 1991). The effect of light on orchid seed germination may be genus or species specific (Stewart and Kane 2006). Initiation of photosynthesis occurred in in vitro raised Habenaria repens seedlings when exposed to light for 1 week. Similarly, exposure of Habenaria quinqueseta seeds to 33 days light increased its germination percentage (Stewart and Zettler 2002). Seedlings of Platanthera praeclara in stage 5 (formation of root initial) developed their first green leaf only after exposure to illumination for 30 days. The leaves grew up to 8 cm upon exposure to light for 50 days (Sharma et al. 2003). Transfer of H. macroceratitis seeds from dark to a 15 h photoperiod enhanced seed germination and early protocorm development (Stewart and Zettler 2002). The influence of photoperiod on seed germination is evident from the fact that exposure of H. macroceratitis seeds to 16–24 h of photoperiod increased the seed germination than those exposed to continuous dark. Further, a 16 h photoperiod appear to be optimum for symbiotic seed germination of H. macroceratitis (Stewart and Kane 2006). In contrast, protocorm development tends to peak under dark conditions (Stewart and Kane 2006). However, protocorms of Eulophia alata developed into plantlets only on exposure to 16 hour photoperiod (Johnson et al. 2007). Takahashi et al. (2000) failed to find any significant effect of light on the germination of Habenaria radiata seeds exposed to either continued darkness or light conditions.

8 Role of OMF Specificity in Orchid Rarity

All orchids depend on mycorrhizal fungi during seed germination and adulthood in the wild (Porras-Alfaro and Bayman 2007). As most of the orchid seeds are microscopic (Kull 2002), they lack stored nutrients to support germination (Rasmussen 2002). When the orchid seeds are colonized by compatible fungi, the germination of seed is initiated by utilizing the fungal sugars and this myco-heterotrophic condition is retained into adulthood in many orchids (Gill 1989). Therefore, mycorrhizal associations are a prerequisite for seed germination and seedling growth of orchids in natural habitats.

The rarity of many orchid species around the globe could be attributed to the decline in the occurrence of mycorrhizal fungi, as most of the orchids appear to have certain degree of specificity for certain mycobionts at the time of germination and during later life stages (Stewart and Kane 2007). A study on the specificity and preference of mycorrhizal associations in two species of the genus Dendrobium suggested that D. officinale associate with a wide range of basidiomycetes, while D. fimbriatum had a high degree of specificity toward Tulasnella (Xing et al. 2013). The rare status of the Florida terrestrial orchid Spiranthes brevilabris could be due to the high mycobiont specificity of this orchid (Stewart and Kane 2007). Tropical epiphytic orchids found that I. utricularioides was highly specialized in effectively utilizing a specific fungal clade, Ceratobasidium (Otero et al. 2004). In contrast to these, the wide distribution of S. cernua can be partially associated with its low-species specificity (Zettler and McInnis 1993).

The orchid-fungal specificity may be at genus or even at species levels. In the terrestrial orchid Cypripedium, high mycobiont specificity occurs at generic level (Shefferson et al. 2005). Of the seven species of Cypripedium examined, five species had mycobionts belonging to Tulansnellaceae (Shefferson et al. 2005). Dactylorhiza majalis predominantly associated with Tulasnellaceae showed occasional colonization by members of the genus Laccaria (Kristiansen et al. 2001). Irrespective of the nutritional mode, genus level specificity was seen in three varieties of non-photosynthetic orchid Hexalectris spicata with Sebacinaceae, whereas, species-specific mycobiont specificity was also reported in Hexalectris (Taylor and Bruns 1999). The mycorrhizal fungi may limit orchid distribution if they had genus or species specific for certain mycorrhizal fungi.

Fungal specificity is also influenced by certain factors like growth conditions, variety, and life stage of orchids. The fungi that can associate with an orchid in natural habitat may not necessarily germinate orchid seeds in vitro (Masuhara and Katsuya 1994). The fungal specificity in natural conditions is termed as ecological specificity and in vitro as potential specificity. Fungi may also differ in their ability to grow under different ecological conditions. A study on the tropical orchids, Tolumnia variegata and I. utricularioides, showed that orchid species with overlapping habitat preferences may differ in mycorrhizal specificity which could influence their distribution (Otero et al. 2004). The varied mycorrhizal diversity and specificity among the different taxa of Lecanorchis could be due to the diverse climatic conditions in which they exist (Okayama et al. 2012). Therefore, the patchy distribution of orchids in nature may be due to the presence or absence of the specific mycorrhizal fungi essential for their survival.

The mycorrhizal fungi colonizing the protocorm and the adult roots might not be the same. For example, Milligan and Williams (1988) found that Epulorhiza sp., initiated germination and E. repens was seen associated in the later stages of Microtis parviflora. Pre-infection with one fungus appears not to preclude colonization of a second endophyte. Even though adult Tipularia discolor had multiple fungal partners at the adult stage, its protocorm stage is more fungal specific associating with only taxa belonging to Tulasnellaceae (McCormick et al. 2004). But in Liparis liliifolia, both protocorms and adult plants fail to associate with multiple mycobionts (McCormick et al. 2012). Bidartondo and Read (2008) hypothesized from their studies on Cephalanthera and Epipactis that the mycobiont specificity is high during early stages of seedling development compared to more promiscuous germination and mature stages of plants life cycle. Isolation of four distinct types of fungi from H. spicata var. spicata and H. spicata var. arizonica gives evidence for the contribution of mycobiont specificity to the evolutionary diversification in orchids (Taylor et al. 2003). But the orchid Drakaea which is widely distributed in different environments, in spite of its preference for Tulasnella sp. (Phillips et al. 2011), raises the question of host-fungus specificity within the Orchidaceae which has been a point of contention for many years.

9 Importance of OMF in Conservation and Restoration of Orchids

Threat to the survival of orchids in nature is alarming, in spite of the significant advances in our understanding of the ecology of orchids (Gale et al. 2018). It is important to note that only 3.3% of the global natural orchid flora has been subjected to IUCN Red List assessments and 56.5% of these are under one or the other threat categories (Govaerts et al. 2017; IUCN 2017). Some of the common threats to natural populations of orchids include deforestation, expanding agricultural and forest plantations, and unsustainable exploitation of plants for food, medicine, and ornamental purposes (Gale et al. 2018). Reintroduction of orchids into their natural habitats is a strategy often adopted in the conservation of the threatened species (Vallee et al. 1997). Although orchid seedlings from asymbiotic process have been transferred to ex vitro conditions, they rarely survive due to the biological and ecological specificities of the taxa. The long term survival of orchids in managed or restricted habitats require the presence of appropriate fungi for seedling recruitment and plant nutritional support (Zettler and Piskin 2011).

Asymbiotically raised economically important endangered orchid Vanda coerulea exhibited a high mortality (>90%) when introduced into their natural habitat (Aggarwal et al. 2012). But, a successful protocol for the reintroduction of V. coerulea seedlings into their natural habitat was developed using Rhizoctonia isolated from the same orchid. Anderson (1991) found a 100% survival for all the symbiotic seedlings of Spiranthes magnicamporum upon their transfer to soil compared to the 5% survival for the seedlings raised asymbiotically (Anderson 1991). Successful application of symbiotic seed germination using Ceratobasidium for reintroduction of endangered terrestrial orchid, Dactylorhiza hatagirea has been reported (Aggarwal and Zettler 2010). The reintroduction of endangered orchids, S. brevilabris and H. repens was highly successful when mycorrhizal with Epulorhiza sp. (Stewart et al. 2003). So the symbiotic technique has practical merit for the conservation of highly important and endangered orchids.

Ecological changes at designated orchid locales may destroy the target mycorrhizal species and the compatible fungi may not be present at the site if the orchid itself is not present. So the symbiotically associated seedlings can serve as both plant material and a source of mycorrhizal inoculum for reintroduction efforts. Reintroduction of orchid seedlings from different habitats could be detrimental as this could alter the gene pool of the resident orchids which could eventually initiate anthesis (Zettler et al. 2005). In a recent study, the success of the out planted in vitro raised Cypripedium calceolus seedlings in the conservation programme in England was shown to be dependent on establishment of mycorrhizal symbiosis by the introduced plants and the presence of suitable mycorrhizal fungi at the introduced sites (Fay et al. 2018).

10 Conclusion

OMF colonize orchids and make the orchids survive better in various habitats. Terrestrial orchids show high diversity of OMF fungi, likewise epiphytic orchids are colonized by diverse groups of fungi. OMF is a crucial factor in the reintroduction of orchids and restoration of orchid populations in their natural habitats. So, identification of OMF diversity at specific sites provides the potential OMF which stimulates seed germination and seedling recruitment. This should be used for orchid conservation program in particular habitats.