The obligate and apparently universal association with fungal endophytes, at least during the seedling stage, seems to be an ancestral character in the orchids. These plants differ from the rest of the angiosperms in a number of vegetative and floral features, and in having an unusual physiology, seed structure, and germination patterns. All these characters hint at a relatively closely-knit nature of orchids as a phylogenetic group. The consistence with which the mycorrhizal associations are developed is an added evidence to their monophyletic nature (45).
The orchids lack charactes found in vesicular arbuscu1ar mycorrhizae (VAM) but have endomycorrhizae in which the basiodiomycetous taxa comprise the fungal component. This unique mycorrhizal system carries considerable information concerning the evolutionary history of orchids.
According to Currah (45), the family Orchidaceae evolved from a primitive lily-like ancestor that moved from a soil based environment to one based on the bark and litter material on the surface of branches and stems of trees. During this process, the orchid ancestor lost its soil dependent Zygomycotina endophyte. Ecological and evolutionary success was obligatory to finding a suitable mycorrhizal partner adapted to live on woody substrates. Most ofthese endophytes belong to a relatively simple and primitive series of wood inhabiting heterobasidiomycetous fungi (l08).
After establishing in their epiphytic environment and after undergoing considerable speciation, some taxa are believed to have moved back to terrestrial mode, taking with them a simple absorptive system of thick, fleshy, and velamen coated roots, and their unusual basidiomycetous mycorrhizal endophytes. The velamen probably developed to prevent desiccation and absorb moisture and nutrients from the bark.
So mycotrophy is an ancient trait in orchids and it had adequate time to evolve a variety of expressions. No other flowering plants utilize fungi for nutritional purposes as extensively and in so many divergent lineages.
Almost half of all flowering plant genera that contain achlorophyllous
S. P. Vij, T. N. Lakhanpal and Ashish Gupta 421 mycotrophs are orchidaceous. Sometimes entire tribes (Epipogoneae, Gastrodieae) can be chlorophyll free.
Orchids with transitory achlorophyllous stages vastly outnumber the holoheterotrophs. This is good evidence that in the evolution of mycotrophy by orchids, juveniles preceded adults in fungal exploitation (10). The ontogeny of orchid seedling also reflects the obligate nature of the mycotrophic way of life. Most significantly, a radical is not formed (138).
In the micropylar end ofthe embryo, where a meristem could have been formed, there is instead a tissue that is specialized for holding and digesting hyphae. This pole of the germinating embryo remains stationary in the soil, unable to explore new areas for nutrient, ion and water. Transformation of the radicular meristem into mycotrophic tissue is a major evolutionary sacrifice, since it prevents immediate, efficient absorption of water by the seedling (120).
If the association with and/or dependence on fungi first appeared in seedlings, complete dependence on a symbiont probably evolved through a progressive setback in the development of autotrophy until the latter was lost altogether. Somewhere along this sequence or evolutionary total reliance path, on carbon from an exogenous source must have become established because it was advantageous to both juvenile and adult stages Whatever selective forces promoted the achlorophyllous state in some orchids, evidence of graded effect exists. There are groups of related taxa with progressively diminished leaf surfaces. Many have far too little photosynthetic foliar tissue. Orchids occasionally also fail to produce carbon fixing tissues to account for their total bulk some long-lived individuals maybe able to alternate between auto and heterotrophic states on a fairly regular basis (10).
Among species which have relatively long, but transitory, heterotrophic stages, temporary reliance on fungi as a source of mrtrition can vary initially.
Nevertheless, habitat preference is a stronger predictor of nutritional energy than is systematic position. The epiphytic orchids though also dependent on mycorrhiza in nature, usually become autotrophic at an early stage in their development than the terrestrial orchids (92).
According to Arditti {l0), common origin and function in Orchidaceae couples very small seeds and symbiotic germination. Survival and performance of orchid seedlings are less dependent on habitat, exposure, availability of water, and fertility all of which factors dictate the size of
422 Techniques in Mycorrhizal Studies
food reserves in the seeds of other plants. Fungal hyphae serve as highly efficient absorbing organs during the early stages of development and provide organic nutrients, mineral ions etc. It is, therefore, not necessary for orchid seeds to contain as many reserves as in other plants. Nor is it necessary for the seedling to develop extensive root systems during the early stage of germination and growth (10).
16. Interaction of Soil Bacteria, Mycorrhizal Fungus, and Orchid Seeds
It is well known fact that fungi develop a symbioticrelationsbip with orchids.
Endotrophic bacteria have also been seen to colonize Gastrodia sesamoides rhizomes, roots, and shoots. They are closely associated with the mycorrhizal fungi in underground tissues and their abundance varies with the season and the orchid species particularly the extent and age of their mycorrhizal roots (163). There is a limited specificity of bacterial groups with orchid taxa. In orchids, mycorrhizal symbiosis is a fine balance of controlled occupancy of cells of the root/tuber cortex by the fungus; the extent of occupancy by, and vitality of fungus varies between the species (118). Since there is a close association between the fungus and bacteria, the seasonal fluctuation is indicated in mycorrhizal infection.
The bacteria may also be involved in the recognition between the orchid plant and the appropriate mycorrhiza. However, the response of the mycorrhiza and the plant to the bacterial presence may simply be nutritionally based. For adult herbaceous perennial terrestrial orchids, bacterial re-infection may occur rapidly each season. Further studies are required to understand seasonal fluctuation in mycorrhizae and bacterial infection, and the co-symbiotic effect of bacteria in pre- and post- germination syndromes and adult plant growth (163).
17. Techniques Used for The Study ofOrthid Mycorrhiza 17.1 Isolation and culture
Smith (133) outlined a procedure for isolation offungal components of orchid mycorrhiza. In this procedure, the fungal endophytes are isolated from mature orchid roots, for which purpose, the roots are cut into 5 mm
S. P. vij, T. N. Lakhanpa/ and Ashish Gupta 423
pieces, surface sterilized with mercuric chloride in 20% (w/v) ethanol, rinsed with sterile distilled water, and plated into peptone-dextrose agar (PDA) at pH 4.5-5.0. The fungal hyphae emerging from the roots can be subcultured and maintained indefinitely on PDA (66, 137).
For determining their nitrogen requirement, the fungi are transferred to Pfeffer mineral salt medium (137). They grow well as pure cultures, in media with asparagine, arginine, and urea but not ammonium ions as source ofnitrogen(69, 137).
Infection intensity of mycorrhiza in young protocorms and mature plants is assessed by the number of infected cells as a percentage of cells available for infection (73). The length and breadth of protocorms inoculated with orchidaceous mycorrhiza is assessed using five protocorms in each ofthree replicate tubes per treatment (81, 132). No field and pot trials have been reported for orchid mycorrhiza.
17.2 Inoculation procedure
For testing the ability of an endophyte to develop a symbiotic relationship with the orchid seed, the seeds are sterilized in 50% bleaching solution, long enough to bleach the seed coat for 20-60 min. (81) and sown into pfeffer agar slopes (66). Mycelial plug (4 mm diameter) on PDA (storage medium) or water agar (preparation medium for symbiotic testing; (69) is placed next to the seed on agar slant a few days after seed sowing (66).
Symbiotic test cultures are maintained at 20°C in darkness. Singh and Varma (131) used Pirijormospora indica for the inoculation of seeds of Dactylorhiza purpurella and D. maculata. They surface sterilized the seeds and placed them on modified oat medium (44) and inoculated with P. indica.
17.3 Processing the material for microtome section and preservation F or locating the fungus, microtome sections of roots, tubers, stems!
pseudobulbs, leaves and inflorescence axes are obtained. The sections are mounted on slides and stained with saffernin-fast green or saffernin- crystal violet. Hand sections can also be cut and stained in cotton blue to locate the fungal components (73). Only roots of mature orchids are sectioned. For microtomy following procedure is followed:
424 Techniques in Mycorrhizal Studies
17.3.1 Collection of material: Orchids have delicate root system and therefore, the plant should not be pulled forcibly to avoid damage to the root system. The material is collected from nature and possibly from different ecological niches so that diversity in root system and mycorrhizal association is reflected therein. The material is transported in polythene bags, thoroughly washed to remove the dirt etc., subdivided into pieces of desired size and preserved in a fixative. Fixation is normally done in formaldehyde-acetic acid alcohol [FAA; ethyl alcohol (70%) - 90 ml, acetic acid (glacial) - 5 mI, formalin (40% commercial- 5 mI] for 4-5 days and then the material is stored in 70% alcohol till use.
17.3.2 Dehydration, embedding, and staining: Ethyl alcohol is the most commonly used dehydrating agent and the dehydration is started at the concentration in which the material is stored. Therefore, the material is first stored dehydrated in 70% alcohol followed by 90% and then absolute alcohol; two changes are given in absolute alcohol to ensure that no traces of water are left since the material would be transferred to xylene. Infiltration in paraffm is begun slowly at room temperature or it is kept in a hot air oven 40-50DC to hasten infiltration. The paraffin dissolves in xylene and gradually replaces it; the process is repeated with a nuclear change of pure paraffm till smell of xylene disappears. Subsequently, the material is embedded in porcelain trays or paper folded trays, made into blocks and sectioned on a microtome at 10-15 J.UD (28).