The world of fungi is intimidating for the novice. In the following paragraphs is a very short briefing on the subject to get you going.

Fungi are not plants, they're a kingdom apart just like animals. As heterotrophic organisms they require organic molecules built by other organisms to grow and develop. In other words, they are unable to synthesize organic, carbon based compounds from an environment that is purely inorganic. Some species are single cells but the vast majority are multicellular. The cells themselves are stretched as filaments, branched, and the main mode of vegetative growth - we call them hyphae. The total mass of thread-like hyphae is the mycelium - the vegetative part of the fungus which can reproduce asexually through fragmentation or via vegetative spores. Fungi can also reproduce sexually via a different set of spores, often produced in a fruiting body called the sporocarp. The fruiting body can appear below ground or above ground. The latter is called mushroom if it's visible to the naked eye.

Fungi have been determined (named) by the asexual reproductive stage (anamorph, often mold-like) or by the sexual reproduction stage (teleomorph, in most cases a mushroom). This has led to a double standard: a single species can have multiple valid names, only when the link between the two stages in the life-cycle is made in the lab can we link the different names. When it's observed that a known anamorph leads to a known teleomorph, the anamorph name is replaced by the teleomorph. 1)
Unfortunately, it's not that simple: there are fungi that are known to never produce a fruiting body (fungi imperfecti) and there are a lot of anamorphs that are suspected to produce a teleomorph but can't be bothered to show it in controlled culture. Next to that you should note that fungus species come in different shades and variations which make identification a complex matter. Identification used to rely on visual inspection of the hyphae under a microscope, in recent years genetic analysis made this proces less subjective. All these factors have lead to an array of synonyms and undetermined cultures.

The fungus relies on other organisms to provide their basic building blocks: fruit rotting in a fruitbowl, rings on a loaf of bread or wallpaper and circular patches in the lawn are all examples of a mycelium that is extracting these ingredients from plant material. There is a whole spectrum of possible interactions between plants and fungi in their attempt to develop. On one end of this scale are fungi that extract or exchange organic molecules via the plant's roots. We call the fungus mycorrhizal if the relationship between a fungus and roots of a plant is symbiotic in nature. Symbiotic does not mean that both partners benefit, in a lot of cases the mycorrhiza is mildly pathogenic.
In 1876 A. de Bary had noticed that the roots of Neottia nidus-avis were covered with a fungus, but he could only speculate on their function and came up with the term "symbiont". Mycorrhiza were discovered around the end of the 19th century by researchers that had seen that roots completely covered with mycelium were completely healthy. They coined the term mycorrhyza: fungusroot. The French botanist Noel Bernard discovered they were the missing key in orchid seed germination, after he turned over a rotting log in the Fontainebleau forest on the 3rd of May 1899 and found orchid seedlings in moulding Neottia nidus-avis seedpods. He made the link and published it on the 15th of the same month 2). Up to today nobody has succeeded in isolating the microbiont of Neottia nidus-avis in order to test the claims of both de Bary and Bernard. In the meantime it's discovered that the impact of mycorrhizae becomes better visible on poorer soils. The reason for this is that the hyphae are far more effective than plantroots for tracing scarce elements like phosphor. The demand for knowledge on mycorrhyzae has drastically increased over the past years: restoration or creation of forests, horticulture, impact of environmental pollution and reintroducing species all depend strongly on the mycorrhizal habitat. Agriculture can employ them to reduce fertilisers and increase resistance to heavy metals, poorer soils, temperature fluctuations and unstable pH. An estimated 90% of all plants have mycorrhizae in one of its many forms.

Not all fungus-plant relationships are the same. They can be categorised by looking at how the connection is established.

The prefix ecto- means "outside", and refers to the fact that the hyphae form a network (the Hartig-net, named after Robert Hartig) between the cells in the plant's root cortex. EM is in most cases characterised by an envelope of fungal tissue that completely wraps the (finer) roots, greatly increasing the resulting root's contact surface. This marriage is found between woody plants (shrubs and trees) and mainly fungi from Basidiomycotina and Ascomycotina.

The fungus initially moves between the root cortical cells, but then invaginates them to form typical arbuscules (branching) and vesicles (a sac-like compartment) structures … these are endomycorrhizas. The term invagination is important: neither the plant- nor fungal cell wall is broken, one cell wraps around the other to create a large inner pocket with a space (apoplastic space) between the two. The confined fungal structures have a short life-span in the order of a few weeks. Most of the fungi in this marriage are found in the order Glomales.

Ericaceous refers to the host plant, belonging to the order Ericales. The hyphae can actually breach and penetrate the plant cells bu doesn't form arbuscules. There are further subdivisions which are beyond the terrorchid scope: Ericoid, Arbutoid and Monotropoid.

This type of mycorrhizal relationship is different from the others mentioned above. The zygote that resulted from pollination has to undergo a number of divisions to create the embryo. This first division normally traverses the length of the seed, creating two poles: one at the top where growth happens and one at the bottom with looks like a stalk. This stalk is called the suspensor and its main job is absorbtion and the manufacture of nutrients from the endosperm - the nutritious food reserve that envelopes the embryo in "non-orchid" seeds. The vast majority of orchids don't have an endosperm (except Bletilla striata & Disa uniflora), but they do grow suspensor cells.

When the fungus breaks into the testa it penetrates the embryo via the (large) suspensor cell, growing its hyphae inside the inner embryonic cells as little coils called peletons. The orchid simply digests these peletons and thus receives nutrition. With this newly acquired source the orchid embryo increases in mass to form the protocorm. Meanwhile the number of hyphae entering the seed increases and when a tipping point is reached the protocorm develops a shoot with roots and leaves. At this point the fungus invades the outer (cortical) cells and starts producing peletons there. When the roots become larger they grow root hairs to increase the contact surface between root and soil (and fungus).
The fungal part of this partnership is typically a species of Rhizoctonia (basidiomycota) that play a large role in decomposing cellulose and organic forest soil 3). It's difficult to determine what the fungus actually benefits from its relationship with the orchid, for this reason it is often said that the orchid is a parasite on the fungus. The orchid digests the peletons as a carbon source until the chlorophyl in the leaves have developed, this can take from months to an eternity as some orchids never build cholorphyl. Dependancy on the fungus can decimate once the orchid reaches maturity. Especially epiphytic orchids are known for losing the need for peletons, terrestrial orchids typically keep the relationship going until death does them part.4)5)

All terrestrial orchids are characterised for having a relationship with fungi that live in the soil. The hyphen contact the roots without destroying them and there's an exchange of organic building blocks - mainly carbon and nitrogen. 6) This is initiated in seed germination and continues into adulthood.
As with just about anything in nature, there is a gradient. Gastrodia sesamoides (the Potato Orchid), Dipodium punctatum (the Spotted Hyacinth Orchid) and the two species of the genus Rhizanthella (Rhizanthella gardneri and Rhizanthella slateri) all lack chlorophyll. This makes it impossible for them to capture the energy of sunlight directly via photosynthesis, they can't tap the energy source other plants rely on for using CO₂ as a source of carbon. These species achieve this by relying on a fungus for this, making them 100% dependant on their partner for their entire life. The Orchidaceae contain about 200 achlorophyllous species, which obtain their C and mineral nutrition completely from symbiotic fungi, and are therefore called myco-heterotrophic 7). Initially, nearly all orchids are myco-heterotrophic, but some never develop leaves and loose the photosynthetic function:

• Aphyllorchis 15 species of SE Asia, Indomalaysia.
  • A. caudata
  • A. prainii
  • A. unguiculata
• Corallorhiza 15 species of north temperate regions; Europe (1 sp.), E. North America to Guatemala
  • C. maculata
  • C. mertensiana
  • C. striata
  • C. striata var. vreelandii
  • C. trifida
  • C. wisteriana
• Cymbidium One mycotrophic species, C. macrorhizon of Japan
• Cyrtosia 5 species of Indomalaysia (related to Galeola)
  • C. javanica
• Cystorchis 8 species of China and Asia. Only C. aphylla mycotrophic
• Didymoplexis 10 species of Old World tropics except W. Africa and India
  • D. pallens Japan
• Cephalanthera 1 species, C. austinae A. Heller. [= Eburophyton; = Chloraea].
  • C. austinae
• Epipogium 3 species of temperate Eurasia.
  • E. aphyllum
  • E. roseum
• Eulophia Ca. 200 tropical species, some mycotrophic.
  • E. zolingeri Japan
• Galeola 10 species of Madagascar, Indomalaysia to Australia. G. altissima (Bl.) Reichb. large!
  • G. nudifolia
  • G. septentrionalis
• Gastrodia 35 species of E Asia, Indomalaysia to New Zealand, Australia. Some mycotrophic species.
  • G. confusa Japan
  • G. elata
  • G. lacista
  • G. nipponica Japan
  • G. procera
  • G. pubilabiata Japan. From the CalypsoLip web site.
  • G. sesamoides
  • G. siamensis
• Hexalectris 7 species of the US and Mexico.
  • H. nitida
  • H. spicata
  • H. spicata
  • H. warnockii
• Lecanorchis 20 species of Indomalaysia to Japan. Some species mycotrophic.
  • L. nigricans
  • L. trachycaula
  • L. trachycaula
  • L. sp. 1. Japan
• Limodorum 1 species of the Mediterranean and Europe to Iran: L. abortivum.
  • L. abortivum
  • L. trabutianum
• Neottia 9 species of temperate Eurasia
  • N. nidus-avis
• Pterostylis 70 species of Australia, Malaysia to New Caledonia. Only some mycotrophic.
• Rhizanthella 2 species of subterranean orchids from SW and E Australia. R. gardneri occurs on Melaleuca uncinata
  • R. gardneri
  • R. slateri
• Stereosandra Acc. to Mabberley, 1 species, S. javanica, of SE Asia and W Malaysia.
  • S. javanica
• Stigmatodactylus 4 species of E Asia to Malaysia.
  • S. sikokianus
• Wullschlaegelia 2 species (W. aphylla, W. calcarata) of tropical America.
  • W. calcarata
• Yoania 2 species of Himalayas and Japan.
  • Y. amagiensis
  • Y. japonica

A few species of Disa from South Africa on the other hand can be germinated on pure sphagnum moss, and bread without a fungus. In other words, there are terrorchids that couldn't be bothered less about fungi, there are those that couldn't live without, but most of them fall somewhere in-between. This bizar relationship is considered to be one of the reasons why orchids produce a humongous amount of tiny dust-like seeds:

• since the fungus provides the seedling with nourishment, the plants aren't required to pack the seeds with starches and sugars like in other plants to get their offspring through the first days of germination. Other plants need to provide this reserve so that the seedling can develop its first leaves and roots, from that point on the seedling relies on its roots and leaves to get organic resources and energy. Orchid seeds are tiny and lack the built-in nutrition of bigger seeds; orchids then pass through a nongreen ("achlorophyllous") developmental stage when they cannot use fats, break down starch, obtain phosphates or photosynthesise, and therefore rely on an external source. This is provided either by man in the form of simple carbon-containing foods in sterile seed germination, or by a fungus which breaks down complex compounds into simpler ones in symbiotic germination. The fungal hyphae penetrate the testa of the seed and enters either through epidermal hairs or the suspensor of the undifferentiated embryo via the base end of the seed. Through invagination of the plasmamenbrane the hyphae enter the cells and coil into structures called pelotons Germination of the seed into a protocorm follows. The cells eventually digest the pelotons8)9), but occasionally the fungi become parasitic and destroy the protocorm.
• there are staggering amounts of fungi in the soil, all competing with one another. There's only a small amount of them that are able (willing) to associate with an orchid without destroying it. Fungi can be found literally anywhere: on every surface, in every microscopic crack and even in the air that you breath. So how do the seeds get to a place where the right partner is? The answer: simple dumb luck ! That's why the orchid produces seeds in the thousands - even in the millions for some species.

from the NZNOG Journal 66:

In some species (Gastrodia, Danhatchia and Corybas cryptanthus in New Zealand) chlorophyll never does develop, so the orchids rely for all their lives on associations with fungi. In others, the leaf-size is too small to support the rest of the orchid, and the orchid continues to rely partly on the fungus for its nutrition (Corybas cheesemanii for instance); such plants have been called saprophytic, but that is an incorrect application of the term. Some plants of the European Spiranthes spiralis pass alternate seasons underground, apparently fully nourished by their fungus during that time; some NZ orchids do not appear above ground every year and may do the same.
Most terrestrials seem to thrive better in the wild than in pots (some cannot be cultivated "artificially" at all), probably because they must have access to at least some of their nutrition via their association.
In different terrestrial orchids the fungi penetrate the stems, tubers or root hairs, via epidermal ("skin") cells after hyphae have spread over the root surface 10). Pelotons are formed, and eventually digested.
"Symbiosis" suggests mutual benefit and indeed Cymbidium and its fungus each require the vitamin thiamine, made up of thiazole and pyridine; the fungus supplies the thiazole and the orchid supplies the pyridine 11). Most orchid-associated fungi can, however, live without the orchid, and it seems that whereas the fungus supplies the orchid with a range of nutrients and stimuli, the orchid usually provides little in return.
Many orchids have "host" cells that store fungus, and adjacent digestion cells that break the fungus down by means of substances known as phytoalexins. The partnership between orchid and fungus has been called symbiosis (a 'Swan situation' as the politicians say in Wellington these days), or a "delicately balanced mutual antagonism' 12), or plain parasitism (of the orchid on the fungus, that is.
Fungi that are apparently symbiotic can turn nasty and attack the orchid; furthermore the fungi of epiphytes may invade the orchid's host tree to the tree's (and ultimately the orchid's) detriment.

The chances that an orchid seeds lands on a spot which provides the correct habitat for plant, fungus and has the fungus present is rather low. This probability is countered in a simple way: since the seeds are so small there is place for many of them in a seedpod. Making them light enables dispersal by wind, increasing the orchid's action radius. Some seeds land near the motherplant (where the fungus is most likely to be present) and some cross oceans, drastically improving the chance of finding a partner and conquering new territory.
To make these travels possible the dustlike seeds are equiped with a tough seedcoat - the transparent testa - which houses the embryo comprising of a few dozen to a few hundred cells. Each embryonic cell contains a certain amount of reserves in the form of lipids and starches, but this only lasts for a few days of slow consumption. Most plant seeds have enough reserve to grow roots, a stem and a couple of small seed leaves (cotyledons) but the orchid does not pack enough for this job. The first stages of germination and development require an external energy source.

It's not so that every species of orchid has a fixed fungal partner. There has been a lot of discussion about this in the past decades, here are the conclussions from a study done by Taylor and Bruns on Corallorhiza and Cephalanthera published in "A View of Specificity in Orchid Mycorrhizae Using Molecular Symbiont Identification"

• Several fungal entities were associated with each orchid studied
• Neighboring orchids of different species never shared the same symbiont
• There was no overlap in fungal symbionts of the four target orchids over the entire range sampled
• RFLP patterns generated with one enzyme were often identical in different fungal types associated with a single orchid species
• There were no identical enzyme patterns in fungi associated with different orchids
• There was no seasonal turnover of symbionts in one population of Corallorhiza maculata
• Multiple fungal types were rarely found associated with a single orchid individual

1. Hypothesis 1, that a particular orchid species will associate with different fungi when growing in different habitats, is supported by our results
2. Hypothesis 2, that neighboring orchids of different species will share the same fungus, is strongly contradicted by our results

In other words: it's not a 1-on-1 relationship. Although overall mycorrhizal specificity in the orchid family is narrow, variation in specificity among orchid species is high. 13)
In culture we make a difference between ecological symbionts and physiological symbionts. The first are the fungi that are found on the orchid roots in the field. The fungi extracted from roots of plants in the wild are determined by the ecology: the place we found them are pointed out by the orchid, but the fungi were there before the seed came in. The latter are fungi that are not naturally occuring symbionts but appear to do the job in germination and raising the plants. It should become apparent that this is very usefull information: with a library of fungal cultures we can germinate seeds by trying different well-chosen candidates in several cultures.

There are a number of facilities available 14):

• The Hardy Orchid Society in the U.K. has a Fungus Bank available for members
• CentraalBureau Schimmelcultures, (Central Office fungal cultures) in the Netherlands
• Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, (German Collection of Microorganisms and Cell Cultures). Orchid fungi deposited at the DSZM in the 90's originated from experiments for the thesis of Heinrich Beyrle, now owner of www.myorchids.de
• Mykoflor in Poland provides fungi isolated from epyphitic orchids from the botanic garden in Kiev
• Don't hesitate to contact a university or researcher with background in mycology of IV.

From Mycorrhizal functioning, an integrative plant-fungal process, Chapman & Hall, p394:

Specificity patterns in orchid mycorrhizae are complicated in that 2 types of colonization may occur: primary, involving the germinating seed and seedling, and secondary, involving new roots 15). A fungus efficient in the adult phase may not be so for the seedling germination phase. Patterns of specificity - if existing - should consequently reflect different selective pressures for different growth phases of orchids.

Another level of complication is the number of orchid species involved, well in excess of 17500 species according to Mabberley 16), and the number of fungal species interacting with them, both ascomycetes and basidiomycetes. A comprehensive and updated list of demonstrated and putative orchid endophytes is highly desirable if patterns of specificity are to emerge, but it is hampered by the difficulty of growing many orchid species in vitro and by the omnipresent task of isolating and linking anamorph and teleomorph stages for numerous endophytes. At least 30 species of orchid endophytic Rhizoctonia species have been described. Recent work by Currah 17), Currah et al. (198718), 198819), 199020)), and Moore (1987) detail recent changes occuring in orchid endophyte taxonomy.

Ecological relationships with other plants, e.g. parasitism and saprophytism, of many orchid mycrobionts further compound the problem of recognizing specificity patterns. For instance, some orchid mycrobionts are parasitic (Armillaria mellea, Rhizoctonia solani, R. cerealis, see Alconero, 1969 21); Smreciu & Currah, 198922); Harley & Smith, 198323)), saprophytic (Coriolus versicolor, see Harley & Smith, 1983), and even symbiotic with EM hosts, such as Sebacina vermifera (teleomorph basidiomycete) on Melaleuca uncinata (Warcup, 1988).

In Australian orchids, Warcup (1981, 1971) indicated that orchid specificity occurs at different levels, from species to subtribe; he notes some reservations about the subtribe level because of difficult orchid classification. Clearly, it may be premature to assign ranges of specificity that would apply to this vast group of orchid mycobionts. However, given the spectrum of restricted habitats throughout the world, many close functional relationships (with restricted host ranges) may have evolved.

Well, apart from the ability of being able to obtain seeds, plants and guidelines, the fungi are considered to be the determining factor as to what can be germinated by hobbyists these days. If you have access to seeds there are three ways of getting them germinated:

• asymbiotic in-vitro culture
• symbiotic in-vitro culture
• symbiotic in situ culture

The first technique requires a good deal of know-how in biochemistry, access to chemicals which are sometimes difficult to obtain or expensive and a trade-off between patience/luck/experience.

The second technique resembles the first, but you're replacing a homebrew cocktail of chemicals with a fungus. It still requires patience/luck/experience but it's less expensive to keep a fungus alive than it is to maintain or build a library of chemicals. You can off course replace those concoctions with mixes of patatoe, banana, coconutmilk, … but there are limits. And there's also a large variation in potatoes.
The third option applies to sprinking of seeds near motherplants, water-sowing them in the garden or germination of the very easy starters like Bletilla striata or Disa uniflora.
Generally speaking: dependancy on the fungus is something we would very much like to get rid of, but it can be treated as a friend instead of a foe. Germination is currently being considered "accessible for beginners" when you're using fungi but it's wishfull to leave it at the stage of germination and not need to consider the underground partner when you're watering and replanting your collection.

So far, we've dealt with germination. Mycorrhiza don't stop there, I'm afraid. A large percentage of the terrestrial orchids remain dependant on the mycorrhiza for the rest of their life and the current consensus is that a number of those plants will not be cultured in pots, but in your backyard at best. If the climate suits them, that is.

So were does that leave us, are there plants we actually can grow you ask? The answer is that there are a huge amount of terrorchids that have been introduced into culture, and more are added every week thanks to hobbyists that share and explain. It's good to know mycorrhiza exist and if you don't want anything to do with them you'll still be able to successfully grow terrorchids. But if you take them into account and learn to work with them, a world of experiments in hybridisation and exotic looking unknown orchids will open up. You can wait for others to open up culture for a genus or species or you can do it yourself.

On this link you'll have access to the fungi that are known to be helping in terrestrial orchid germination and growth. The goal is to offer a reference point for those that want to try mycorrhizal germination of orchid seeds. We should also provide you with the information on the process of isolating and growing your own fungi. We know that fungi isolated from species on a different continent can aid in the culture of new species.

Terrestrial orchids have a wide range of underground configurations with or without tubers, fleshy roots or rhizomes. The best place to find the fungus for isolation is the root - the tuber or rhizome should be ignored. Amputating a root will normally not lead to a weaker plant and can be done without disturbing the plant and soil. A few tips:

• Genera like Ophrys are reduced to a tuber when dormant, you should not expect to successfully extract fungi from such a tuber. These plants grow new roots every year and they become impregnated with the fungus as they contact the soil - hyphae activity is at its peak when the plant is at its vegetative peak.
• Rhizomatous genera such as Bletilla and Cypripedium use the roots during vegetative growth for storing and extracting starch, it's only after the leaves have withered that the hyphae re-enter the cells to produce peletons.
• Always chose the largest, fastest growing plants for fungal isolation. They yield the best results for mycorrhizal germination
• Always get permission form the land-owner on private property, or from the ruling authority on public domain. Sampling the plant for mycorrhizae is an invasive procedure that is in most cases forbidden by law. Most terrestrial orchids enjoy a protected status, but even for the unprotected ones you can't just barge in and start taking root cuttings.

24)

Below are a number of recipes for media preparation in a setup using kitchen utensils and ingredients available in your corner shop 25). Hygiene is a very important factor in success-rate and steps requiring sterilisation can't be skipped. Fungal spores and bacteria are in the air we breathe and on every surface around us, this means working in a sterilised environment such as a home-built glove box. Since the media is designed for optimal growth of a fungus you can expect large amounts of failure due to contamination by unwanted organisms. These should be removed and destroyed as quickly as possible. A good culture produces a single color (which can literally be anything and can change when the growing medium changes) and contamination can be recognised as spots or local gradients of a different colour or shade 26). If you have a digital camera don't hesitate to document and share your experience.

A few tips for Fungus Kitchen Culture

1. safety first : the various stages of sterilisation use heat-treatment from pressure-cooking or flaming the utensils. Use your brain to avoid wounding yourself or setting the place on fire. Especially the first time do a dry-run of you procedure: find out the best place to temporarily put everything. Reduce the amount of running around.
2. Try to experiment with variations in your ingredients, the media below are general purpose and can be tuned. The ideal medium is transparent, making it easy to see what's happening in the container.
3. Keep the metrics simple: ingredients are often in mixed units of grams, liters or teaspoons. Work them out before you start to avoid guessing. We normally mix everything to result in a liter of medium - when a recipe tells to add 3 grams, it means 3 grams for every liter of finished medium. One liter (1000ml) of water ways 1 kilogram (1000g) so when adding 10 grams of something else you could try to think of it as 1% of the resulting mix.
4. Sometimes we need to filter liquid from a broth - use a handkerchief or some thick clean fabric, not cheesecloth 27)
5. We often need a gel to solidify the medium, the most commonly used is agar. Agar is extracted from a seaweed and is sold as dry flakes or strings that dissolve in hot water and gel up below around 45°C. Use just the amount needed to keep the medium solid - normally around 8g for soft and 14g for hard agar. You can buy it in medium to large sacks, most often in shops for Asian food. Check the label to see if it's pure, we don't want additives (such as fungicides). The purer the better, agar from food stores contain a large amount of minerals and this is known to be problematic for germinating Drakaea and Paracaleana 28).
6. The acidity of a medium is denoted as pH (power of Hydrogen). You can find strips for testing the pH at the drug store, DIY (there are kits for testing pH of tap water, garden soil or pond water) or you can make it yourself: with red cabbage juice 29). Use Vinegar to adjust the pH down, sodium carbonate or bicarbonate to increase pH 30)
7. avoid reheating the medium after the pH has been lowered as this can lead to the agar not gelling when cooling.
8. Only use materials that can stand the temperatures of a pressure cooker (for sterilisation). Use undamaged metal utensils and baby food jars that can stand the temperature.
9. keep containers and utensils covered during procedure - dust always moves the air but it generally falls down.
10. don't smell the cultures, spores of unfriendly fungi can get into your lungs.
11. components such as oat, starches, … are best bought from bio-shops or with a bio-label to avoid fungicides and preservative chemicals.

The media requires three components for the fungi to grow on:

• nitrogen : can come from peptone, yeast extract, malt extract, amino acids, ammonium or nitrate compounds.
• carbon : from sugars such as glucose, fructose or mannose. Sucrose (regular sugar) can be added in some media but we normally extract the sugars from an organic source
• vitamins: fungi have a natural deficiency for vitamins, but these are provided by the same organic ingredient we extract the sugars from.

Most media will be cloudy. Mix the ingredients of the medium you choose and keep stirring to avoid burning at the bottom of the pot. You can use a clean cloth to filter it after cooling, filter it in a measuring beaker so you can add water to compensate for the loss in boiling and filtering - add water so you have 1 liter. A small note on filtering: it's best to first scoop out visible chunks with a mesh and then run it through a cloth. This avoids the cloth from clogging. Now pour it back in the cleaned cooking pot and add the agar while reheating. Keep stirring as the agar can stick to the bottom and burn. It'll take a few minutes before all of the agar has dissolved, but don't let the mix boil - otherwise it'll foam. The jars in which you'll dispense should be prepared and waiting on the table on a clean cloth. It's absolutely necessary that the jars are squeaky clean. When you cleaned them it's important you removed all residues from previous content, as well as paper and glue on the outside and lids. After cleaning rinse with tap water to remove the last traces of the detergent (they're designed to kill fungi and bacteria). Dispense the medium in the jars while it's hot, the agar solidifies when it cools. Pour about 1 cm in each jar and keep it clean: don't spill medium on the rim or wall of the jar (both inside and outside). The lid should have a small punctured hole in the middle, a few millimeter diameter is enough. Screw it on and wrap Aluminum foil over the lid till a centimeter below the top. Now we need to sterilise the jars in the pressure cooker. Pressure cooking isn't optional, under pressure the temperature of the water and steam will rise above 100 °C and kill all spores of contaminating bacteria and fungi. You can wait a day between preparing the jars and cooking them, but it's better to do it in one go. Please follow the instructions that came with the cooker, these things can explode when used inappropriately. After the cooker has reached pressure start the timer for 20-30 minutes. After this time cut the heating and let the cooker cool. Your jars are now ready to be inoculated, but keep them stored at 20-25°C for a week. If a spore of contamination got through it'll grow immediately and spread, these jars are useless and should be cleaned as soon as possible - you can re-use the jar after they're cleaned. You never know what kind of infection got in, don't sniff or touch it but dump it in the bin.

Keep the foil on the jars, it's a cover that stops spores getting through the small hole in the lid. Take a minute to look at your jars. A perfect one has a uniformly clouded gel without droplets on the inside of the glass. Droplets aren't really a problem, but you should avoid a layer of water on top of your medium, this is normally due to either too less agar or because of taking the jars out of the cooker too soon. Droplets can collect on top of the medium, don't swirls the jar but let it rest for a few days - small amounts can still be absorbed by the medium.

If your cloth or filter is too rough or if you squeezed the cloth to force out all the liquid you'll have a bottom layer which is a tad darker than the rest of the medium. This is not bad but you should go for a uniformly colored medium.

Potato Dextrose Agar is the most widely used medium for growing fungi and bacteria which attack living plants or decay dead plant matter. 31) PDA is a very rich medium which causes excessive mycelial growth and inhibits the formation of spores on most fungi, use it for rapid multiplication. The medium is commercially available from a large number of suppliers.

1. Boil 200g of peeled sliced potatoes in 1 liter of water until the potatoes are soft.
2. filter the extract in a container and add water to get 1 liter
3. add between 10g and 20g of dextrose
4. and 12g to 17 of agar, try a batch in advance to get an idea - it depends on the source of the agar how much is needed to solidify
5. bring this mixture to a gentle boil with constant stirring. Then dispense in cleaned and rinsed babyfood jars or jam-jars that can stand the heat of a pressure cooker.
6. place the punctured lids on the jars and wrap/cover them with aluminum foil
7. cook in the pressure cooker at 15 PSI for 20 - 30 minutes and let everything cool before removing the lid of the cooker

Corn Meal Agar and Oat Meal Agar also do well for growing most fungi, but sporulation is faster. The carbohydrates in oat and corn meal are more difficult to digest, leading to a normal potency compared to PDA. There's also a variation on the pH called ACMA (Acidified CMA) which has a very low pH (around 3.5), stopping bacterial growth. The disadvantage of ACMA is that it can not be reheated (sterilised) after pH correction - requiring a very sterile environment to mix. ACMA is often used as short for "Alkaline CMA" (a very high pH) or "Antibiotic CMA" (CMA with antibiotics) - two different formulations.

CMA:

1. Add 60g freshly ground cornmeal to 1 liter of water
2. heat to boiling and simmer gently for 1 h
3. filter through cloth and add water to get 1 liter
4. add 15g agar on the fire and bring this mixture to a gentle boil with constant stirring. Then dispense in cleaned and rinsed baby food jars or jam-jars that can stand the heat of a pressure cooker.
5. cook in the pressure cooker at 15 PSI for 20 - 30 minutes and let everything cool before removing the lid of the cooker

OMA:

1. measure 1 liter of water and add 30g oat flakes or oat powder
2. bring it to a boiling point and lower the temperature so that it simmers for 2 more hours
3. filter the flakes (not required for powder) and add water so it's 1 litre again
4. add 15g agar while still hot and keep stirring
5. dispense in the jars and add the punctured lid, seal of with aluminium foil
6. sterilise for 20 minutes in the pressure cooker at 15 PSI

Water Agar is a weak medium containing nothing more than water and agar to solidify. It's sometimes used for initial isolation of aggressive fungi: a surface-sterilised sample of orchid tissue is introduced and a few fungi will start to grow. The medium doesn't allow fast growth and samples from different species can be taken to a new WA or a richer medium 32)

1. Agar 20 g
2. Distilled water 1000 ml
3. mix, stir, heat, dispense and sterilise

Potato Carrot Agar is weaker than PDA (and maybe CMA) but is a valid alternative when CMA is not doing well.

1. wash, peal, chop and boil 40g of carrots in 1 litre of water for 10 minutes
2. filter the extract
3. wash, peal, chop and boil and 40 g potatoes in 1 litre for 10 minutes
4. filter the extract
5. add 250ml carrot extract, 250 ml potato extract and 500ml of water together and add 15g agar whilst stirring on a fire
6. dispense and sterilise 20 minutes at 15 PSI

The Mycorrhiza Literature Exchange
http://www.pfc.cfs.nrcan.gc.ca/biodiversity/bcern/manual/index_e.html
http://mycology.cornell.edu/listings.aspx?findex=Discussion
http://www.indexfungorum.org/Names/NamesRecord.asp?RecordID=251038
http://www.ionopsis.com/sainsburyp.htm

Mycorrhizal specificity in endemic Western Australian terrestrial orchids (tribe Diurideae): Implications for conservation
PhD Doctorate, Penelope Sarah Hollick
Peterson RL, Farquhar ML. Mycorrhizas - integrated development between roots and fungi. Mycologia 1994; 86 (3): 311-326.
Ramsay RR, Dixon KW, Sivasithamparam K. Patterns of infection and endophyte associations with Western Australian orchids. Lindleyana 1986; 1: 203-214.
Hijner JA, Arditti J. Orchid mycorrhiza; vitamin requirements and production by the symbionts. Amer.J.Bot. 1973; 60: 829-835.
Arditti J, Fundamentals of orchid biology. Wiley, New York, 1992. p445.
Lancaster T.L. Preliminary note on the fungi of the New Zealand epiphytic orchids. Trans.N.Z.L 1911; 43: 186-191.
Campbell E.O. The mycorrhiza of Gastrodia cunninghamii HookF. Trans.Roy.Soc.N.Z 1962; Bot 1: 289.
Campbell E.O. Gastrodia minor Petrie, an epiparasite of Manuka. Trans.Roy.Soc.N.Z 1963; Bat 2: 73.
Campbell E.O. The fungal association of a colony of Gastrodia sesamoides R.Br. Trans.Roy.Soc.N.Z. 1964; Bot 2: 237.
Campbell E.O. The Fungal Association of Yoania australis. Trans.Roy.Soc.N.Z. 1970; Biol.Sci. 12: 5-12.
Campbell E.O. The Morphology of the Fungal Association of Corybas cryptanthus. J.Roy.Soc.N.Z 1972; 2: 43-47.
Dixon K. Seeder/clonal concepts in Western Australian orchids. In Population ecology of terrestrial orchids. Eds T.C.E. Wells and J.H. Willems. J.H. SPB Academic Publishing: The Hague, 1991, ppl11-124.
Warcup J.H. and Talbot P.H.B. Perfect states of Rhizoctonias associated with orchids I-III. New Phytologist 1967; 66: 631-641; 1971; 70: pp35-40; 1980; 86: pp267-272.
Warcup J. H. Specificity of mycorrhiza association in some Australian orchids. New Phytologist 1971; 70: pp41-46.
Warcup J. H. Symbiotic germination of some Australian orchids. New Phytologist 1973; 72: pp387-392.
Warcup J.H. ne mycorrhizal relationship of Australian orchids. New Phytologist 1981; 87: pp371-387.
Warcup J. H. Mycorrhiza. In Orchids of South Australia. Eds R.J. Bates and J.Z. Weber. Flora and Fauna of South Australia Handbook Committee, Adelaide, 1990. pp21-26.
Ramsay R.R. Sivasithamparam K. and Dixon K.W. Anastomosis groups among Rhizoctonia-like endophytic fungi in south western Australian Pterostyis species. Lindleana 1987; 2: pp161-167.
Matsuhara G. and Katsuya K. In situ and in vitro specificity between Rhizoctonia spp. And Spiranthes sinensis. New Phytol. 1994; 127: 711-718.
Perkins A.J. Masuhara G. McGee P.A. Specificity of the associations between Microtis parviflora and its mycorrhizal fungi Australian J. Bot. 1995; 43: pp85-91.
Perkins A.J. and McGee P.A Distribution of the orchid mycorhiza1 fungus, Rhizoctonia solani, in relation to its host Pterostyis acuminata, in the field. Australian Journal of Botany 1995; 3(6): pp565-575.
Clements M. Orchid mycorrhizal associations. Lindleyana 1988; 3: pp73-86.