Mycorrhiza in Orchids
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The orchids are a highly medicinal and floriferous assemblage of flowering plant species. Each orchid fruit encloses thousands of dust like minute and highly reduced seeds. Due to lack of endosperm and presence of complex carbohydrates, these seeds require specific fungal partner to accomplish germination in nature. The fungus plays a crucial role in the germination of the orchid seeds and their growth and development in mature orchid plants. In this manuscript, some intricacies pertaining to mycorrhizal interactions are being discussed.
KeywordsEndomycorrhiza Monocot Orchid seeds Root cortex Symbiosis
The orchids are highly evolved group of angiosperm plants belonging to monocot family Orchidaceae. In the world, a total of 28,000 species are confined to 763 orchid genera . Despite of being cosmopolitan in distribution, they do not appear as dominant vegetation in any part of the world.
Shows endophytic fungi promote enzyme activity in orchid species
Name of the endophytic
Enhances activities of following the enzymes: chitinase, β-1,3-glucase, phenylalanine ammonia-lyase, polyphenol oxidase
Epulorhiza sp., Mycena anoectochila
Enhance enzyme activities
Epulorhiza sp., Mycena sp., Tulasnellales, Sebacinales, Cantharellales
Enhance the absorption of nutrients in plants, promotes seed germination of the host plant
Epulorhiza sp., Fusarium sp.
Enhance the absorption of N, P, and K elements in plants promoting the seed germination of host
2 Geological Location and Environment
Some orchids are extremely specific in selecting their symbionts, as they prefer a single genus of fungi. Corallorhiza maculata, a myco-heterotroph, associate only with Russulaceae irrespective of their geological location and presence of other orchids in its vicinity .
Certain orchid species change their symbiotic fungal partner in response to environmental stress with regard to variations in altitude from tropical to temperate regions . Goodyera pubescens associates only with one fungal mycobiont, if not subjected to changes in the environment, like drought, etc. The orchids with high degree of specialization have fewer fungal associations . Some other orchid species, for instance, Chloraea collicensis and Chloraea gavilu, make symbiotic association with only Rhizoctonia .
3 Mycobiont Invasion in Orchid Tissues at Different Stages of Development
The fungus invades into the tissues of orchid at its different stages of development. Fungal hyphae penetrate inside the testa of seed through the opening and into the parenchymatous cells of germinating orchid seeds, protocorms, and cortex of the roots. Certain physiological and cytological changes also take place at the time of the invasion of fungus into parenchyma cells. Increased number of mitochondria and few vacuoles enhances metabolic activity of the parenchyma cells of embryo. Fungal mycobiont enter into the protocorms through the chalazal end of the embryo [16, 17].
4 Root Cortex and Fungal Pelotons
In orchids, two types of host cells such as digestive cells and host cells are involved in nutrient transfer . The digestive cells are engaged in dense peloton development followed by digestion and subsequent reinvasion, whereas the host cells contain live hyphae in pelotons, which are not digested, or at least not as readily . There is an additional mode of nutrient transfer known as phytophagy. It involves lysis of fungal cells. The complete fungal hyphae are not digested, but only the growing end (tip region) is degraded, and the cell contents are released into interfacial space between the plant and hyphal membrane and are provided to the plant .
5 Fungal Members as Orchid Mycorrhiza
5.1 Rhizoctonia Fungi
The fungi that act as orchid mycorrhizae belong to class basidiomycetes. These basidiomycetous fungi include certain genera of fungus, namely, Rhizoctonia, Sebacina, Tulasnella, and Russula species. Most of the orchid species build up their association with saprotrophic or pathogenic fungi, whereas a few show preference for ectomycorrhizal fungal species. The orchid species show association with different fungal partners at different developmental stages in their life cycle. These associations could be at the time of either seed germination or protocorm development or could be throughout the life cycle of an orchid plant species . Moreover, different orchid species show preference for their symbiotic fungi depending on the type of environmental niches in which they thrive, whether terrestrial or growing on other plants as an epiphyte .
5.2 Basidiomycetous and Ascomycetous Ectomycorrhiza
Few species of basidiomycetous fungi are also reported to be making symbiotic association with orchid species, but they do not belong to Rhizoctonia. Specific myco-heterotrophic orchids are also reported to be associated with ectomycorrhizal basidiomycetes that belong to genera such as Thelephora, Tomentella, and Russula. Ascomycetous fungi are rarely seen establishing symbiotic connections with orchid species. A terrestrial orchid species Epipactis helleborine has a specific association with ectomycorrhizal ascomycetes in the Tuberaceae.
6 Orchid Specificity for a Symbiont
At successive developmental stages, orchid species show preference for their fungal mycobionts. Terrestrial orchid species build symbiotic association with members of family Tulasnellaceae, yet a few autotrophic and saprophytic orchids make association with several ectomycorrhizal fungi also [28, 29]. Few clades of fungus Rhizoctonia also show association with some epiphytic species . Rhizoctonia fungi can form symbiotic relationship with either an epiphytic or terrestrial orchid, but very rarely, they associate with both . It has been shown through seed baiting techniques that the seeds of Dendrobium aphyllum germinate when they make symbiotic association with Tulasnella, but do not germinate when treated with Trichoderma isolated from orchid plant, which strongly advocates about the specificity of symbionts to different developmental stages. The preference for symbiosis with a fungal partner differs at various developmental stages of an orchid species [31, 32]. With the advancing age of an orchid plant, the fungal associations also become more complex. Cephalanthera longibracteata, a mixotrophic orchid species, symbiotically associates with numerous fungal species belonging to family Russulaceae, Tricholomataceae, Sebacinales, and Thelephoraceae .
7 Peloton Formation and Mycophagy or Necrotrophy
Metabolically active and live pelotons facilitate the transfer of nitrogen and carbon; however, when fungal pelotons get digested, most of the nitrogen and carbon is absorbed by the plant itself by the process of mycophagy [25, 34]. Shortly, after the invasion of fungus into the cortical tissues and peloton formation, their lysis starts . Pelotons are formed in a unique way. At the time of their development, a thin membrane surrounds them, which eventually acts as endoplasmic reticulum surrounded by Golgi apparatus. Afterward, inside this cover, digestive enzymes are secreted into the space between the plant membrane and peloton to digest them . Further, the digestion of pelotons starts; at the same time, a secondary membrane is also formed around the fungal peloton which is a large vacuole and allows the degradation of the peloton in isolated manner . Additionally peroxisomes accumulate within the digestive plant cells and undergo exocytosis into the newly formed vacuole; a number of enzymes concentrate such as chitinases, uricases, peptidases, oxidases, and catalases are secreted which ultimately, breaks down the peloton [35, 36].The fungal remnants are consumed by the plant itself, thus, transferring the nutrients to the host plant .
Few experimental studies using stable isotope imaging technique reveal that C13 and N15 when applied to mycorrhizal hyphae got readily transported to the host plant through fungal pelotons, leading to an inconsistent quantity of these isotopes inside the peloton containing plant cell and the peloton itself. It had also been observed that the senescing pelotons contain higher concentrations of C13 and N15 isotopes than in live pelotons .The fungal hyphae also undergo morphological change; they swell before collapsing, most probably due to the increased load of nutritive compounds . Once the pelotons are completely digested, reinvasion into the digestive cell occurs shortly after, and a new peloton starts forming again . Reinvasion and digestion occur cyclically throughout the entire life span of the symbiotic association .
8 Orchid Mycorrhiza and Nutrient Transport
Orchid mycorrhizal associations involve a variety of nutrient transport systems, structures, and phenomenon which are specifically found in the family Orchidaceae. These interactions are observed between basidiomycetes and almost all species of Orchidaceae . In basidiomycete, the fungi Rhizoctonia is generally found associated with orchid species. Rhizoctonia is known for its saprophytic abilities also and establishes anomalous associations . The orchid plant species which inhabit dense and highly shaded forest areas depend completely on their mycorrhiza for nutrition such as carbon [35, 38]. Orchid seeds being extremely reduced and non-endospermic show an obligatory parasitic stage during germination and draw nutrients with the help of mycorrhizal fungus . After the orchid seed germination, the orchid fungal interactions become specific to utilize the carbon and available nutrients. These associations are often governed by the orchid plant itself . Orchid mycorrhizal interactions could either be completely parasitic on the fungal associate or show mutualistic interaction, thus, establishing bidirectional nutrient flow between the plant and mycorrhizal fungus . In the natural habitats, orchid mycorrhiza shows a specific mycorrhizal nutrient transfer interaction upon which the diversity of the orchid genera depends .
9 Nutrient Transfer Mechanism in Orchids
Orchid mycorrhizal interactions show unique flow of nutrients. In the arbuscular mycorrhizal associations, it is observed that plant species channelize unidirectional supply to fungus with carbon swapping with either or both, phosphorus or nitrogen, depending on the environment [41, 42]. There occurs bidirectional flow of carbon between the fungus and plant, besides flow of nitrogen and phosphorus from the fungus to plant. Nearly 400 plant species are not able to provide carbon to their system. In fact, all of the nutrients of the plant are supplied by the fungus . However, in these interactions, carbon gain by the plant is positive in the majority of the observed interactions . Peloton starts forming shortly 20–36 hours, after the invasion of fungus . The plasma membrane of invaded parenchyma cells also assists with the fungal infection and its further growth . For exchange of nutrient materials between pelotons and surrounding plasma membrane, extensive surface area is created. The invaginated plasma membrane surrounds the growing pelotons and creates vast surface area from which nutrients can be exchanged. The pelotons are highly coiled fungal hyphal mass as compared to endomycorrhiza of arbuscular mycorrhiza [35, 44, 45]. During fungal invasion, increase in ribosomes also takes place. Plasma membrane participates in the exchange of nutrients between plant and fungus besides the enzyme excretion inside the space termed interfacial apoplast [40, 46].
The pelotons are not permanent structures; these are swiftly digested within a few hours of their formation in orchid parenchyma cells. The digestion of pelotons is a universal feature observed in almost all endomycorrhizal associations; in case of orchid species, these coiled structures get digested sooner after their formation .
10 Transport of Phosphorus
Phosphorus is a macroelement which is required by all plants. Phosphorus is taken up by the mycorrhizal fungus from the soil particles in three distinct forms: inorganic phosphorus, organic phosphorus, and phosphate. Deficiency of phosphate in soil allows the formation of a symbiotic relationship between plant and fungus. Mycorrhizal fungi are capable of increasing soil surface area besides initiating the secretion of a variety of enzymes [41, 42, 47]. Inorganic phosphate is transferred either through active transport as phosphate through Pi transporters (inorganic phosphorus) and is moved out of the fungal hyphae into the interfacial apoplast, where it forms dihydrogen phosphate and then subsequently gets transferred by active Pi transporters into the plant cell or it depends upon passive efflux of Pi out of the fungus and active absorption by the plant [41, 47]. For efficient transport of phosphorus, these pathways depend on comparative high concentrations of Pi inside fungal cell and low concentration of Pi inside the plant cell. The second method is more dependent on this condition. Certain genes which are Pi transporter genes such as MtPT4 and StPT3 are known to regulate the exchange of phosphorus in orchid plants along with H+ ATPase transporters . In orchid species, once mycorrhizal symbiotic connections are established afterward phosphorus is obtained by the plant only through the metabolically active pelotons of fungal tissue; once the digestion of pelotons starts degrading simultaneously, the flow of phosphorus also ceases .
11 Transfer of Other Micronutrients
Mostly, passive transport helps in the transfer of micronutrients across the cell membranes, both during absorption from soil by fungi and further from fungi to the host plant . But under specific conditions, active transport of micronutrients takes place as well . The upregulation of cation transporters is seen in orchid D. officinale symbioses, suggesting that fungi make possible the transfer of nutrients from fungi to plant . Cation, such as iron, mostly found adhered tightly to the organic substrates and remains out of reach of plants and fungi. There are certain compounds, for instance, siderophores (small molecule which have high affinity for Fe3+ utilized by fungal species), which are secreted into the soil by fungi to acquire these cations . These cations are liberated into the soil around the hypha and absorb iron from the soil. These siderophore molecules are reabsorbed into the fungal mycelium where the iron has to be dissociated from the siderohore and quickly utilized . The orchid species possess siderophores in association with mycorrhizal fungi within the genus Rhizoctonia which can utilize the siderophore “basidiochrome” as the major iron-chelating compound . Apart from these known chelating compounds, other vital nutrients may also be transferred between mycorrhizal fungi and orchid plants through specialized methods also.
12 Transport of Nitrogen
Nitrogen transport is an equally important and essential process that often occurs through mycorrhizal associations . Nitrogen is abundant and much easy to obtain as compared to phosphorus. Mycorrhizal interactions give a significant benefit in the allocation of nitrogen. Bioavailable nitrogen (nitrate and ammonium) is absorbed from the soil media by the mycorrhizal fungi and further assimilated into the amino acids . There are a few proposed mechanisms by which nitrogen is transferred to the host plant. These pathways are biotrophic; a significant amount of nitrogen may also be transferred necrotropically but through a distinct process .
In pathway-1, the amino acids get transferred into the extra-radical mycelium where these amino acids are broken down. The amino acid such as arginine is synthesized and transferred intra-fungally and later on is catabolized into ammonium ions and is moved into the interfacial space between peloton and surrounding plant membrane and later on transported into the plant cell through ammonium transporters and incorporated into the plant .
In the transportation of nitrogen primarily as ammonium, certain ammonium transporter genes get regulated in the plant and mycorrhizal fungal associate which further regulate a class of genes called “protease genes” along with other three transporters such as an external amino acid permeases, nitrate transporters, and ammonium transporters [41, 46]. Nitrogen may also be transferred in the form of other amino acids, namely, arginine, glycine, and glutamine into the cell through specialized amino acid transporters. The mycorrhizal fungus T. Calospora in symbiotic association with orchid plant is able to regulate the expression of SvAAP1 and SvAAP2. These genes encode amino acid permeases that strongly support amino acids to be important molecules involved in nitrogen transport [34, 46, 47]. The transport of inorganic nitrogen in the form of ammonium and the transport of organic nitrogen as amino acids occur simultaneously . In fact, it has been proved through isotope (C13 and N15) studies that amino acids may be the primary nitrogen compound transferred in the orchid .
13 Transport of Carbon
As soon as the fungus gets carbon, it converts it into sugar mainly trehalose and assimilates into the fungal mycelium. The transport of carbon from fungal partner to plant cells occurs in one of two forms primarily trehalose, but carbon can also get converted into glucose and sucrose or as an amino acid arginine but can also be converted into glycine and glutamine [34, 40, 52].The transport of these molecules occurs through specialized amino acid permeases and carbohydrate transporter protein molecules. These molecules are fixed into the fungal peloton membrane, into the interfacial space where they get absorbed into the plant cell by similar transporter protein molecules in the orchid cell endoplasmic reticulum membrane surrounding the hyphal coils [34, 52].
The active transport of carbon from symbiotic fungal partner to plant cell is a biotrophic phenomenon. A significant amount of carbon gets transferred inside plant cell when the fungal pelotons are degraded and digested. Genes encoding the transporter proteins get regulated, simultaneously, both in plant and fungi, in the similar fashion as nitrogen and phosphorus compound transporter genes during symbiosis .
Orchid mycorrhizal interactions consist of various symbiotic fungi, ranging from myco-heterotrophic plants (Monotropa uniflora) to chlorophyllous orchid species such as Goodyera repens [40, 52, 53]. As mentioned earlier in the text that during symbiotic interactions in orchid mycorrhiza carbon is translocated readily from fungi to the plant tissues. Conversely, this may or may not occur with the transfer of carbon from plant to fungi [25, 52, 54]. Orchid mycorrhiza is more or less considered to be showing partial myco-heterotrophic interactions . In myco-heterotrophic orchids, carbon is taken up, by the fungi (basidiomycetes), in the form of molecules of peptide and carbohydrate [35, 55, 56]. Specific genes that codes for proteases and cellulose, lignin digestive enzymes, as well as oligopeptide and carbohydrate transporters get regulated mycelium present in soil to support enhanced carbon uptake .
Interestingly, it is also observed that apart from orchids, myco-heterotrophic fungi interact with roots of beech trees as well . Few studies report that myco-heterotrophic fungi are also involved in the translocation of photosynthates from tree to the fungus and then to orchid, but a thorough investigation is required to unravel such intricacies in orchid fungus interactions .
The orchids have meticulously evolved to survive in nature in spite of having highly reduced seed structure. It becomes imperative to understand orchid fungus intricacies for conservation purpose. A few studies have been reported about orchid fungus interactions. The knowledge of species-specific as well as developmental stage-specific fungal symbionts would be a step toward saving them from getting extinct in nature. More morphological and molecular studies are required for the identification of fungal symbionts that inhabit at every stage of their development. It would prove useful in replenishing their natural resources, thus saving them for future use.
English language assistance from Dr. H.S. Sekhon is acknowledged with deep gratitude.
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