mycorrhizal Cultivation Idea

[themushroombloke]

I've used a few different techniques meshed together. I'll embed 2 diagrams to explain my idea.

the basic idea is to plant a seed in a hole in the top of a mushroom (a small hole with soil in it, then bury the mushroom in some moist soil. Hopefully the mycelium will recover and connect with the roots of the seedling. The other part of the idea is to water the seedling with a spore solution.

It requires:

a mycorrhizal compatable tree seed

2 mycorrhizal mushrooms of the same species (1 for spore print one to put the seed in)

a needle

a jar with injection port (synthetic ear plug)

a pressure cooker

I realise it's a pretty crazy Idea but I thought I'd put it out there, see if I cant refine the idea.

thoughts....................?

[Marcel]

I love the mushroom fruit as jiffy-pot idea! You definitely get an A++ for ingenuity!

As for the spore-water syringe, wouldn't all the sterile technique be useless if you're eventually going to pour that stuff all over soil, etc?

[occidentalis]

I love the diagram, but there's an easier way.

1. Crush up mushroom in water and create a spore/mushroom slurry. Filter out the big bits.

2. Sterilise soil and pots.

3. Plant seed into sterilised soil.

4. Water with spore slurry.

Bingo

I think the problem with planting a seed into the mushroom is that the mushroom will start to rot, and that may end up rotting the seed/seedling.

[shortly]

I recon Occidentalis is on the money, except i wouldn't sterilize the soil.

I would pasteurize it the same as you would with a bulk media.

[obtuse]

Loving this.

i have been contemplating similar ideas such as, somehow, making a substrate w/- soil mix, then using hydrogen peroxide to sterilise the surface of the seed, and then trying to gerimate them in the mycelium soil mix.

In the case of boletus, my understanding is that the main problem is that other ectomycorrhizal or endomycorrhizal fungi take the opportunity to form the symbiosis with the plant before the boletus does, and therefore the relationship is not formed, i.e. the need for sterile soil in order to minimise this risk.

So in the case of boletus, the mushroom slurry is a great idea, but will not be sterile so in artificial culture arent their too many risks of contaminants. But then there is also talk of the need for bacteria present in the soil to help facilitate the process, so pasteurisation would have to be the go. again i have concerns here regarding potential contamination issues when done artificially.

anyway, im just going blah, so keen to see what ideas other have regarding this.

Cheers, Obtuse.

[shortly]

I wonder if you could grow your seedling invitro (in a really tall flask) on an agar based media long enough for the fungi to make itself at home?

As a rule i've found vinoxide that is commonly used in htc is far better for surface sterilization of the majority of seeds.

http://ahjs.biz/pdfs/Vinoxide-HTC.pdf

Edited June 7, 2011 by shortly

[themushroombloke]

I wonder if you could grow your seedling invitro (in a really tall flask) on an agar based media long enough for the fungi to make itself at home?

As a rule i've found vinoxide that is commonly used in htc is far better for surface sterilization of the majority of seeds.

http://ahjs.biz/pdfs/Vinoxide-HTC.pdf

 

Yeah i think you're onto something there I saw this picture

this uses some sort of gel, however i did find a product called "plant gel"

this is a just add water product.

What if the water was a mushroom/spore slurry??

now I think we're getting somewhere.

let's face it people if we can crack this nut then we could all have a little mushroom orchard!!

here's the plant gel product

let's keep this ball rolling

[shortly]

I recon you might be better to try to germinate the two separately so as to isolate out all the contams.

Otherwise i recon you might need several hundred flasks of seedlings to get one or two comtam free.

The other thing to remember is your gonna need to have your seeds pre germinated unless your thinking of removing the seedcoat & a GA3 dip to speed things up.

Because just mixing a spore slurry & gel will most likely give you spores germinating well before your seeds, and correct me if my memory is faulty but i recall that with some mycorrhizals there is only a smallish window of opportunity for the fungus to unite with a root hair.

That gel is fun to play with but its more prone to metabolite build up than agar so keeping your seedling alive long enough might be a battle. Its normally used with throwaway gimmick plants or open jars that can be "washed".

activated carbon will help with the metabolites but i don't know if the fungus would like it or not? And you definitely wouldn't be able to see what was going on with the roots.

[themushroombloke]

I recon you might be better to try to germinate the two separately so as to isolate out all the contams.

Otherwise i recon you might need several hundred flasks of seedlings to get one or two comtam free.

The other thing to remember is your gonna need to have your seeds pre germinated unless your thinking of removing the seedcoat & a GA3 dip to speed things up.

Because just mixing a spore slurry & gel will most likely give you spores germinating well before your seeds, and correct me if my memory is faulty but i recall that with some mycorrhizals there is only a smallish window of opportunity for the fungus to unite with a root hair.

That gel is fun to play with but its more prone to metabolite build up than agar so keeping your seedling alive long enough might be a battle. Its normally used with throwaway gimmick plants or open jars that can be "washed".

activated carbon will help with the metabolites but i don't know if the fungus would like it or not? And you definitely wouldn't be able to see what was going on with the roots.

 

here's my next Idea based on your advice

[obtuse]

Nice

Love it. The only thing left then is the isolation then maintenace of the spores and mycelium of the species with which to set up the symbiosis. And then the maintenance of the sybiosis once in soil.

I am very keen on hearing how you go with this, and using same or simlar techniques.

Cheers, Obtuse.

[occidentalis]

I did a project a bit like this at uni, although the aim was slightly different and I wasn't trying to grow any particular type of fungi - just to see if any grew.

I don't think you really need to worry too much about contams if you are using a whole mushroom, because the number of mushroom spores will greatly outnumber any contaminant spores. You might get a few colonies of contaminants forming, but you're not going to grow the plant in vitro until maturity, so you'll need to expose it to contamination at some point.

[shortly]

Given that i suspect your going to have to keep the seedling & fungi together invitro for at least weeks if not longer to be certain the relationship between them has formed i thing it a huge ask for both the myc & more importantly the seedling to overwhelm any & all contams.

Bearing in mind that the protocols generally used for germinating seeds invitro would have to be altered since many contain fungal inhibitors(all the ones i have recipes for do), so the risk of contamination from that point is significantly higher as well.

I would at least hedge your bets & do some where you germinate your spores in separate flasks, isolate some clean myc & inoculate the seedlings with that.

Your other hurdle will be to find a nutrient mix that suits both. Trust me using a premixed plant media (couldn't find my glasses) with mushrooms = dead myc

Does anyone know if it must be a seedling that is used as a host & if so why.

Or can a TC'd plant still in culture or even a rooted procorm be used?

Assuming of course that the TC media itself doesn't inhibit or kill the myc.

Edited June 7, 2011 by shortly

[NSF]

Boletus Edulis was mentioned before and i wanted to add something. So it seems B.e is weak and others colonise roots better BUT i've read that B.e only grows on trees that are 25-30 years old.

Has anybody else heard this?

And if yes, what would be the reason it only grows on mature trees?

[obtuse]

Does anyone know if it must be a seedling that is used as a host & if so why.

Or can a TC'd plant still in culture or even a rooted procorm be used?

Assuming of course that the TC media itself doesn't inhibit or kill the myc.

 

Yes, i was starting to wonder about tissue culture too as a prefferred way to set this up.

Boletus Edulis was mentioned before and i wanted to add something. So it seems B.e is weak and others colonise roots better BUT i've read that B.e only grows on trees that are 25-30 years old.

Has anybody else heard this?

And if yes, what would be the reason it only grows on mature trees?

 

I dont see why it would have to be a 25 - 30 year old tree, as surely by then the plant has initiated whatever symbiosis they need. But having said that, some plants dont sexually mature till they are 50, and i was at a lecture recently where it was discussed how some plants begin with an arbuscular (endomycorrhizal) symbiosis, and then as they mature change to an ectomycorrhizal relationship, why? im not sure. And i think its an area of further research due to lack of understanding. I would presume there is some expression of enzymes by the host tree that initiates the change in symbiosis, so maybe this is the case for Boletus edulis, where it is the ectomycorrhizal fungi that takes over from the arbuscular fungi, this is only based on assumption atm.

This ian area i need to do some more reading on, and in time do more reasearch on so im keen for more info.

Cheers, Obtuse.

[themushroombloke]

I dont see why it would have to be a 25 - 30 year old tree, as surely by then the plant has initiated whatever symbiosis they need. But having said that, some plants dont sexually mature till they are 50, and i was at a lecture recently where it was discussed how some plants begin with an arbuscular (endomycorrhizal) symbiosis, and then as they mature change to an ectomycorrhizal relationship, why? im not sure. And i think its an area of further research due to lack of understanding. I would presume there is some expression of enzymes by the host tree that initiates the change in symbiosis, so maybe this is the case for Boletus edulis, where it is the ectomycorrhizal fungi that takes over from the arbuscular fungi, this is only based on assumption atm.

This ian area i need to do some more reading on, and in time do more reasearch on so im keen for more info.

Cheers, Obtuse.

 

Ok so I'm not sure about this one, but I have a theory: Maybe tree's that Live for a long time need a boast inially to get establish which requires a certain chemical (provided by one fungus) then at a certain age a message is sent to say "you're no longer needed, dont let the door hit your arse on the way out" Then the boletus spores are just chillin waiting for the go ahead, then they're let in. The boletus probably is the tree's second partner and is the secret in living for a long time.

just an idea...

Also forgive my ignorance but I dont fully understand thsi "Or can a TC'd plant still in culture or even a rooted procorm be used? " can anyone explain?

also I found this havn't read it yet but it's alot to take in

click here to read document!

I think that the first species I'd try cultivating is Saffron milk cap as it's relatively easy to find and pine tree's a usually quick growing so it maybe easier than tackling Porcini straight away.

Edited June 8, 2011 by themushroombloke

[themushroombloke]

and this link looks good also click here

Edited June 8, 2011 by themushroombloke

[shortly]

Sorry wrong forum for those acronyms.

try can a tissue cultured plant still in a culture jar/flask

"rooted procorm" sorry typo, Protocorm in this contex is a lump of rapidly dividing callus tissue created buy treating an explant with plant growth regulators invitro. Often used to make LOTS of a paticular clone but prone to senescence.

Edited June 8, 2011 by shortly

[themushroombloke]

Sorry wrong forum for those acronyms.

try can a tissue cultured plant still in a culture jar/flask

"rooted procorm" sorry typo, Protocorm in this contex is a lump of rapidly dividing callus tissue created buy treating an explant with plant growth regulators invitro. Often used to make LOTS of a paticular clone but prone to senescence.

 

so if i may dumb down what you're saying:

getting a sterile freshly germinated seed, taking a tiny piece of that and cloning it to create another plant.

Is this all done under sterile conditions?

Iam starting to think it would be better to take the contents of a whole petri dish of chosen mushroom species then put it on top of some of that plant gel stuff. Then make a hole in the mycelium, plant the protocorm and let it grow down into the gel (this is all done in sterile conditions of course). This way it may be possible to have a slightly different growing media under the petri dish agar.

Am I going in the right direction here??

[themushroombloke]

Ok So i found this handy french document and converted it to english with google translate!!

if we could pull it apart and make sense of it we could unravel the mycorrhizal secrets.

I have changed the colour of the most interesting text in this document.

Also here's a link to the original document it has some interesting pictures!! click here for link

CONTROLLED TO INOCULATION

OF EDIBLE MUSHROOMS AND BOLETS?

J.-M. OLIVIER - J. GUINBERTEAU - J. Rondet - Michele MAMOUN

Among the edible mushrooms, porcini represent species that everyone wants to see

domesticated. The French Confederation of Canned and FNSACC (National Federation of

Agricultural unions Mushroom Growers) in 1995 provide estimates of the

collection of 2 500 tonnes of mushrooms and the manufacture of 1 200 tons of canned food. It is quite

likely that these values ​​from official statistics are incomplete or mixing several

types of mushrooms are very underestimated. The study by J. Rondet (1990) gave evidence

precise estimation of crops in the region Limousin-Auvergne. Their transposition

nationally led some observers to estimate the pickup between 6 and 9 000 tonnes of

fresh porcini mushrooms, depending on the year. Housing market and the information given by the canners

suggest that imports are of equal importance. The total tonnage of fresh mushrooms

marketed fresh or processed in France therefore oscillates between 10 and 15 000 tonnes,

is the turnover of the industry around 1.5 billion francs. In some regions like

Limousin, the income from the collection and gathering of mushrooms has become a

important to the local economy, where other sources of income are reduced. It is common

Now that production is raised during mycological reforestation projects. All

These reasons have led INRA and its partners to consider domesticating mushrooms with a

two-pronged approach: firstly understand, as much as possible, the natural growth source

current collection, on the other hand, longer term, to build a system

production recalling what has been done for truffles, mushrooms and yellow milk caps.

THE CONCEPT OF MUSHROOMS AND ECOLOGY OF SPECIES

The term "mushroom" has received a legal sense (Decision No. 64, Official Journal of 21 November 1971);

it limits its commercial use in two species Boletus edulis (porcini Bordeaux) and Boletus

aereus (mushroom black head). This definition seems too restrictive, since two other species deserve

to be included in the "mushrooms" and are actually almost on the market: Boletus aestivalis

= Boletus reticulatus (porcini mushroom summer or crosslinked) and Boletus pinicola Boletus pinophilus =

(Mushroom mountain). Boletus mamorensis is also present on the French market from imports

Morocco (Redeuilh 1978; Redeuilh and Simonini, 1993). The bay boletus (Xerocomus badius)

no longer classified in the genus Boletus and, despite its taste was not retained as

"Mushroom" in the legislation.

222

• Boletus edulis is a ubiquitous species, collected from almost all French territory, having

generally a period of fruiting in late summer and autumn. The species is associated with

hardwood (oak, beech, chestnut ...) or softwood (spruce, fir, pine hook ...).

The species is described in both plain that on average over a large mountain

diversity of soil, sand Landes granite or shale Morvandiau Brittany.

• Boletus aereus, with dark fruiting bodies, is relatively thermophilic, so in most southern

its distribution and is common in oak or chestnut.

• Boletus aestivalis, paler than the cap Boletus edulis, grows earlier in the season, hence its

common name for mushroom summer. Its ecology is similar to that of Boletus edulis, with a preference

stands for Chestnut Oaks or low or medium altitudes.

• Boletus pinophilus is primarily montane species, rarer in the lowlands, often

associated with pine and spruce but sometimes encountered in hardwood (Beech).

This brief overview highlights the complexity of the situation when considering a

possible domestication, including several species associated with a variety of hosts capable of

grow in different soil and climatic contexts. Foresters will notice that it was not mentioned

Douglas (Pseudotsuga menziesii) as associated gas. Ceps under crops

this species are rare and often open to interpretation on the origin of delicate

fruiting bodies of fungi.

The application possibilities for improving the production of edible mushrooms

223

Rev. For. Fr XLIX - No sp. 1997

Common Names Species Ecology Breakfast

Boletus edulis Cep Bordeaux Conifer Species ubiquitous

Picea abies rather fall

Abies alba Hygrophila

Abies grandis Plain and average

Pinus uncinata altitude

Hardwood

Quercus spp.

Castanea sativa

Fagus sylvatica

etc..

Cep Boletus aereus blackheaded ubiquitous Hardwood Species

or head negro mostly summer and fall

or cep tan (Quercus spp. rather xerothermophilous

Castanea sativa) Plain / hill

Boletus aestivalis "Cep" Summer hardwood species dominated

(= Reticulatus) (porcini or crosslinked) mainly Atlantic

(Quercus spp. Springtime or Summer

Castanea sativa, Thermophile

Fagus sylvatica) Plain / hill

Boletus pinophilus "Cep" Pine Hill Conifer Species

(= Pinicola) mainly mountain and middle mountain

(Abies spp. Hygrophila Picea

abies, Pinus uncinata, Autumnal Late

Pinus sylvestris)

(Occasionally

chestnut)

Boletus Boletus mamorensis of Mamora Hardwood Species in Africa

mainly North

(Quercus suber) and Colline average

mountain

Table I. General data on the ecology

four species of Boletus species and associated forest

224

1

April 3

2

Photo 1

Draft fruiting of Boletus aestivalis formed

Pachlewska on agar amended.

Photo 2

Natural fruiting of Boletus edulis in a

oak regenerated network.

Photo 3

Mycorrhizae formed by Boletus edulis on roots

Pinus attenuata x radiata.

Photo 4

Chestnut breeding greenhouse cloned and mycorrhizal

by Boletus edulis.

Photos J. GUINBERTEAU

EXPERIMENTAL APPROACH TO THE ECOLOGY OF MUSHROOMS

It is obvious to any observer that there are good and bad years for fruiting

ceps. Among the explanations advanced by some tasty collectors (and Larrère

La Soudière, 1987), the relationship with the local climate emerges as a constant. To go

Later in the analysis of the phenomenon, a device for data collection was agrometeorological

put in place in Aquitaine, Midi-Pyrenees, Limousin, Poitou-Charentes. Fifty plots

were chosen primarily for sites known to be fruiting bodies of Boletus edulis

Boletus aereus or / and Boletus aestivalis. They are followed by trained personnel, hence the use

with organizations like the Chambers of Agriculture, the Regional Property

Forest (CRPF), the National Forestry Board (NFB) or agricultural colleges. The majority of sites

is equipped with small items of Agrometeorology, rain gauges, thermometers (ground and air), blood ...

a minority is equipped with recording stations, now type

Humicro 2000. Besides the climate record and analysis of soil, the sprouts mushrooms

are noted (dates and quantities, identification, location). Context (preceding crop, flora

underbrush, other fungal species, state of forest stand ...) been identified

regular.

It is too early to give definitive results, the initiation of the network, its gradual extension

and training of relevant officials who need 5 years. Relatively few data

accurate, however, can be presented.

In Figure 1 (below) appears quite clearly the events that led to the fruiting

two species (Boletus Boletus aestivalis and aereus) in an oak forest of the Landes. In July,

a rainy period (80 mm) of one week is followed by a decrease in soil temperature (- 10 cm)

The application possibilities for improving the production of edible mushrooms

225

Rev. For. Fr XLIX - No sp. 1997

Rain + irrigation

Soil temperature (- 10 cm)

Cumulative production Boletus aereus

+ Boletus reticulatus

July August September

100

C

mm

20

20

40

10

80

60

40

20

Kg

July August September

July August September

Figure 1

EXAMPLE OF RELATIONSHIP

BETWEEN FRUIT

AND PARAMETERS OF MUSHROOMS

CLIMATE

(Landes Plot 1)

5.6 ° C; basidiocarps appear first 7 days after, the growth continuing for

12 days (with high rainfall). A neighbor scenario repeats itself at the end of August. In

both cases, a rise in temperature coincided with the cessation of fruiting bodies. Data

collected in other regions (Dordogne, Lot, Gironde, Tarn et Garonne) confirm these observations

for Boletus edulis. Simply, for this case a little later, down the

temperature during the development phase of basidiocarps can finalize the

production. The results can now be stripped summarized as follows:

- High rainfall, exceeding 50 mm over a decade, provides constant moisture

in the soil (saturated blood pressure over 5 days);

- A fall of temperature, air and soil leads to soil cooling in the first 10

centimeters in most cases, the induction of fruiting bodies is linked to a difference of 5 ° C.

compared to the average initial temperature and the number of hours during which this difference manifests itself

should be, cumulatively, in 20 hours can be spread over three to five nights; situation analysis

leads to the concept of cumulation of hours "cold";

- No fruit appears if soil temperatures are above 20 ° C or below

10 ° C for Boletus edulis, at 12 ° C for other species;

- In general, thermal shock occurs during or after the period of rain but it

were observed in cases where the rain was held from 3 to 5 days after the temperature drop;

- The first basidiocarps were observed 6-10 days after the thermal shock if the temperature

exceeds 15 ° C, 8 to 15 days if less;

- Further lowering the soil temperature of at least 3 ° C during the period

fruiting stops the process.

During the fruiting season in late summer or autumn, it was not possible to distinguish

events clearly inducers depending on the species of mushrooms and their possible thermophilic.

However, Boletus Boletus aestivalis aereus and begin fruiting earlier, from June to mid-August,

the role of heat shock seems to be smaller, the system appeared to operate according to the model

Suillus (see below) with the major effect of water (rain or watering).

The above data should be regarded only as a basic need to refine.

However, a comparison is possible with data Suillus granulatus (porky

yellow) for which observations were conducted in experimental plots after mycorrhization

artificial (Poitou et al., 1983) and with irrigation as controllable parameter. This species grows

April to November, the basidiocarps appear when the soil temperature is above

9 ° C and below 20 ° C. The trigger is a rain or irrigation of at least 10 mm

likely to generate lasting moisture in the litter. The use of irrigation has

build a predictive model (Poitou et al., 1989). Sporocarps are detected at least 10 days

after the rain (or irrigation), their maturation may require another 4 to 10 days depending on the

temperature. Unlike Boletus, there is no need for Suillus a real shock

heat is the limiting factor then the availability of water (in a temperature range

large). In the case of Suillus, a relaxation period of 10 to 15 days is significantly

observed (the time required before induction of new growth over the same area). With

Boletus, the existence of such a period of rest has not been precisely determined, because most

often there was only one series of favorable events per plot for a given species

(Succession of two different species of mushrooms is often observed in cons

the same plot). The use of irrigation to supplement rainfall should help, as

Suillus, to clarify the events triggering fruiting of Boletus.

The soil of each plot is analyzed. For example, Table II (p. 227) gives the ranges

identified for different analytical elements in 23 sites.

J.-M. OLIVIER - J. GUINBERTEAU - J. Rondet - Michele MAMOUN

226

A twenty-fourth parcel out clearly from these bands: C / N> 20, organic matter> 50,

~ 2 total nitrogen, total P = 0.7 and very high fraction of fine silt> 50%. It should therefore be wary of

any generalization.

The majority of these plots is relatively poor acid soils. These values ​​cover

data Poitou et al. (1982). The same authors analyzed the areas of fruiting

Boletus edulis, associated with Pinus uncinata in the Pyrenees, in this case, soils were

more acidic with C / N below. However, the soil type areas "on mushrooms" is much

less restrictive than those on the truffle.

The plots for the shoots of wild mushrooms are the subject of further studies on the flora

undergrowth and experiments on the maintenance of it and it is not enough to decline

conclude, for example, in the interest of maintaining associated plants such as Calluna, the Bruyeres

Ferns or fescue. The fungal flora of the parcels is identified each year

determine the species of the ecosystem descriptrices, indicators related to the growth of mushrooms or

potential competitors. The procession met in a chestnut fungus has been described

(Chauvin et al., 1988), similarly for the Spruce (Le Tacon et al., 1984) or the Pin Hook (Poitou

personal communication). Table III (p. 228) gives the example of the fungal flora observed

in three oak woodlands adjacent to the coastal area of ​​the Landes. Note the great diversity, with

some species often associated with mushrooms as Amanita muscaria or Amanita spissa. The

Clitopilus (miller) is given to precede the appearance of mushrooms, there is no evidence

now assign a different role than that of phenological indicator. By comparison,

in the case of tests with Suillus, it was shown that there may be competition between

Suillus granulatus (introduced) and Suillus bovinus (native) while Suillus granulatus

has lived for 14 years with Lactarius deliciosus on twenty trees observed. In

all cases, the experimental trees from mycorrhizal proved attractive to

monitor the dynamics of root colonization by native species.

Controlled mycorrhization

Controlled mycorrhization with porcini mushrooms has motivated many studies, mycorrhizal

were obtained by different authors as Melin (1923), Tozzi et al. (1980), Molina and Trappe

(1982), Ceruti et al. (1983, 1985), Zucherelli (1988), Meotto and Pellegrino (1989). INRA Poitou

successful mycorrhization of Pinus radiata (1982) and Pinus uncinata (1994) with Boletus edulis.

Bawadikji (1993) highlights the heterogeneity of results in its own testing of mycorrhization

Pinus sylvestris with Boletus edulis as in those of the authors cited above.

Mycorrhizae have been obtained easily with Suillus (Poitou et al. 1982; Chevalier and

Detolle, 1984), milk caps (Poitou et al., 1989) or relatively easily with Tuber (Chevalier and

Grente, 1979), it is clear that this is not the case with fungi of the genus Boletus. Will

The application possibilities for improving the production of edible mushrooms

227

Rev. For. Fr XLIX - No sp. 1997

Table II ranges of values ​​found in the analysis of soil (depth (20 cm)

of 23 experimental plots Cep (Landes, Dordogne, Tarn et Garonne)

Expressed in ‰ ms (except pH and C / N)

Ph .. . . . . . . . . . . . . . . . . . . . . . . Total P 4.9 to 5.6. . . . . . . . . . . . . . . . . . . . . 0,3-0,5

C / N. . . . . . . . . . . . . . . . . . . . . . . 15.0 to 17.7 K2O. . . . . . . . . . . . . . . . . . . . . . . 0,07-0,11

Organic matter. . . . . . . . . . . . CaO 24.6 to 39.5. . . . . . . . . . . . . . . . . . . . . . . 0,3-0,9

Total N. . . . . . . . . . . . . . . . . . . . . MgO 0.8 to 1.2. . . . . . . . . . . . . . . . . . . . . . . 0,13-0,27

P (Dyer). . . . . . . . . . . . . . . . . . . . Clay from 0.001 to 0.05. . . . . . . . . . . . . . . . . . . . . . 92-184

Fine silt. . . . . . . . . . . . . . . . . 115-305 Coarse sand. . . . . . . . . . . . . 315-400

Coarse silt. . . . . . . . . . . . . 147-220 Fine sands. . . . . . . . . . . . . . . . . . 153-238

J.-M. OLIVIER - J. GUINBERTEAU - J. Rondet - Michele MAMOUN

228

not treated here the phenomena of fruiting of the mushrooms without symbiotic association and these cases

are seen relatively often, but reproduction of the phenomenon, beyond the work of

Granetti (1992), has not been published (photo 1, p. 224, young boletus Boletus aestivalis

having reached 7 mm high on agar PLKO at INRA Bordeaux).

Table III An example of fungal community to accompany Cep

Case of three neighboring parcels with as dominant species Quercus robur

Genus / Species Common Name tree-host presumed

Mycorrhizal

Amanita Amanita asteropus bulb Star Oak or Pine

Amanita citrina Amanita citrine or Pin Oak

Amanita excelsa or Pin Oak

Amanita muscaria fly agaric Oak, Pine, Birch

Amanita rubescens Amanita blushing or Pin Oak

Amanita Amanita spissa thick English Oak

Amanitopsis vaginata var. Amanita fulva vaginée fawn or Pin Oak

Boletus edulis Cep Bordeaux English Oak

Cantharellus cibarius Chanterelle Girolle or Oak or Birch

Chanterelle Cantharellus tubiformis Tube Pin

Clitopilus prunulus Meunier Oak

Cortinarius Cortinarius cinnamomeus cinnamon leaf or Pin Oak

Cortinarius Cortinarius paleaceus glitter or Pin Oak

Cortinarius torvus Oak

Hydnum repandum Foot Sheep Oak

Inocybe Inocybe asterospora spore star Oak

Laccaria amethyst amethystina Laccaire Oak

Laccaria laccata Laccaire lacquered oak or pine

Lactarius camphoratus Lactaire camphorated Oak

Lactarius chrysorrheus Lactaire milk yellow oak

Lactarius hepaticus Lactaire color liver Pin

Lactarius Quietus Lactaire quiet Oak

Lactarius subumbonatus Lactaire wrinkled Oak

Paxillus involutus Paxille wound Oak or Birch

Pisolithus tinctorius pisolites dyers Pin essentially

Russula atropurpurea = krombholzii Russule burgundy Oak

Russula cyanoxantha Russule coal Oak

Russula emetica var. sylvestris Russule emetic Oak

Russula fragilis Fragile Russule Oak

Russula nigricans Russule blackening Oak

Russula vesca Russule edible Oak

Scleroderma citrinum Scleroderma Oak

Tricholoma Tricholoma album White Oak

Tricholoma fulvum Tricholoma flavobrunneum = tan oak or birch

Tricholoma Tricholoma sulphureum sulfur Oak

Xerocomus badius Bolet bai * Oak / Pine essentially

Saprophytic

Caloceras viscosa Calocère Pin essentially viscous

Chlorociboria aeruginascens Oak

Collybia butyracea collybia butyracée or Pin Oak

Collybia maculata Spotted collybia Oak or Pine

Gymnopilus penetrans Pholiota penetrating Pin

Pholiota spectabilis Gymnopilus remarkable Pin

Hypholoma Hypholoma fasciculare tufted or Pin Oak

Birch essentially Leotia lubrica

Lycogala epidendron Pin essentially

Psathyrella hydrophila Psathyrella hydrophilic or Pin Oak

* Saprophytic or mycorrhizal.

The process requires controlled mycorrhization phases:

- Isolation, characterization and multiplication of the mycelium used as inoculum;

- The propagation of the host tree, if possible, trying to reduce the heterogeneity of plants,

hence the use of selected seeds, cuttings or micropropagation where the

possible;

- The association of two partners in controlled conditions, with the aim of obtaining

a maximum of mycorrhizae.

Moreover, with the mushrooms, it is for late fruiting species in a stand

forest (at least 15 years, Le Tacon et al., 1984), in case of mycorrhizal tree planting, he

will therefore ensure the sustainability of the association over a long period. In the case of Suillus granulatus

and Lactarius deliciosus, mycorrhization continues 17 years after planting, we know

Now cases of truffle mycorrhizal with trees planted 20 years with sustainability

of mycorrhization. Even if we should not unduly close slightly different systems,

sustainability should be possible to provide, all this depends, among other things, the quality of mycorrhization

initial and potential competitor of the native fungal flora of the nutritional status of the medium,

the speed of root system development, etc..

The fungus

The mycelium of four species of mushrooms can be obtained by cutting meat basidiocarps

young or by isolation from mycorrhizae; germination of spores is useless so far

on a practical level, lack of reliable method. It is the same for the regeneration of

protoplasts obtained from areas hymenial.

Identification of the material is made by classical taxonomy (Alessio, 1985) when available

of basidiocarps. In general, the criteria are reliable in distinguishing among species.

However, there are sensitive cases and is also common to have to check the mycelium derived from collections

or isolates from mycorrhizae. The use of protein markers has long been

the best recourse. Figure 2 (p. 230) (Fabre, 1994) gives the typical profiles obtained for

distinguish the four species of mushrooms from proteins with a beta glucosidase activity. These

Profiles can be completed using esterase activity. Currently, molecular markers

are good tools. The method is to amplify regions of the genome encoding

Nucleic acids comprising the ribosome, then cutting the amplicons with the enzymes with

Specific sites of clashes. Differences in the sizes of fragments are revealed by electrophoresis

and distinguish the species from the structure of this part of the genome.

To be more precise, in the case of mushrooms, it is to amplify the ITS regions of the genome called

(Internal Transcribed Spacer) 1 and 2, then using the enzymes Alu I, Cfo I. .. Other

techniques based on amplification of random fragments of the genome called RAPD (Rapid

Amplified Polymorphic DNA) also give good results (Cameleyre, 1996).

At the intraspecific level, enzyme activities (eg, phosphoglucomutase in

Boletus edulis) or molecular markers show strong differences. There is therefore a

variability within a given species, but these genetic differences can not today

be related to differences in behavior or ecological specialization and Breakfast.

The proliferation of the mycelium to produce an inoculum can be produced on solid medium

(Agar), liquid or mixed (medium perlite or vermiculite and / or peat moss with nutrient solution). In practice,

three methods succeed or complement during the procedure (Poitou et al., 1981,

1982, Chauvin et al. 1988; Bawadikji, 1993 ...). Figures 4 (p. 230) and 5 (p. 231) show that

problem is not simple and there are strong differences in behavior at both

interspecific qu'intraspécifique.

The application possibilities for improving the production of edible mushrooms

229

Rev. For. Fr XLIX - No sp. 1997

J.-M. OLIVIER - J. GUINBERTEAU - J. Rondet - Michele MAMOUN

230

100

80

60

40

20

18 ° C 25 ° C 18 ° C 25 ° C

7 d

35 d

70 days

Boletus edulis

Boletus aestivalis

Diameter (mm)

Figure 4

Mycelial development

Gelose (PLKO)

Figure 2

COMPARISON OF ENZYME PROFILES

(I glucosidase) for the four species,

Boletus edulis (Be), Boletus aestivalis (Ba)

Boletus aereus (Bar) and Boletus pinophilus (Bp)

Bars represent proteins with

i glucosidase activity and revealed

electrophoresis according to their speed

migration.

Figure 3 RAPD AMPLIFICATION OF DNA of Boletus edulis (BE) and Boletus aestivalis (BA)

(Bands from major Cameleyre, 1996)

Primers P3, P4 and P7, Kit P. Bioprobe. M = size scaling (* = 3 and 0.5 respectively KPB).

The bars represent the nucleic acid fragments revealed by electrophoresis based on their migration speed.

Species

Bp Ba Be Bar

P3 P4 P7

Ba Ba Be Be M M M Ba Be

In the example shown in Figure 4, the strain of Boletus aereus was not made because it does

hardly grows. The two isolates of Boletus edulis growth rates that vary

practically double. For isolates of Boletus aestivalis, the effect of "temperature" is

remarkable.

Figure 5 shows different kinetic (dry weight) obtained in liquid medium with growth rates

of 14.7 mg / day and 8.2 mg / d for both Boletus edulis and 10.9 mg / d for Boletus aestivalis

(Fresh weight). The practical conclusion is that each strain (or group of strains) must

adapt a method of inoculum production.

The application possibilities for improving the production of edible mushrooms

231

Rev. For. Fr XLIX - No sp. 1997

The host tree

For plants likely to be colonized, seeding has been widely used and continues

to be for guests not reproduced by other methods (Abies grandis, Pinus uncinata,

for example). Besides heterogeneity, the main problem is the disinfection of seeds

solved by soaking in hydrogen peroxide and / or by treatment with ultrasound. Cuttings is a

operation with low yields for species of interest but the efficiency was significantly

improved for Norway spruce (CEMAGREF), chestnut (Nurseries grout) or

Oaks (INRA Clermont-Ferrand). Micropropagation in vitro for mycorrhization was

obtained for Chestnut (Strullu et al. 1986; Chauvin et al., 1988) and the English Oak

(Bawadikji, 1993).

The association

In general, the plants are used in vitro or in non axenic conditions must

there induction of secondary roots before introduction of the inoculum. It is also there

compatibility between rooting conditions and fungal activity (Strullu et al., 1986). Mycorrhization

So after transplantation occurs in an aerated medium (perlite or perlite + peat) with

a nutrient solution adapted. All authors stress the need at this stage, control

strict levels of free sugars, nitrogen and phosphorus. The work of Chauvin et al.

(1989) or Bawadikji (1993) illustrate the technical difficulties with Boletus when

compared on the same plant material, with other fungal species (Amanita,

Paxillus, Terfezia ...). The choice of isolate Boletus is paramount. The specialization of a strain

fungal species to a specific host is suggested (Poitou et al., 1982, 1984) but was not

shown. It is also interesting to note that the same strain will regularly mycorrhizal

good yield in a series of tests and will not be in another series. Differences

not controlled in the manufacture of the inoculum may explain this fact.

0,4

0 14 24 35 44 55

0,3

0,2

0,1

Days

Boletus edulis 1

Boletus edulis 2

Boletus aestivalis

Dry weight (g)

Figure 5

Mycelial development

ROUGH IN LIQUID CULTURE

At this stage, experiences from in vitro plants or from seedlings conditions

protected only rarely yielded results greater than 50% of inoculated plants carrying

mycorrhizal Boletus after one year. To this must be added the risk of contamination by

mycorrhizal exogenous for raising seedlings in the greenhouse. Through our own experience,

loss rate by these competitors is greater than what was obtained for milk caps or

truffles. On softwood, it is found that Suillus spp. can be formidable competitors

compared with Scleroderma, Thelephora or Hebeloma.

Mycorrhizal controls are made between 6 and 9 months after adding the inoculum. The

mycorrhizae (Fig. 3, p. 224) appear to pearl white. The mycelium can be isolated from it

and controlled (see above). Mycorrhization rates are expressed as the number of mycorrhizal plants

by Boletus (typically <30%) and percentage of mycorrhizal roots per plant bearing

mycorrhizal Boletus (rarely> 10%). The only exception is Pinus uncinata mycorrhizal

Boletus edulis by mycorrhization rate with> 90%. Note for this association there has

had a significant effect of mycorrhization on the growth of the tree in the greenhouse and field

(Poitou et al., 1984). With Chestnut, no similar effect was observed with wild mushrooms

net while he was with Paxillus involutus or Amanita muscaria. By cons, a strain of Boletus

aestivalis stimulates rooting of Castanea sativa clone S17 (Chauvin et al., 1988).

Regarding tests of INRA, plantations of trees from the following mycorrhizal

:

- 3 plots (Pyrénées-Atlantiques) totaling 0.8 ha of grasslands with montane Pinus uncinata

associated with Boletus edulis planted since 1984, 80 trees in place. The effects mentioned

above on tree growth were confirmed in situ and to date, no fruiting

mushroom has been registered;

- 1 plot (Dordogne) with clones of Castanea sativa and hybrids crenata-sativa (CA15,

CS74 ...) with mycorrhizal Boletus edulis, Boletus aestivalis, 50 trees (planting on young

Previous cereals, 1994-1995).

Added to this are limited trials with Pinus radiata, Pinus radiata x attenuata, Abies, Quercus,

Chestnut. The ecological basis presented in the first part of the article will be applied

in these plots to help fruiting.

CONCLUSIONS

The current situation leads to some caution in predicting the domestication

ceps. The acquisition of ecological data and is certainly more accurate physiological

help take into account the mycological component in the management of areas with a strong

forest, as the Limousin, Auvergne, Morvan, the Vosges ... Tree planting

mycorrhizal remains the experimental field. Yields obtained by mycorrhization

different authors are fairly low, although each laboratory is progressing in a certain

discretion to apply the results more economically. Anyway, the choice of

strains, the quality of the inoculum, the conditions of realization of the association are elements

improvement. Any relationship with microorganisms in the rhizosphere (BAM, cf. Article

Garbaye et al. in this issue, p. 110) were not specified for the mushrooms. Finally, virtually

nothing is known about the relationship between the fungus introduced early and train fungal

which is obtained after planting. However, the domestication of the mushroom is only of interest with the fruiting

expected 10, 15 or even 20 years after planting.

J.-M. OLIVIER - J. GUINBERTEAU - J. Rondet - Michele MAMOUN

232

Associations established in various departments of the South West with the help of local

authorities are active supports for these studies. The agreement between INRA and mycoplasma

(Comprising three nurseries) will allow the optimization and scale changes on

mycorrhization. A large experimental effort is essential and requires the cooperation of all

potential partners.

The application possibilities for improving the production of edible mushrooms

TO THE INOCULATION OF MUSHROOMS AND CONTROLLED boletes? (Abstract)

The mushrooms have a dominant role in the activity of the French sector collection-processing-marketing

wild edible mushrooms. The ecology of major species of mushrooms is presented through the

mycological data and an experimental approach implemented in the Southwest of France. The importance of

Lowering the temperature in the process of induction of fruiting bodies is highlighted. The first results

for controlled mycorrhization are presented: the inter-and intra-species is analyzed and the

multiplication operations and association hosts under controlled conditions. The first tree planting

mycorrhizal with porcini mushrooms (mainly chestnut and pines) are under observation. It remains

However, important work to be done to improve mycorrhization, estimate tracking the fungus on the ground

and come to fruition in plots planted.

IS ARTIFICIAL INOCULATION OF THE EDIBLE SPECIES Boletus FEASIBLE? (Abstract)

The Boletus species Is One Of The Major edible forest fungi In The French mushroom gathering / selling / processing Sector. The

Ecological environment for The Main Varieties In The SI species Presented On The Basis of Mycological year experimental data and

system set up in south western France. The Significance of a temperature drop in Bringing about fruiting IS Highlighted.

Initial Findings Regarding artificial inoculation are Described - inter-and intra-specific diversity, multiplication and host association

Under controlled conditions. The first of Boletus mycorrhized trees plantations (Mainly chestnut) are Under observation.

More research required to Improve IS mycorrhization quality, estimate fungal survival in the Field and Enhancement in fruiting

Planted Stand With inoculated material.

Edited June 13, 2011 by themushroombloke

[obtuse]

yoiks bloke!

look forward to reading through that, when i find some free time at the end of the week.

thanks.

Cheers, Obtuse.

[themushroombloke]

I think this video demonstrates what would be necessary to pull off the plant component of this idea.

IS THIS A PROTOCORM PEOPLES?????

 

 

Edited June 20, 2011 by themushroombloke

[themushroombloke]

So i've definately decided to go with The saffron milk cap mushroom (Lactarius deliciosus) as my first candidate for growing mycorrhizal mushrooms.

here's a link to some people who are already doing it in New Zealand CLICK HERE

[NSF]

I had a look at that link to the NZ farm but their website reveals nothing about what they are up to. It certainly doesn't discuss how they are doing it. Did i miss a page??

EDIT: Ah the newspaper article about them features a little more information.

It'd be great to know where they are sourcing their trees from.

Maybe they are getting them from the Forest Institute

http://www.sciencedirect.com/science/article/pii/003807177990021X

Oops, no, more than likely they are getting them from here:

http://www.trufflesandmushrooms.co.nz/Lactarius%20deliciosus%20web.pdf

Edited June 22, 2011 by NSF

[NSF]

This is a pretty useful list of trees that can be near truffles and those that can't.

http://www.trufflesandmushrooms.co.nz/A%20list%20of%20ecto-%20and%20arbuscular%20mycorrhizal%20plants%20plus%20families.pdf

We can also use it to determine trees we should innoculate and those we shouldn't consider.

[gecko]

When inoculating trees with T.melanosporum (usually Oak, sometimes Hazelnut, though there are many host trees),

the trees are allowed to grow in pasteurised potting media for few months at least,

before spore slurry is introduced.

this is done in order for there to be a half decent root system for the

germinating spores and resultant mycelium to team up with.

Micofora in Spain inoculate pine seedlings with milk caps (L.deliciosus and maybe L.sanguifluus) and say that you can expect to have them produce after as little as 3 yrs.

Truffles can fruit that early too, but usually take a little longer.

They also produce and sell pellet spawn (spores in a pelletized dehydrated gel or water crystals afaict) of Boletus spp.(I think it's B.edulis, not sure)

It's recomended that you dig holes or a furrow 10-15cm deep around the dripline of a suitable host tree (beech, oak, chestnut, pine) 20yrs or older.

backfill, water and leave alone.

fruit can be expected after 2yrs,

but no guarentees, so repeat every 2 yrs.

Some plants I've inoculated in tubes with various species have fruited

with species I didn't put in there (Poison pie Hebeloma crustuliniforme ).

the spores or mycelium either has been present in the potting mix or has blown in.

it happened on Chestnut seedlings and Valley white oak (Q.lobata) seedlings,

both less than 2yrs old in forestry tubes.

I tend towards non-sterile aproach, because as obtuse mentioned there may be other factors

needed to get the target species going (esp. Boletus spp. or Cantharellus spp.)

Also, the edno- ecto- thing with trees, changing from one to the other throughout the course of their lives.

Eucalypts do it.

The way I understand it is that they can start of in some pretty hostile sites

and therefore may only have endomycorrhizae to work with if spores or hosts are already present.

If not there, they usually turn up some how.

The tree grows, kills major grass (endomycorrhizal hosts) competition,

then the tree shifts conditions to favour ectomycorrhizal species

and the biology becomes more complex and fungaly dominant soil develops.

If you want to get really serious about it, I recomend This book

It deals with ecto- and endomycorrhizae .

there are agar recipes for sterile culture of ECMs

inoculation methods etc. drawing on a lot of work in West Aus. and some from forestry projects in Asia.

Here is a handy site , mostly native species but not all.

Of the Boletus spp., I'd get B.aereus if I could, as it grows in warmer parts of Nthn. hemisphere.

B.pinophilus for its love of Pine trees... if it could establish in P.radiata plantations,

you'd never pick a Slipery jack again

Well you wouldn't not use any of them really, B.edulis, B.aestivalis.

B.edulis var. grandedulis (Nth Amer.), B.barowsii etc.

I have pines (P.ponderosa, P.patula,) in tubes that are showing signs of ECM activity now

after inoculating last year with a mix of species.

It's a long term project....