5 years research on black truffle mothers and fathers


mothers come from mycorrhizae but truffle father from other sources

For the first time it has been possible to study genetically the female and the male factors in black truffles. In a study of our researcher Herminia de la Varga, while she was in a postdoc in the INRA, shows us the results after genetically analyzing for 5 years, 241 truffles and 475 mycorrhizae plus so many soil samples.

To understand how she recognized mothers and fathers, using molecular markers she read the mother’s DNA in the veins of the gleba. Later analyzed the spores and obtained two readings, that of the mother (which was the same as that of the white veins of the gleba) and a second reading that by discarding, was the father’s.

Although we have already advanced some conclusions of this extensive work, let us write in this post some more:

truffle inner structure male and female parts

The majority of parents (75%) were transient (they were only in a single truffle), but 16 parents were found in different truffles and in subsequent years and also they were the ones that produced more truffles (21% and 37%). One of them produced just himself 18 truffles (8.7% of the produced ones), and was found in truffles of two different trees 20 meters apart. So these fathers who produced truffles in different years can not come from spores who germinated.

Only 3 of these fathers were found as hermaphrodites in the mycorrhizae. From all this it is concluded that the fathers have little capacity to associate to the host tree and that there must be a mycelium that persists in the soil, sometimes associated with herbaceous plants and becomes a pool of fathers. However most fathers come from spores that germinate, and this explains the positive effect of the spanish wells with spores in the plantations.

It would be very interesting to isolate and study these persistent parents, who are at the same time the most producers.

Only 3 individuals (1.5% of those analyzed) found themselves acting both father and mother (hermaphrodite), although producing 55 truffles (27%), while most were detected acting only as father or mother (you will see in this and other articles as “dioecy” or “trioecy” when hermaphrodite is taking into account).

Reference:

I leave you a photo of Herminia de la Varga giving one of the invited lectures at ICOM9 two weeks ago in Prague, about the results of this work:

Cheers,

Marcos S. Morcillo

herminia de la Varga at ICOm9.jpg-large

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Black and summer truffle live as well associated to Herbaceous plants


black truffle new life cycle

July has been crazy and I could not find time to write in this Blog. Yesterday I flu from Barcelona to Cape Town and instead of watching films, I read some pending papers 😉 here some comments from them:

A french work of 2016 opens a new vision to the already complex biological cycle of the black truffle. We know that the truffle often fruits under certain shrubs (juniper, rosemary, thyme …) and that there are several herbaceous plants that tolerate brules. Now it´s been found with DNA techniques as black truffle mycelium is able to colonize the roots of certain plants and shrubs with which it does not create true mycorrhiza.

In this work, when analyzing a wild truffiere of Quercus ilex, they detected with PCR mycelium of black truffle in 74 roots of herbaceous plants, corresponding to half of the analyzed samples.

The majority of this mycelium (65%) found in herbal roots is the same as the maternal part of the truffles that were harvested around it, but there is a 25% that is not found in the collected truffles.

Grinder already found something similar for summer truffle. See reference.

They found as well something quite funny: truffle may have a single mother, but several parents! you probably have found sometimes huge truffles that looks like small single ones stuck to each other. They observed that these different parts of the truffle come from a different “father”, although they share the mother. It is as if different truffles together merge in their development. Not only did these “multilobulated” truffles have different parents, but several of the “mono lobular” (normal round truffle) presented different paternal individuals. Each lobe could be a different fertilization of the same mother!

This study also finds a high consanguinity in the truffles found in the same truffiere. Possibly because either the truffle is dispersed by an animal or because it rots in the soil, this causes that in the same place we find the two sexual types (maternal and paternal) coming from the spores that germinate as a maternal part or paternal part.

This work also shows how gene flow seems to be limited to the size of the brule. So mothers and fathers come in wild truffieres from physically close individuals. This would weaken the theory that some parents might come from “mobile spermatia”, such as conidia or spores of asexual reproduction found in some truffles like Tuber borchii, or maybe these conidia have a weak dispersion.

However these works are done in wild truffieres, and possibly as we expand these studies and sample sizes some of the results could change. Also, in plantations where the soil is worked and we add spanish wells with spores we could find different data or less consanguinity.

Cheers from South Africa, where we are for some projects 😉

Marcos S. Morcillo

References:

 

 

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update on truffle growing in America


 

truffle growing in Chile Nothofagus x Tuber

A couple of news have been just released that make a nice overview of the truffle farming industry in Chile and USA. I thought it could be nice to share it here:

Why Haven’t American Truffles Taken Root Yet?

and the Chilean one, where they describe the truffle industry in Chile for the last 10 years. Though it is in spanish language, but worth try the translator:

 «Una década del cultivo en Chile: la trufa comienzaa oler bien»

BTW, I´ll be in California again from 23rd august to 5th september 2017, just in case any local grower need anything…

Cheers,

Marcos S. Morcillo

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how Quercus ilex tolerates cold climate?


holm oak mycorrhized with black truffle

Last winter in Catalonia we had a whole month with temperatures below 0ºC and with constant fog. Frost accumulated day after day left holm oaks like the one of the picture above (left).

We have been sampling these orchards in late spring and saw like all Quercus ilex have grown normally. Not even the leaves dropped after ice melt. (right above pic in same plantation).

I saw while I was in Virginia (USA) last year problems with this host tree. Lack of growth that growers attributed to cold. I suspect that those Quercus ilex where Q. ilex ssp. ilex, less tolerant to cold than its brother Q. ilex ssp. ballota (=Q. ilex ssp. rotundifolia).

Searching on bibliography found several data:

In a spanish paper found that winter temperatures at the nursery and therefore the hardening during the first year will condition the tolerance to the cold in the field.

Late frost may damage flowers as bloom comes when temperatures between march and may raises al average of 20 °C and 10 hours of daily sunlight, after a period of stress.

About acorn germination, although there is high variability from seed origin, if temperature drops to -12ºC during one nigh, acorns do not germinate.

They found no relationship between rate of growth of the holm oak seedlings and cold tolerance.

In another study named “Variation in cold hardiness and carbohydrate concentration from dormancy induction to bud burst among provenances of three European oak species” found that Mean cold hardiness values in January were –56, –45 and –27 °C for Q. robur, Q. pubescens and Q. ilex, respectively. Holm oak tolerates temperatures of -22ºC without lethal damage in 50% of the tissues, so pretty good resistance to cold.

Quercus ilex cold tolerance graphic

Studies reported that seedlings of Q. ilex ssp. ballota may be hardy to temperatures between –8 °C and –15 °C. Same authors found their results were consistent with studies on adult trees of North American Quercus species, showing maximum cold hardiness values up to –60 °C. Calmé et al. (1994) assessed the cold hardiness of seedlings of Quercus rubra (a species that can be considered ecologically comparable with Q. robur) originating from latitude 45° N and found cold hardiness values of between –10 and –20 °C in autumn.

And how does this cold tolerance works?

As oposite to deciduous oaks, in the evergreen species, Q. ilex, the total carbohydrate concentration increased continuously from October to March, suggesting that winter is a favorable period for evergreen Mediterranean species to accumulate carbon, indicating that mobilization of starch to sucrose invariably accompanies the development of freezing tolerance.

Total carbohydrate concentration is related to maximum cold hardiness. Total carbohydrate concentration represents the pool of carbon that can be used either to enhance cold hardiness via hydrolysis to soluble carbohydrates, or to support metabolic processes. At the time of maximum risk (January) of freezing injury, most of the total carbohydrate pool was hydrolyzed to soluble carbohydrates, whereas there was a much smaller proportion of total carbohydrates occurring as soluble carbohydrates at times of lower risk of freezing injury (e.g., October), suggesting that soluble carbohydrates serve in processes other than those associated with growth.

 Cheers,
Marcos S. Morcillo

 

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We just published a new paper on Tuber borchii growing with stone pine


bianchetto truffle farming

The last edition of the Montes review (just in spanish edition, sorry for that) has published our latest paper titled: “Viabilidad y rentabilidad de una plantación de pino piñonero (Pinus pinea) micorrizado con trufa bianchetto (Tuber borchii), por M. Morcillo, E. de Paz, X. Vilanova y M. Sánchez”

This is “feasibility and profitability of a stone pine plantation (Pinus pinea) mycorrhized with bianchetto truffle (Tuber borchii)”.

 

bianchetto made in Spain

We keep on working with this truffle trying to get a better understanding on its biology, life cycle and fruiting triggering factors. As an example studying the diversity of bianchetto in Spain and Portugal. The ones on the pic above harvested in north Barcelona, DNA confirmed as Tuber borchii.

Cheers,

Marcos S. Morcillo

 

 

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Bookings open for the new editions of the Barcelona Truffle Tour


truffle farming seminars in Barcelona

We just have new dates for the coming editions of the only 2 truffle growing seminars in english language we will organize in Barcelona:

11th-16th december 2017  and  12th -17th february 2018

One of the features this year is the workshop about aromatic profiles and post harvest treatments that Dr. Pedro Marco Montori will develop. He´s a researcher at Zaragoza University. His PhD was focused on post harvest truffle treatments. Here you´ll brush, clean and package in modified atmosphere your own truffle. Compare liofilizated vs. heated vs. wax coatings on truffles, analyze how truffles breathe at different temperatures, and learn and understand the complexity of truffle aroma.

Note group size is limited to 20 people.

And if you want to see what we do in these seminars, just get in http://barcelonatruffletour.com

See you in Barcelona!

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How 2016/17 black truffle season has gone in France & Spain?


import - export black truffle in France, marketing and prices for black truffle

In France producers and traders estimate for the 2016/17 harvest the volume of French truffle production to represent 30 tonnes of Tuber melanosporum and 25 tonnes of Tuber aestivum var uncinatum and mesentericum. A half-tinted campaign (44.8 tons for 2015/16), which took place more favorably in irrigated truffles.
The federation of nursery/truffle tree producers and the regional federations of truffle growers estimated the professional plantations at 1,200 hectares for the year 2016.

FranceAgrimer just published a great document with lots of interesting data on truffle market, prices and volumes, exports and imports:

http://www.franceagrimer.fr/content/download/51148/491610/file/Truffe%20-Présentation%20Commerce%20extérieur%20au%2010052017.pdf

The truffle production during the last season was bad in Spain, in fact, there were no truffle at all on the wild in most regions, however, is the first season that I can remember – from the past 20 years that I´ve been working with truffles- that even being such a bad season, total quantities (wild + farms) harvested in Spain were higher than last year. This is because every year we have more and more plantations with irrigation. Prices were higher in average than other years so growers (who could water) were quite happy.

Prices paid at harvester (dirty and all qualities mixed) in the largest black truffle market (Teruel) has been between 400-750€/kg.  In Catalonia every year more restaurants use truffles in their menus, this made that local truffle harvesters start selling their best truffles directly to local restaurants and leave the worst truffles to be sold at the weekend truffle market. This has lead to drop prices at local markets (due to low quality) and even some traders are not interested to drive to these markets to buy anymore, so some markets are disappearing. This is of course a pity as we loose a tradition but at the same time means that truffle consumption is growing in Spain and this is something to congratulate.

Cheers,

Marcos S. Morcillo

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Where and when males come from to fertilize truffles?


two-year-old-oak-producing-its-first-black-truffle

In the following paper we hypothesize about a new theory we created related with black truffle life cycle and when spores germinate, if they do it as males or females. We are working in order to prove it in coming research… I would love to hear your comments…

Cheers,

Marcos Morcillo and Xavier Vilanova

________________________________________________________________________

New considerations of Tuber melanosporum life cycle

Xavier Vilanova1, Marcos Morcillo1, Mónica Sánchez1, Herminia De la Varga1

1 Micologia Forestal & Aplicada. Camí d’Alfou sn, 08459 Sant Antoni de Vilamajor, Barcelona, Spain.

Corresponding author: xaviervilanova@micofora.com

 

Introduction

The black truffle, Tuber melanosporum Vittad., is an ascomycete fungi that produces ectomycorrhizal associations with different trees and shrubs (Mello et al. 2006). The black Périgord truffle is known by the organoleptic properties of their fructifications, that are naturally harvested under mediterranean regions, but also cultivated all over the world in orchards planted with inoculated seedlings. Since 2015 it is considered as a perennial culture for the European Union, although the production of inoculated seedlings is done since the 70s (Murat 2015), and thus the importance of study the species. After the publication of T. melanosporum genome (Martin et al. 2010), in the last years a huge progress has been done to unravel truffle life cycle (Le Tacon et al. 2016, Taschen et al. 2016, De la Varga et al. 2017). Nowadays we have more information about how the fungus live, in symbiosis with plants, and in soil. We have information coming from scientific research, from experimental truffle sites, wild forests and established truffle plantations.

Regarding research publications, we have an approximation to T. melanosporum life cycle. It is a heterotallic specie, mating is controlled by two idiomorphs called MAT 1 and MAT2 (Rubini et al. 2011). Truffles are the result of sexual reproduction, we know that a female individual (that can be either MAT 1 or MAT 2), found always as mycorrhiza, has to meet with a male one of the opposite mating type (whose origin can be different). Most of individuals acting as fathers come from germinating ascospores, as its principal function is the sexual reproduction (De la Varga et al. 2017, Taschen et al. 2016).

In different publications, it has been described that it exists, at root level, a kind of progressive exclusion of the female individuals harboring the mating type opposite to the dominant one, leading to have plantations with a patchy distribution of the mating types at root level, having each tree roots with individuals of only one mating type (Murat et al. 2013, De la Varga et al. 2017).

One of the practices that is widespread among truffle growers, specially in Spain, is to make the called “truffle traps”. This technique consists on doing holes or trenches around the trees and to introduce a substrate with a mixture of spores. In those substrates, the spores come from a mixture of different truffles, so a mixture of both mating types. In a recent publication (Murat et al. 2016) it was tested the efficiency of this technique and showed how truffle production could be promoted and localized in a few centimeters, opening also the possibility to better understand the role of ascospores.

Discussion

In our experiences along the years, when we observe the behavior of the spores throughout the different steps in the process of truffle production in managed plantations (plant production, plantation establishment and management techniques – substrate addition to increase truffle production – ) we have seen that:

  • For the production of melanosporum mycorrhized seedlings, the inoculation takes place in march-april (in the northern hemisphere) observing the first mycorrhizae in October-November (observing the roots under a microscope). Moreover, using molecular techniques, as the Real-Time PCR (Parladé et al. 2013) it can be detected an increase of germinated mycelia, coming from the spores, from July. (unpublished data)
  • Periodically, inoculated seedlings are analyzed to check the level of mycorrhization of the roots. When doing those analyses in nursery plants, but also in two years old trees, that have been planted in the field, non-germinated spores are detected. They remain in the substrate, near the roots, and apparently, they are viable spores.
  • As it has been noticed in field experiences (data not shown) and in other publications (Murat et al. 2016), in the places where substrate has been applied truffles fructify 2 years after the application. The results of adding spores to the soil surrounding the trees are reflected as an increase of the production of truffle ascocarps, that are localized in the place where the substrate has been applied (Murat et al. 2016).

All those evidences make us hypothesize that the first germinating spores form the ectomycorrhiza, so that they could be “predetermined” to act as mother/female individuals, or they “choose” to germinate and act as mother/female elements and form the ectomycorrhizae. The other spores remain in the soil waiting to germinate next year or later. This could also explains why the results of applying substrates with spores are not seen after 2 years. Indeed, it has been reported the fructification of truffles in plantations with inoculated trees of two years old (data not shown). In this case, it could be explained by the germination of the spores that remained in the substrate, and that they act as male elements for the sexual reproduction.

This simple hypothesis could explain the behavior observed in the spores at the different stages of truffle life cycle. Currently, the mechanisms that determine that one individual acts as female or male element are unknown (genetics, environmental, molecular, etc.), but probably this function is determined. It could be that in the genome of each individual it was determined so that the first spores to germinate act as female element, that could entail an energy saving for the fungus, as well as an explanation to the rates of consanguinity described in some publications (Taschen et al. 2016).

The fact that T. melanosporum fructifies in traps with added spores after 2 years, and in young two years old trees planted in orchards, could be explained by this hypothesis. Both kind of fructifications could be promoted by the late germination of the spores that will act as male elements. In the example of truffle traps these male partners will come from the spores added to the substrate; for the young seedlings, they may come from the bank of spores remaining in the substrate of the plant. The question is that if this is like that, we should expect to have fructification in older plants maintained in nurseries. This has never been reported, probably the germination of the ascospores that will act as male elements is triggered by environmental signals and that is why they remain non-germinated as a bank of spores.

Conclusion

The last years research has advanced to unravel how is the life cycle of the black truffle (Murat et al. 2013, Le Tacon et al. 2016, Taschen et al. 2016 and De la Varga et al. 2017), but there are still some points to be clarified. More research needs to be done to explain how and why one individual act as female or male element, especially how this is regulated. Those findings would have an impact in truffle industry, as methodologies to promote mating will lets us to have more control on the production of truffle ascocarps.

References

De la Varga, H., Le Tacon, F., Lagoguet, M., Todesco, F., Varga, T., Miquel, I., Barry-Etienne, D., Robin, C., Halkett, F., Martin, F. and Murat, C. (2017), Five years investigation of female and male genotypes in Périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies. Environmental Microbiology. Accepted Author Manuscript. doi:10.1111/1462-2920.13735

Le Tacon, F., Rubini, A., Murat, C., Riccioni, C., Robin, C., Belfiori, B., Zeller, B., De la Varga, H., Akroume, E., Deveau, A., Martin, F., Paolocci, F. (2016) Certainties and uncertainties about the life cycle of the Périgord black truffle (Tuber melanosporum Vittad.). Annals of Forest Science, 73: 105. doi:10.1007/s13595-015-0461-1

Martin, F., Kohler, A., Murat, C., Balestrini, R., Coutinho, P.M., Jaillon, O., et al. (2010) Perigord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis. Nature. 464(7291):1033–8.

Mello, A., Murat, C., Bonfante, P. (2006). Truffles: much more than a prized and local fungal delicacy. FEMS Microbiology Letters 260, 1–8.

Murat, C. (2015) Forty years of inoculating seedlings with truffle fungi: past and future perspectives. Mycorrhiza 25, 77–81.

Murat, C., Rubini, A., Riccioni, C., De la Varga, H., Akroume, E., Belfiori, B., Guaragno, M., Le Tacon, F., Robin, C., Halkett, F., Martin, F. and Paolocci, F. (2013), Fine-scale spatial genetic structure of the black truffle (Tuber melanosporum) investigated with neutral microsatellites and functional mating type genes. New Phytologist, 199: 176–187. doi:10.1111/nph.12264

Murat, C., Bonneau, L., De la Varga, H., Olivier, J.M., Sandrine, F. and Le Tacon, F. (2016) Trapping truffle production in holes: a promising technique for improving production and unravelling truffle life cycle. Italian Journal of Mycology 45. doi: 10.6082/issn.2531-7342/6346

Rubini, A., Belfiori, B., Riccioni, C., Tisserant, E., Arcioni, S., Martin, F., and Paolocci, F. (2011) Isolation and characterization of MAT genes in the symbiotic ascomycete Tuber melanosporum. New Phytologist 189: 710 722.

Taschen, E., Rousset, F., Sauve, M., Benoit, L., Dubois, M.-P., Richard, F. and Selosse, M.-A. (2016), How the truffle got its mate: insights from genetic structure in spontaneous and planted Mediterranean populations of Tuber melanosporum. Molecular Ecology, 25: 5611–5627. doi:10.1111/mec.1386

 

Here a link for the pdf of this paper: https://www.slideshare.net/micofora/new-considerations-of-tuber-melanosporum-life-cycle-1

 

 

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how to decide a layout for truffle farming in the USA?


The last two weeks we have been visiting 8 truffle farming projects in the USA with @xvilanovasola, mostly in California and in North Carolina. Let me comment some of the issues we mostly find and to compare the climates and soils.

In Ashville (NC) is probably where we find temperatures more similar to the ones in Catalonia. i.e. 23,7ºC as average of the hottest month (July) and 2,8ºC as average in the coldest month (january). Here black truffle will enjoy direct sunlight to the ground, so better make layouts with rows North-South with low density (150 trees/acre).

Ashville 2ashville 1

In this area it rains all year round, with 70-80mm every month, so some orchards would not even need irrigation during fruiting stage.

On the other hand, in California temperatures are quite higher, specially the average of the maximum in the hottest month. The amplitude of temperatures during day & night are huge, so truffle will probably look for some shade in some areas, so we can design layouts a little more dense or with rows East-West.

sonoma climate truffles micoforaSonoma valley truffle growing Morcillo

Another issue that may raise is when truffle season ends in mid march, we have to do all tasks in a truffle orchard, this is to prune, chop branches, add truffle spores or spanish wells and rip or till before spring starts. But in CA most trees already move/sprout in late march, we also found mycorrhizae were active in most samples in early april. This means that the “window” to do more tasks is shorter than in Spain. 2 weeks in CA while 4-5 weeks in Spain. And in north California rains in march so soil may be too wet to work before spring comes…

Here two pics one with old Quercus suber and another with Q. robur & pubescens with black truffle. The first with an excessive canopy and the second will close soon if trees are not heavily pruned:

quercus suber produces trufflesquercus robur mycorrhized with black truffle micofora

Rain is one of the main problems in the area north of San Francisco, like Napa and Sonoma Valley, specially in winter where the amount of water is pretty high at the time that truffle are harvested. This will create problems in the orchards in the valleys, with clay soils and bad drainage. Ripping or improving drainage (french drains) will be a must to avoid truffle rot.

We found several orchards with excessive tree density that need to be thinned, trees toped or heavily pruned:

hazel mycorrhized with black truffle micoforaXavier Vilanova samples a filbert truffle orchard

Cheers,

Marcos S. Morcillo & Xavier Vilanova

 

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Truffle Growing in México


truffle growing in México

I attach a picture of the 1300 Pinus pinea that we planted in Zumpango, México in 2015 and their evolution since planted.
In another pilot plot more to the north we have 1 hectare with 250 stone pines plus another with Pinus cembroides. This other pine also produces edible nuts. In the same plantation we have some Mexican native oaks mycorrhized as well.

The mycorrhizal levels of the samples sent to our lab in Barcelona are very good and we will try this spring to quantify the concentration of mycelium of Borchii with the molecular technique Real Time PCR.
pino micorrizado borchii México
We are considering the possibility to graft these pines with highly productive clones of pine cones, to shorten the waiting time to pine cones production and increase nuts yield.
Another key factor for these plantations to enter into production will be to add special substrates with bianchetto spores to provide the “male factor” that can fertilize the maternal part found in the soil and mycorrhizae, as we do with the spanish wells with the black truffle.
Piñonero Zumpango trufa Méjico
These pilot plantations will be the first to produce truffles and European pine nuts jointly, two non-timber products of high market value.

If you want to know more about the reproduction of this truffle:

Characterization of the reproductive mode and life cycle of the whitish truffle T. borchii

Cheers from California, where my partner Xavier Vilanova and I are visiting several truffle projects. Will keep you posted!

Note the next International Workshop on Edible Mycorrhizal Mushrooms  IWEMM9 will be this summer in Mexico

 Marcos S. Morcillo

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