Mycorrhizal fungi

Author: András Geösel

Mycorrhiza is a symbiotic association between two organisms: fungi and plants. Mycorrhizal associations between the mycelia and the root of the plants are generally mutual. The two types of mycorrhizas: endo- and ectomycorrhiza. From the mushroom industry and mushroom consumption point of view, ectomycorrhizas are more important. The association between fungi and plants was first discovered in the XIX. century and by now it became one of the most significant research field of mycology. These connections are so common in the nature that nowadays a plant without a fungi partner is considered to be a peculiarity.

Fungi partners can either be sac fungi (Ascomycota) or basidiomycota species, many of them are valuable edible mushrooms. Some species of the Basidiomycota division (Boletus spp., Xerocomus spp., Leccinium spp., Suillus spp. and the chanterelle – Cantharellus cibarius) are highly symbiotic, which means that, unlike button mushrooms or oysters, we are yet unable to cultivate these species. The highest amount of information and practical experience is available in case of truffles. Truffles are hypogeic ascomycetes fungi. They are ectomycorrhizas and associate with pine trees and other (mainly woody) plants. Because of their characteristic flavor, aroma and odor, truffles have been important elements of gastronomy for centuries. As they are sold on a very high price on the market, average consumers usually cannot even afford these unique fungi.

Arbuscular mycorrhiza formation

Mycorrhiza research resulted in important, practical information. Forestry trees planted with mycorrhizal fungi on their roots are more likely to survive and develop dynamically. These forests are more drought resistant and tolerate soils with lower nutrient content. More and more mycorrhizal trees are used all over the world. Most of them are inoculated with truffles.

Mycorrhizal association

Mycorrhiza-like formations were found on fossils originating from the Devonian period. According to a group of scientists, mycorrhizal associations made possible that the first plants could colonize land. This special type of connection between plants and fungi proved to be persistent throughout evolution. By the help of the symbiotic association, the “world wide web” evolved: all woody plants of the vegetation are connected by the hyphae of mycorrhizal fungi. Throughout this connection, trees are able to exchange nutrients.

Plant-fungi nutrient and water exchange

There are two types of mycorrhizas: the vesicular-arbuscular mycorrhiza (VAM) and the ectomycorrhiza (EcM).

The vesicular-arbuscular mycorrhiza (VAM)

The vesicular-arbuscular mycorrhiza is an endomycorrhizal association, which means that the hyphae enter into the plant cell and grow intracellularly. This type of mycorrhiza produces structures that are either balloon-like (vesicles) or dichotomously branching invaginations (arbuscules). The fungal hyphae do not in fact penetrate the protoplast (i.e. the interior of the cell), but invaginate the cell membrane. The structure of the arbuscules greatly increases the contact surface area between the hyphae and the cell cytoplasm to facilitate the transfer of nutrients between them.

VAM symbiosis developed prior to ectomycorrhizal associations, and it can be found in 80-90% of the plant families. The fungi that form VAM are typically hypogeic species, they usually do not have any fruitbodies and they belong to a few families. Horticultural companies use VAM forming Glomus species to produce soil conditioning formulas. These products improve soil life by facilitating nutrient transport, thus boosting yields of horticultural crops.

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Top half: root without mycorrhiza. Bottom half, left: ectomycorrhizal association. Bottom half, right: endomycorrhizal association

Ectomycorrhiza

Ectomycorrhizas, or EcM, are typically formed between the roots of woody plants and fungi belonging to the phyla Basidiomycota and Ascomycota. Unlike in case of VAM, the hyphae of EcM do not penetrate the plant cell. It only forms a so called “Hartig net” intracellularly. This is the active surface of the ectomycorrhizal system and this is where transport processes take place. The dense net of ectomycorrhizas covers the root tip like a cape. In some cases hyphae form root-like rhizomorphas. The color and structure of the ectomycorrhiza covering the root are important information for taxonomical classification. The mycelium of the ectomycorrhizal fungi usually inoculates 10-15 cm soil around the root. The exoenzymes and acids produced by the mycelia degrade nutrients, and then the hyphae utilize the macro- and microelements, which are transported to the surface where mineral exchange takes place.

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Ectomycorrhized root tip and the mycelia around it

Mycorrhizal association could break if the plant suffers some kind of a stress (drought, herbicide, high temperature etc.), this case the vital hyphae of the fungi withdraws. Extreme nitrate and phosphorous level could also result in the same effect. Lifecycle of the EcM fungi is mainly haploid.

The lifecycle of Tuber magnatum is shown as an example. The ascus (1) forms in the mature fruitbody (modified apothecium) (6), which could take years to develop. The ascospores sprout (2), this primer mycelia colonize the root tip and form the ectomycorrhiza (3). Since numerous fungi have mycorrhiza, these formations can be found inside the plant. Mycelia of the mycorrhiza and of the intracellular space grow into the soil (4), and – through a less-known sexual process (probably ascogamy) – dicaryotic hyphae are formed. At the end of the reproductive process, the heterokaryotic hyphae (which produce spores) are surrounded by vegetative heterokaryotic hyphae. The mature fruitbodies (ascocarp) develop from the ascus and the sterile hyphae.

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The lifecycle of Tuber magnatum

The mycelia colonize the soil during the spring period. In case of T. melanosporum, the primordia forms in May and June, but the truffles themselves only develop in August. Due to the intense growth of the fruitbody, cracks appear on the soil surface. The truffles mature by winter. As the lifecycle shows, EcM fungi have an independent lifestyle phase besides the symbiotic phase. As saprophytism is not really an option for EcM fungi, these species do not, or only moderately grow in sterile cultures (e. g. tissue cultures). The ratio of the independent phase depends on the species.

Fungi, like T. melanosporum, are obligatory associates of plants, since they cannot synthesize certain enzymes, and they need nutrients from the partner. Because of this characteristic of mycorrhizal fungi (the fact that they need plant associates to form fruitbodies), we are not able to cultivate them with intensive technology. The common grain-based spawn cannot be used in case of mycorrhizal fungi, thus some other kind of propagation is needed.

The succession of the mycorrhizal fungal community of forests is similar to that of any plants. Young and old woods and those with different kind of trees do not have the same fungal species. Inoculation of woody associates with fungi species more typical in young forests (e. g. truffles) had better results, than those cases, when fungi associates of older forests (e. g. boletus or Caesar's mushroom) were used for inoculation and the fungi quickly disappeared from the roots of the trees. Another problem is that the spores of certain species can stay dormant for many years or even for decades.

Biology of truffles

The fruitbody of truffles (ascocarp) have spores inside them. Species are differentiated for example by the color and the pattern of their surface. According to some experts, the intense odor of truffles is caused by the decaying bacteria under the outer layer (peridium) of the fruitbody. The mycorrhizal association does not only affect the tree partner itself, but also other plants around it: in many cases, plants around the colonized trees almost completely disappear: the area appears burnt (truffle burn). It is not yet clear, what causes this phenomenon. One theory is that the mycelium parasites the surrounding plants or it produces some kind of molecules with herbicide effect. Another explanation is that the mycelium-plant root complex absorbs most of the water and nutrients from the soil, this way other (competitor) species cannot survive.

Various biological and ecological factors affect the appearance of the first fruitbodies, but it usually concurs with the first fruiting of the mycorrhizal tree. The fruitbodies are then ready to pick in 2-3 years after the truffle burn appears. The truffles can be collected for decades (or even until the tree dies), if the environment is optimal for the tree associate (e. g. soil type, moisture content, pH, pests, herbicides, fungicides etc). Generally, EcM fungi have a positive effect on the vitality of their tree associates in the early stage of the symbiosis (after planting), as the water and nutrient absorption is more intense in case of mycorrhizal trees than those without fungal partners. Due to certain research data, in some cases, this positive effect on vitality cannot be observed. Later on, the fungi could switch to saprotrophic life cycle and they could start degrading old or dead tree roots.

The most important indigenous species of the Carpathian Basin

In the following chapter, those (potentially valuable) truffle species are presented, which have already been included in Hungarian researches with practical results. Summer, winter and sweet truffles are quite common in Hungary. 6-7 thousand tons are picked each year. The smooth black truffle is also presented below, although it has lower gastronomical value and is not as important from the cultivation point of view as other truffles.

Summer (burgundy) truffle – Tuber aestivum Vittad. (= Tuber unicantum Chat.)

Although they were considered to be distinctive, molecular analysis proved that summer and burgundy truffles are the same species. The differences in odor, taste or spore shape are due to environmental factors. Summer truffles are collected from May to August, while burgundy truffles are picked in October and November.

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Summer truffle is common all throughout Europe, except for areas with heavy and frequent rains, heavy soils and neutral pH. The size of the fruitbodies varies between 2 and 9 cm, but a 1 kg sized truffle has also been found. Lighter (sandy) soils result in round truffles, while elsewhere amorphous, brown or black fruitbodies with scales form. The scales are polygonal, with a sunken part in the middle. The odor of the ripen truffles are similar to that of cooked corn. Summer truffles have a number of host plants, for example: oak (Quercus), nut (Coryllus), hornbeam (Carpinus) or beech (Fagus), but they can also associate with pines, poplars and chestnut.

Winter truffle / Black winter truffle – Tuber brumale Vittad.

The distribution and of T. melanosporum and T. brumale are similar. Although winter truffle prefers heavier, nutrient rich soils with low lime content, it can tolerate less optimal environments as well. The host plants are also the same in case of the two species.

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The fruitbody is round or amorphous. The black (or sometimes brownish-red) scales are 1-3 mm in diameter and they can easily be removed by washing. The flesh is white or grayish. The mature truffle has a strong, distinctive odor similar to that of muscatel wine. In Hungary, winter truffles are picked from October until March. Since it easily associates with the host plants, using this species for inoculating young plantations is quite cost effective. In Hungary, winter truffle plantations have established with nut and oak associates.

White desert truffle / Sweet truffle / Sand truffle – Mattirolomyces terfezioides /Mattir./ E. Fischer

White desert truffle is found in the sandy areas of the Carpathian Basin, where the lime content is lower but the soil is nutrient rich. In Hungary, it can be picked from August till November. The white-grayish fruitbody is round, 1-12 cm in size, with a usually smooth or sometimes cracked surface. The flesh is light colored, pinkish with fine white lines. Raw white desert truffle is poisonous, but after heat treatment (for example cooking) it becomes edible. Due to the sweet taste, it can be used as an ingredient of desserts.

Garlic truffle / Smooth black truffle – Tuber macrosporum Vitt.

This truffle species is quite common in most parts of Europe, except for the Mediterranean region. It prefers forests with higher moisture level. The truffle can be picked from September till January. In Hungary this species can be found mainly in the northern mountainous regions, but it also grows in certain (mostly southern) parts of the Great Plain. The fruitbodies are usually walnut-sized and the color is dark brownish or black. The surface of the truffle is covered by warts. The grayish-brown flesh is tough. Garlic truffles have lower culinary value, because of the slight garlic smell of the fruitbody. Besides, even the younger truffles are firm. This species is not as important from the cultivation point of view.

Besides the above mentioned species growing in Hungary, there are other truffles that can be found on the market and are included in cultivation experiments and research in Europe. For example the white truffle (Tuber magnatum Vittad.) is one of the most valuable and expensive truffles, which can be found in the forests of the Istria peninsula. Great attempts have been made to find a successful cultivation technique for this species, but so far the plantations have very low (if any) yields.

The black or Périgord truffle (Tuber melanosporum Vittad.) is found in the Mediterranean regions of Europe. It has a strong, characteristic aroma, which is not favorable for all consumers, besides it is slightly bitter and has a long aftertaste. Both the cultivated and collected truffles are sold on a high price on the world market under the name black truffle. The cultivation of this species dates back to almost two centuries, which explains why have black truffles been planted in the largest scale and why is it the most well-known amongst truffles. Since the mycelia of black truffles can easily be damaged by low temperatures, in Central-Eastern Europe, the cultivation has a higher risk due to cold winters.

The whitish truffle (Tuber borchii Vittad.) is a tolerant species with white peridium. It is popular especially in Italy, which is why the first plantation was established here. The young fruitbodies taste very good; both the odor and the aroma resemble garlic. It forms mycorrhizal association in an early stage, thus it can be cultivated successfully in plantations.

Cultivation of mycorrhizal fungi

The cultivation technologies of various truffle species are slightly different. It is quite common for species of the same genus to have different needs regarding substrate and ambient conditions for cultivation. The specific and detailed growing technologies of truffles are available in many sources, thus this chapter contains only the basic steps and information on how to establish a plantation.

Truffle growing has a great tradition in France. J. Talon planted the acorns of truffle “producing” oaks in areas where truffles were found, then removed the seedlings, which after a couple of years started to “produce” truffles. This method spread quickly, but nowadays it is considered more of a traditional, but not a very modern technology.

By using more intensive technologies, the plantation yields earlier, but at the same time other mycorrhizal species could appear, with which for example the black truffles cannot compete. That is why various factors (e. g. species, plant partner, soil type, area of production) have to be taken into consideration when developing a cultivation technology for a truffle species.

Preparation of the production area

The soil of the production area should contain a low number of EcM or species with poor competitive ability. Assessment of the flora of the area and molecular biology methods also aids evaluation and identification of mycorrhizal fungi present in the soil, which is crucially important information in planning. In order to prevent EcM contamination, areas with mycorrhizal plants should be avoided. Meadows, grasses, vines and fruit trees are generally arbuscular mycorrhizal associates. The presence of Acer, Fraxinus and Juglans species and members of the Rosaceae family do not influence mycorrhizal fungi composition of a production area. Planting young trees to replace older ones is not recommended due to the high number of mycorrhizal (and potentially competitive) species, which can be lowered by 3-5 years of intensive cultivation.

The previously intensively cultivated areas with high nutrient (especially nitrogen and potassium) and organic matter content are not sufficient for mycorrhizal fungi. Different truffle species need various water amounts. Those species that require higher water content is the soil, should be grown in cool valleys or along rivers. Swampy or too dry areas with low humus content are not recommended for establishing plantations. Summer truffles prefer harder soils or those with higher lime and humus level, but at the same time lower phosphor and potassium content.

In case of nutrient rich soils, lower yields can be expected, if fruitbodies even form. Active soil life contributes to the successful establishment of a new plantation, thus any kind of soil preparation should be done with special care.

Preparation of seedlings

The quality of the inoculated seedlings is a crucial point. New cultivation technologies and developments made significantly higher yields possible. The centers of truffle research in Europe are France and Italy. Due to practical experience, they could provide us with most of the modern technologies and a number of new developments. The first inoculated truffle seedling was produced in France, by INRA (Institut National de la Recherche Agronomique), in 1970. A few years later both their black and summer truffle plantations started producing truffles.

Until now, three main types of inoculated seedlings were used:

  • the seedlings are planted in plots in the originally truffle producing area, where the soil was sterilized and re-inoculated,
  • the seedling are grown in Melfert-pots filled with artificial substrate,
  • the seedlings are planted in special containers, which prohibit the spiraling of roots.

 

Preparing seedlings for inoculation starts with the sowing of the seeds or planting of the vegetative plant parts. Using treated seeds have the advantage of being completely EcM free. Another (and cheaper) method is to sow nuts or acorns, which have been soaked into a suspension containing truffle spores. This case mycorrhiza formation is unsure and the process is not really predictable.

At the beginning, clones of the trees or micropropagated seedlings were used. The high costs and long acclimatization period are the disadvantages of this method, but it is also true that these kinds of plants are completely free from infections. Researchers are continuing to use these seedlings, but only for the purposes of experiments and not for cultivation.

For starting intensive plantations, certified seedlings planted in containers should be used. Using those types of seedlings, which do not have any soil or contained covering or protecting their roots, is not recommended, due to risk of contamination or the likely loss of mycorrhiza.

Inoculated seedlings are produced by specialized companies with well equipped glass houses or modern plastic houses. The substrate used for filling the containers is often a mixture of soil from a truffle producing area and perlite or sand. Mycorrhized seedling producers use other peat or perlite based soil mixtures, containing for example vermiculite or gypsum, but the recipes and ingredients are kept secret for obvious reasons. The substrate is sterilized entirely or partially to eliminate contaminating fungi.

The seedlings can be mycorrhized / inoculated by various methods:

  • the seeds can be soaked in spore suspension,
  • dried and ground truffles or spores can be mixed into the substrate,
  • the seedling can be irrigated by spores mixed into water,
  • the roots of the seedlings can be dipped into spore suspension,
  • the micropropagated seedlings can be inoculated by vegetative mycelia.

For the purposes of making spore suspension, the fruitbodies are first picked, examined (to check the species), then their surfaces are cleaned. The truffles are often cut in half in order to be able to find any sign of contamination, to aid identification of the species and to make sure that the spores are mature. To prevent secondary infestation, any pieces that are damaged by insects are removed and not used for making the spore suspension. The selected and cleaned truffles are than ground in sterile water. The suspension works properly only if the seedlings are carefully isolated. The purpose of isolation (e. g. placing the seedlings on tables or stands and not on the ground) is to keep EcM fungi away, which could contaminate the seedlings and interfere with the establishment of the mycorrhizal association.

Mycorrhization is completed within 5-6 months under optimal ambient conditions. Excessive watering and fertilization should be avoided. In Hungary, the quality control and certification of mycorrhized seedlings is done by NFCSO (National Food Chain Safety Office). During their evaluation, not only the presence and the amount of the mycorrhizal partner on the root of the seedling are checked, but the general condition of the plant is examined as well.

Establishing a new plantation

For mycorrhization, those truffle species should be used that already grow in the area, which means that the partners are more likely to be fit into the ecological system of the land. In Hungary, oak, hazelnut, beech and hornbeam are the most adequate and therefore the most commonly used species. Usually 800-2000 certified seedlings are planted on 1 hectare. The higher number is used in case of summer truffles, which form fruitbodies if the canopies of the trees become closed. A lower number of seedlings is sufficient is the plantation is irrigated.

The trees are planted traditionally in the autumn or early spring. In order to avoid loss of the mycorrhizal fungi on the roots, the trees are irrigated with special care after they are placed into the ground. For further protection, the seedlings are surrounded by fences one-by-one to keep wild animals away.

In the first couple years, the focus has to be put on preserving the condition of the young trees and aiding root development. Irrigation should be done frequently but in smaller amounts. The soil should be loosened around the trees and weeds have to be removed either manually or by using herbicides. Fungicides are to be avoided, or only certain contact plant protection products should be used. The pruning of the trees should be done in a way it is optimal for the truffle species.

Under optimal conditions, the plantation could produce fruitbodies in the 3rd-4th year, but it is more likely to get the first truffles 8-10 years after planting the mycorrhized trees. The truffles are found by specially trained dogs. Picking a truffle has to be done carefully. Only the fully mature fruitbodies can be collected and after the truffles are removed, the remaining holes in the ground should be covered over.

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Certification of truffle hunter dogs

Depending on the species, truffles can be stored fresh, dried or frozen. Creams, desserts and other foods containing truffles are premium products. Recipes of the different truffle specialties can be found in numerous books and other sources. Results of different European truffle researches have already been brought into effect in Hungary. Multi-centralized Hungarian research projects also contribute to the extension of truffle production. High quality mycorrhized seedlings are produced by Hungarian companies as well. Following careful planning, these trees can successfully be used for establishing new plantations. The formerly intensively cultivated areas are suitable for afforestation. This type of utilization of agricultural lands could aid revenue production of the countryside.

Check your knowledge!

  1. Describe the lifecycle of Tuber magnatum!
  2. Which are the most important truffle species of Central Europe?
  3. What are the main aspects of establishing a new plantation?
  4. What are the differences between VAM and EcM associations?
  5. Give a short description of picking truffles!

References

Bagi, I., Fekete, A. (2007): A szarvasgombász mesterség. Trajan Könyvesműhely.

Bratek, Z., Jakucs, E., Bóka, K., Szedlay, Gy. (1996): Mycorrhizae between black-locust (Robinia pseudoacacia) and Terfezia terfezioides. Mycorrhiza, 6, 271-274.

Chevalier, G., Frochot, H., Bratek, Z. (szerk.)(2004): Az európai fekete szarvasgomba (Burgundi szarvasgomba - Tuber uncinatum Chatin). Első Magyar Szarvasgombász Egyesület, Budapest

Gógán, Cs, A., Bratek, Z., Dimény, J. (2007): A new tool for rural development: truffle cultivation. Cereal Research Communications, 35 (2), 413-416.

Gógán, Cs. A. (2011): A nyári szarvasgomba (Tuber aestivum Vittad.) és a nagyspórás szarvasgomba (Tuber macrosporum Vittad.) magyarországi termeszthetőségének vizsgálata. Doktori Értkezés. Szent István Egyetem, Gödöllő.

Bratek Z.: Szarvasgomba-termesztés. In: Győrfi, J. (szerk)(2012): Gombabiológia, gombatermesztés. Mezőgazda Kiadó, Budapest, 324-345.

Hollós, L. (1911): Magyarország Földalatti Gombái. K. M. Természet-tudományi Társulat, Budapest.

Jakucs, E. (2009): A föld alatti gombavilág titkai. Természet Világa, 9: 413-415.

Paolocci, F., Rubini, A., Riccioni, A., Arcioni, S. (2006): Re-evaluation of the Life Cycle of Tuber magnatum. Appl Environ Microbiol, 72 (4): 2390-2393.

Szemere, L. (2005): Föld alatti gombavilág. Mezőgazda Kiadó, Budapest.

Zambonelli, I. A., Iotti, M., Rossi, I., Hall, I. (2000): Interactions between Tuber borchii and other ectomycorrhizal fungi in a field plantation. Mycological Research, 104: 698-702.

Fotó:

http://permitools.wikispaces.com/file/view/mystery+mycorrhizae+and+plants.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449033/

http://www.trufamania.com/Tuber%20aestivum%20English.htm

 

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Utolsó frissítés: 2014 11. 13.